Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali

Association between mutant E6 and E7 Human... | F1000Research "use strict";function _typeof(t){return(_typeof="function"==typeof Symbol&&"symbol"==typeof Symbol.iterator?function(t){return typeof t}:function(t){return t&&"function"==typeof Symbol&&t.constructor===Symbol&&t!==Symbol.prototype?"symbol":typeof t})(t)}!function(){var t=function(){var t,e,o=[],n=window,r=n;for(;r;){try{if(r.frames.__tcfapiLocator){t=r;break}}catch(t){}if(r===n.top)break;r=r.parent}t||(!function t(){var e=n.document,o=!!n.frames.__tcfapiLocator;if(!o)if(e.body){var r=e.createElement("iframe");r.style.cssText="display:none",r.name="__tcfapiLocator",e.body.appendChild(r)}else setTimeout(t,5);return!o}(),n.__tcfapi=function(){for(var t=arguments.length,n=new Array(t),r=0;r 3&&2===parseInt(n[1],10)&&"boolean"==typeof n[3]&&(e=n[3],"function"==typeof n[2]&&n[2]("set",!0)):"ping"===n[0]?"function"==typeof n[2]&&n[2]({gdprApplies:e,cmpLoaded:!1,cmpStatus:"stub"}):o.push(n)},n.addEventListener("message",(function(t){var e="string"==typeof t.data,o={};if(e)try{o=JSON.parse(t.data)}catch(t){}else o=t.data;var n="object"===_typeof(o)&&null!==o?o.__tcfapiCall:null;n&&window.__tcfapi(n.command,n.version,(function(o,r){var a={__tcfapiReturn:{returnValue:o,success:r,callId:n.callId}};t&&t.source&&t.source.postMessage&&t.source.postMessage(e?JSON.stringify(a):a,"*")}),n.parameter)}),!1))};"undefined"!=typeof module?module.exports=t:t()}(); dataLayer = dataLayer || []; // Standard GTM initialization - Google Consent Mode handles consent automatically (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start': new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0], j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src= 'https://www.googletagmanager.com/gtm.js?id='+i+dl+ '>m_auth=hzk0Vc3qFsQYhCrIoHz68A>m_preview=env-1>m_cookies_win=x';f.parentNode.insertBefore(j,f); })(window,document,'script','dataLayer','GTM-MWFK8L5J'); ;window.NREUM||(NREUM={});NREUM.init={distributed_tracing:{enabled:true},privacy:{cookies_enabled:true},ajax:{deny_list:["bam.nr-data.net"]}}; ;NREUM.loader_config={accountID:"438030",trustKey:"438030",agentID:"772317073",licenseKey:"97f8f67f26",applicationID:"772317073"} ;NREUM.info={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net",licenseKey:"97f8f67f26",applicationID:"772317073",sa:1} ;/*! For license information please see nr-loader-spa-1.236.0.min.js.LICENSE.txt */ (()=>{"use strict";var e,t,r={5763:(e,t,r)=>{r.d(t,{P_:()=>l,Mt:()=>g,C5:()=>s,DL:()=>v,OP:()=>T,lF:()=>D,Yu:()=>y,Dg:()=>h,CX:()=>c,GE:()=>b,sU:()=>_});var n=r(8632),i=r(9567);const o={beacon:n.ce.beacon,errorBeacon:n.ce.errorBeacon,licenseKey:void 0,applicationID:void 0,sa:void 0,queueTime:void 0,applicationTime:void 0,ttGuid:void 0,user:void 0,account:void 0,product:void 0,extra:void 0,jsAttributes:{},userAttributes:void 0,atts:void 0,transactionName:void 0,tNamePlain:void 0},a={};function s(e){if(!e)throw new Error("All info objects require an agent identifier!");if(!a[e])throw new Error("Info for ".concat(e," was never set"));return a[e]}function c(e,t){if(!e)throw new Error("All info objects require an agent identifier!");a[e]=(0,i.D)(t,o),(0,n.Qy)(e,a[e],"info")}var u=r(7056);const d=()=>{const e={blockSelector:"[data-nr-block]",maskInputOptions:{password:!0}};return{allow_bfcache:!0,privacy:{cookies_enabled:!0},ajax:{deny_list:void 0,enabled:!0,harvestTimeSeconds:10},distributed_tracing:{enabled:void 0,exclude_newrelic_header:void 0,cors_use_newrelic_header:void 0,cors_use_tracecontext_headers:void 0,allowed_origins:void 0},session:{domain:void 0,expiresMs:u.oD,inactiveMs:u.Hb},ssl:void 0,obfuscate:void 0,jserrors:{enabled:!0,harvestTimeSeconds:10},metrics:{enabled:!0},page_action:{enabled:!0,harvestTimeSeconds:30},page_view_event:{enabled:!0},page_view_timing:{enabled:!0,harvestTimeSeconds:30,long_task:!1},session_trace:{enabled:!0,harvestTimeSeconds:10},harvest:{tooManyRequestsDelay:60},session_replay:{enabled:!1,harvestTimeSeconds:60,sampleRate:.1,errorSampleRate:.1,maskTextSelector:"*",maskAllInputs:!0,get blockClass(){return"nr-block"},get ignoreClass(){return"nr-ignore"},get maskTextClass(){return"nr-mask"},get blockSelector(){return e.blockSelector},set blockSelector(t){e.blockSelector+=",".concat(t)},get maskInputOptions(){return e.maskInputOptions},set maskInputOptions(t){e.maskInputOptions={...t,password:!0}}},spa:{enabled:!0,harvestTimeSeconds:10}}},f={};function l(e){if(!e)throw new Error("All configuration objects require an agent identifier!");if(!f[e])throw new Error("Configuration for ".concat(e," was never set"));return f[e]}function h(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");f[e]=(0,i.D)(t,d()),(0,n.Qy)(e,f[e],"config")}function g(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");var r=l(e);if(r){for(var n=t.split("."),i=0;i {r.d(t,{D:()=>i});var n=r(50);function i(e,t){try{if(!e||"object"!=typeof e)return(0,n.Z)("Setting a Configurable requires an object as input");if(!t||"object"!=typeof t)return(0,n.Z)("Setting a Configurable requires a model to set its initial properties");const r=Object.create(Object.getPrototypeOf(t),Object.getOwnPropertyDescriptors(t)),o=0===Object.keys(r).length?e:r;for(let a in o)if(void 0!==e[a])try{"object"==typeof e[a]&&"object"==typeof t[a]?r[a]=i(e[a],t[a]):r[a]=e[a]}catch(e){(0,n.Z)("An error occurred while setting a property of a Configurable",e)}return r}catch(e){(0,n.Z)("An error occured while setting a Configurable",e)}}},6818:(e,t,r)=>{r.d(t,{Re:()=>i,gF:()=>o,q4:()=>n});const n="1.236.0",i="PROD",o="CDN"},385:(e,t,r)=>{r.d(t,{FN:()=>a,IF:()=>u,Nk:()=>f,Tt:()=>s,_A:()=>o,il:()=>n,pL:()=>c,v6:()=>i,w1:()=>d});const n="undefined"!=typeof window&&!!window.document,i="undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self.navigator instanceof WorkerNavigator||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis.navigator instanceof WorkerNavigator),o=n?window:"undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis),a=""+o?.location,s=/iPad|iPhone|iPod/.test(navigator.userAgent),c=s&&"undefined"==typeof SharedWorker,u=(()=>{const e=navigator.userAgent.match(/Firefox[/\s](\d+\.\d+)/);return Array.isArray(e)&&e.length>=2?+e[1]:0})(),d=Boolean(n&&window.document.documentMode),f=!!navigator.sendBeacon},1117:(e,t,r)=>{r.d(t,{w:()=>o});var n=r(50);const i={agentIdentifier:"",ee:void 0};class o{constructor(e){try{if("object"!=typeof e)return(0,n.Z)("shared context requires an object as input");this.sharedContext={},Object.assign(this.sharedContext,i),Object.entries(e).forEach((e=>{let[t,r]=e;Object.keys(i).includes(t)&&(this.sharedContext[t]=r)}))}catch(e){(0,n.Z)("An error occured while setting SharedContext",e)}}}},8e3:(e,t,r)=>{r.d(t,{L:()=>d,R:()=>c});var n=r(2177),i=r(1284),o=r(4322),a=r(3325);const s={};function c(e,t){const r={staged:!1,priority:a.p[t]||0};u(e),s[e].get(t)||s[e].set(t,r)}function u(e){e&&(s[e]||(s[e]=new Map))}function d(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:"",t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:"feature";if(u(e),!e||!s[e].get(t))return a(t);s[e].get(t).staged=!0;const r=[...s[e]];function a(t){const r=e?n.ee.get(e):n.ee,a=o.X.handlers;if(r.backlog&&a){var s=r.backlog[t],c=a[t];if(c){for(var u=0;s&&u {let[t,r]=e;return r.staged}))&&(r.sort(((e,t)=>e[1].priority-t[1].priority)),r.forEach((e=>{let[t]=e;a(t)})))}function f(e,t){var r=e[1];(0,i.D)(t[r],(function(t,r){var n=e[0];if(r[0]===n){var i=r[1],o=e[3],a=e[2];i.apply(o,a)}}))}},2177:(e,t,r)=>{r.d(t,{c:()=>f,ee:()=>u});var n=r(8632),i=r(2210),o=r(1284),a=r(5763),s="nr@context";let c=(0,n.fP)();var u;function d(){}function f(e){return(0,i.X)(e,s,l)}function l(){return new d}function h(){u.aborted=!0,u.backlog={}}c.ee?u=c.ee:(u=function e(t,r){var n={},c={},f={},g=!1;try{g=16===r.length&&(0,a.OP)(r).isolatedBacklog}catch(e){}var p={on:b,addEventListener:b,removeEventListener:y,emit:v,get:x,listeners:w,context:m,buffer:A,abort:h,aborted:!1,isBuffering:E,debugId:r,backlog:g?{}:t&&"object"==typeof t.backlog?t.backlog:{}};return p;function m(e){return e&&e instanceof d?e:e?(0,i.X)(e,s,l):l()}function v(e,r,n,i,o){if(!1!==o&&(o=!0),!u.aborted||i){t&&o&&t.emit(e,r,n);for(var a=m(n),s=w(e),d=s.length,f=0;fn,p:()=>i});var n=r(2177).ee.get("handle");function i(e,t,r,i,o){o?(o.buffer([e],i),o.emit(e,t,r)):(n.buffer([e],i),n.emit(e,t,r))}},4322:(e,t,r)=>{r.d(t,{X:()=>o});var n=r(5546);o.on=a;var i=o.handlers={};function o(e,t,r,o){a(o||n.E,i,e,t,r)}function a(e,t,r,i,o){o||(o="feature"),e||(e=n.E);var a=t[o]=t[o]||{};(a[r]=a[r]||[]).push([e,i])}},3239:(e,t,r)=>{r.d(t,{bP:()=>s,iz:()=>c,m$:()=>a});var n=r(385);let i=!1,o=!1;try{const e={get passive(){return i=!0,!1},get signal(){return o=!0,!1}};n._A.addEventListener("test",null,e),n._A.removeEventListener("test",null,e)}catch(e){}function a(e,t){return i||o?{capture:!!e,passive:i,signal:t}:!!e}function s(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;window.addEventListener(e,t,a(r,n))}function c(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;document.addEventListener(e,t,a(r,n))}},4402:(e,t,r)=>{r.d(t,{Ht:()=>u,M:()=>c,Rl:()=>a,ky:()=>s});var n=r(385);const i="xxxxxxxx-xxxx-4xxx-yxxx-xxxxxxxxxxxx";function o(e,t){return e?15&e[t]:16*Math.random()|0}function a(){const e=n._A?.crypto||n._A?.msCrypto;let t,r=0;return e&&e.getRandomValues&&(t=e.getRandomValues(new Uint8Array(31))),i.split("").map((e=>"x"===e?o(t,++r).toString(16):"y"===e?(3&o()|8).toString(16):e)).join("")}function s(e){const t=n._A?.crypto||n._A?.msCrypto;let r,i=0;t&&t.getRandomValues&&(r=t.getRandomValues(new Uint8Array(31)));const a=[];for(var s=0;s {r.d(t,{Bq:()=>n,Hb:()=>o,oD:()=>i});const n="NRBA",i=144e5,o=18e5},7894:(e,t,r)=>{function n(){return Math.round(performance.now())}r.d(t,{z:()=>n})},7243:(e,t,r)=>{r.d(t,{e:()=>o});var n=r(385),i={};function o(e){if(e in i)return i[e];if(0===(e||"").indexOf("data:"))return{protocol:"data"};let t;var r=n._A?.location,o={};if(n.il)t=document.createElement("a"),t.href=e;else try{t=new URL(e,r.href)}catch(e){return o}o.port=t.port;var a=t.href.split("://");!o.port&&a[1]&&(o.port=a[1].split("/")[0].split("@").pop().split(":")[1]),o.port&&"0"!==o.port||(o.port="https"===a[0]?"443":"80"),o.hostname=t.hostname||r.hostname,o.pathname=t.pathname,o.protocol=a[0],"/"!==o.pathname.charAt(0)&&(o.pathname="/"+o.pathname);var s=!t.protocol||":"===t.protocol||t.protocol===r.protocol,c=t.hostname===r.hostname&&t.port===r.port;return o.sameOrigin=s&&(!t.hostname||c),"/"===o.pathname&&(i[e]=o),o}},50:(e,t,r)=>{function n(e,t){"function"==typeof console.warn&&(console.warn("New Relic: ".concat(e)),t&&console.warn(t))}r.d(t,{Z:()=>n})},2587:(e,t,r)=>{r.d(t,{N:()=>c,T:()=>u});var n=r(2177),i=r(5546),o=r(8e3),a=r(3325);const s={stn:[a.D.sessionTrace],err:[a.D.jserrors,a.D.metrics],ins:[a.D.pageAction],spa:[a.D.spa],sr:[a.D.sessionReplay,a.D.sessionTrace]};function c(e,t){const r=n.ee.get(t);e&&"object"==typeof e&&(Object.entries(e).forEach((e=>{let[t,n]=e;void 0===u[t]&&(s[t]?s[t].forEach((e=>{n?(0,i.p)("feat-"+t,[],void 0,e,r):(0,i.p)("block-"+t,[],void 0,e,r),(0,i.p)("rumresp-"+t,[Boolean(n)],void 0,e,r)})):n&&(0,i.p)("feat-"+t,[],void 0,void 0,r),u[t]=Boolean(n))})),Object.keys(s).forEach((e=>{void 0===u[e]&&(s[e]?.forEach((t=>(0,i.p)("rumresp-"+e,[!1],void 0,t,r))),u[e]=!1)})),(0,o.L)(t,a.D.pageViewEvent))}const u={}},2210:(e,t,r)=>{r.d(t,{X:()=>i});var n=Object.prototype.hasOwnProperty;function i(e,t,r){if(n.call(e,t))return e[t];var i=r();if(Object.defineProperty&&Object.keys)try{return Object.defineProperty(e,t,{value:i,writable:!0,enumerable:!1}),i}catch(e){}return e[t]=i,i}},1284:(e,t,r)=>{r.d(t,{D:()=>n});const n=(e,t)=>Object.entries(e||{}).map((e=>{let[r,n]=e;return t(r,n)}))},4351:(e,t,r)=>{r.d(t,{P:()=>o});var n=r(2177);const i=()=>{const e=new WeakSet;return(t,r)=>{if("object"==typeof r&&null!==r){if(e.has(r))return;e.add(r)}return r}};function o(e){try{return JSON.stringify(e,i())}catch(e){try{n.ee.emit("internal-error",[e])}catch(e){}}}},3960:(e,t,r)=>{r.d(t,{K:()=>a,b:()=>o});var n=r(3239);function i(){return"undefined"==typeof document||"complete"===document.readyState}function o(e,t){if(i())return e();(0,n.bP)("load",e,t)}function a(e){if(i())return e();(0,n.iz)("DOMContentLoaded",e)}},8632:(e,t,r)=>{r.d(t,{EZ:()=>u,Qy:()=>c,ce:()=>o,fP:()=>a,gG:()=>d,mF:()=>s});var n=r(7894),i=r(385);const o={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net"};function a(){return i._A.NREUM||(i._A.NREUM={}),void 0===i._A.newrelic&&(i._A.newrelic=i._A.NREUM),i._A.NREUM}function s(){let e=a();return e.o||(e.o={ST:i._A.setTimeout,SI:i._A.setImmediate,CT:i._A.clearTimeout,XHR:i._A.XMLHttpRequest,REQ:i._A.Request,EV:i._A.Event,PR:i._A.Promise,MO:i._A.MutationObserver,FETCH:i._A.fetch}),e}function c(e,t,r){let i=a();const o=i.initializedAgents||{},s=o[e]||{};return Object.keys(s).length||(s.initializedAt={ms:(0,n.z)(),date:new Date}),i.initializedAgents={...o,[e]:{...s,[r]:t}},i}function u(e,t){a()[e]=t}function d(){return function(){let e=a();const t=e.info||{};e.info={beacon:o.beacon,errorBeacon:o.errorBeacon,...t}}(),function(){let e=a();const t=e.init||{};e.init={...t}}(),s(),function(){let e=a();const t=e.loader_config||{};e.loader_config={...t}}(),a()}},7956:(e,t,r)=>{r.d(t,{N:()=>i});var n=r(3239);function i(e){let t=arguments.length>1&&void 0!==arguments[1]&&arguments[1],r=arguments.length>2?arguments[2]:void 0,i=arguments.length>3?arguments[3]:void 0;return void(0,n.iz)("visibilitychange",(function(){if(t)return void("hidden"==document.visibilityState&&e());e(document.visibilityState)}),r,i)}},1214:(e,t,r)=>{r.d(t,{em:()=>v,u5:()=>N,QU:()=>S,_L:()=>I,Gm:()=>L,Lg:()=>M,gy:()=>U,BV:()=>Q,Kf:()=>ee});var n=r(2177);const i="nr@original";var o=Object.prototype.hasOwnProperty,a=!1;function s(e,t){return e||(e=n.ee),r.inPlace=function(e,t,n,i,o){n||(n="");var a,s,c,u="-"===n.charAt(0);for(c=0;c 2?n-2:0),o=2;o {r(A[T],e,w),r(E[T],e,w)})),r(l._A,"fetch",y),t.on(y+"end",(function(e,r){var n=this;if(r){var i=r.headers.get("content-length");null!==i&&(n.rxSize=i),t.emit(y+"done",[null,r],n)}else t.emit(y+"done",[e],n)})),t}const O={},j=["pushState","replaceState"];function S(e){const t=function(e){return(e||n.ee).get("history")}(e);return!l.il||O[t.debugId]++||(O[t.debugId]=1,s(t).inPlace(window.history,j,"-")),t}var P=r(3239);const C={},R=["appendChild","insertBefore","replaceChild"];function I(e){const t=function(e){return(e||n.ee).get("jsonp")}(e);if(!l.il||C[t.debugId])return t;C[t.debugId]=!0;var r=s(t),i=/[?&](?:callback|cb)=([^&#]+)/,o=/(.*)\.([^.]+)/,a=/^(\w+)(\.|$)(.*)$/;function c(e,t){var r=e.match(a),n=r[1],i=r[3];return i?c(i,t[n]):t[n]}return r.inPlace(Node.prototype,R,"dom-"),t.on("dom-start",(function(e){!function(e){if(!e||"string"!=typeof e.nodeName||"script"!==e.nodeName.toLowerCase())return;if("function"!=typeof e.addEventListener)return;var n=(a=e.src,s=a.match(i),s?s[1]:null);var a,s;if(!n)return;var u=function(e){var t=e.match(o);if(t&&t.length>=3)return{key:t[2],parent:c(t[1],window)};return{key:e,parent:window}}(n);if("function"!=typeof u.parent[u.key])return;var d={};function f(){t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}function l(){t.emit("jsonp-error",[],d),t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}r.inPlace(u.parent,[u.key],"cb-",d),e.addEventListener("load",f,(0,P.m$)(!1)),e.addEventListener("error",l,(0,P.m$)(!1)),t.emit("new-jsonp",[e.src],d)}(e[0])})),t}var k=r(5763);const H={};function L(e){const t=function(e){return(e||n.ee).get("mutation")}(e);if(!l.il||H[t.debugId])return t;H[t.debugId]=!0;var r=s(t),i=k.Yu.MO;return i&&(window.MutationObserver=function(e){return this instanceof i?new i(r(e,"fn-")):i.apply(this,arguments)},MutationObserver.prototype=i.prototype),t}const z={};function M(e){const t=function(e){return(e||n.ee).get("promise")}(e);if(z[t.debugId])return t;z[t.debugId]=!0;var r=n.c,o=s(t),a=k.Yu.PR;return a&&function(){function e(r){var n=t.context(),i=o(r,"executor-",n,null,!1);const s=Reflect.construct(a,[i],e);return t.context(s).getCtx=function(){return n},s}l._A.Promise=e,Object.defineProperty(e,"name",{value:"Promise"}),e.toString=function(){return a.toString()},Object.setPrototypeOf(e,a),["all","race"].forEach((function(r){const n=a[r];e[r]=function(e){let i=!1;[...e||[]].forEach((e=>{this.resolve(e).then(a("all"===r),a(!1))}));const o=n.apply(this,arguments);return o;function a(e){return function(){t.emit("propagate",[null,!i],o,!1,!1),i=i||!e}}}})),["resolve","reject"].forEach((function(r){const n=a[r];e[r]=function(e){const r=n.apply(this,arguments);return e!==r&&t.emit("propagate",[e,!0],r,!1,!1),r}})),e.prototype=a.prototype;const n=a.prototype.then;a.prototype.then=function(){var e=this,i=r(e);i.promise=e;for(var a=arguments.length,s=new Array(a),c=0;c e())),t};function m(e,t){i.inPlace(t,["onreadystatechange"],"fn-",E)}function b(){var e=this,t=r.context(e);e.readyState>3&&!t.resolved&&(t.resolved=!0,r.emit("xhr-resolved",[],e)),i.inPlace(e,f,"fn-",E)}if(function(e,t){for(var r in e)t[r]=e[r]}(o,p),p.prototype=o.prototype,i.inPlace(p.prototype,J,"-xhr-",E),r.on("send-xhr-start",(function(e,t){m(e,t),function(e){h.push(e),a&&(y?y.then(A):u?u(A):(w=-w,x.data=w))}(t)})),r.on("open-xhr-start",m),a){var y=c&&c.resolve();if(!u&&!c){var w=1,x=document.createTextNode(w);new a(A).observe(x,{characterData:!0})}}else t.on("fn-end",(function(e){e[0]&&e[0].type===d||A()}));function A(){for(var e=0;e {r.d(t,{t:()=>n});const n=r(3325).D.ajax},6660:(e,t,r)=>{r.d(t,{A:()=>i,t:()=>n});const n=r(3325).D.jserrors,i="nr@seenError"},3081:(e,t,r)=>{r.d(t,{gF:()=>o,mY:()=>i,t9:()=>n,vz:()=>s,xS:()=>a});const n=r(3325).D.metrics,i="sm",o="cm",a="storeSupportabilityMetrics",s="storeEventMetrics"},4649:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageAction},7633:(e,t,r)=>{r.d(t,{Dz:()=>i,OJ:()=>a,qw:()=>o,t9:()=>n});const n=r(3325).D.pageViewEvent,i="firstbyte",o="domcontent",a="windowload"},9251:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageViewTiming},3614:(e,t,r)=>{r.d(t,{BST_RESOURCE:()=>i,END:()=>s,FEATURE_NAME:()=>n,FN_END:()=>u,FN_START:()=>c,PUSH_STATE:()=>d,RESOURCE:()=>o,START:()=>a});const n=r(3325).D.sessionTrace,i="bstResource",o="resource",a="-start",s="-end",c="fn"+a,u="fn"+s,d="pushState"},7836:(e,t,r)=>{r.d(t,{BODY:()=>A,CB_END:()=>E,CB_START:()=>u,END:()=>x,FEATURE_NAME:()=>i,FETCH:()=>_,FETCH_BODY:()=>v,FETCH_DONE:()=>m,FETCH_START:()=>p,FN_END:()=>c,FN_START:()=>s,INTERACTION:()=>l,INTERACTION_API:()=>d,INTERACTION_EVENTS:()=>o,JSONP_END:()=>b,JSONP_NODE:()=>g,JS_TIME:()=>T,MAX_TIMER_BUDGET:()=>a,REMAINING:()=>f,SPA_NODE:()=>h,START:()=>w,originalSetTimeout:()=>y});var n=r(5763);const i=r(3325).D.spa,o=["click","submit","keypress","keydown","keyup","change"],a=999,s="fn-start",c="fn-end",u="cb-start",d="api-ixn-",f="remaining",l="interaction",h="spaNode",g="jsonpNode",p="fetch-start",m="fetch-done",v="fetch-body-",b="jsonp-end",y=n.Yu.ST,w="-start",x="-end",A="-body",E="cb"+x,T="jsTime",_="fetch"},5938:(e,t,r)=>{r.d(t,{W:()=>o});var n=r(5763),i=r(2177);class o{constructor(e,t,r){this.agentIdentifier=e,this.aggregator=t,this.ee=i.ee.get(e,(0,n.OP)(this.agentIdentifier).isolatedBacklog),this.featureName=r,this.blocked=!1}}},9144:(e,t,r)=>{r.d(t,{j:()=>m});var n=r(3325),i=r(5763),o=r(5546),a=r(2177),s=r(7894),c=r(8e3),u=r(3960),d=r(385),f=r(50),l=r(3081),h=r(8632);function g(){const e=(0,h.gG)();["setErrorHandler","finished","addToTrace","inlineHit","addRelease","addPageAction","setCurrentRouteName","setPageViewName","setCustomAttribute","interaction","noticeError","setUserId"].forEach((t=>{e[t]=function(){for(var r=arguments.length,n=new Array(r),i=0;i 1?r-1:0),i=1;i {e.exposed&&e.api[t]&&o.push(e.api[t](...n))})),o.length>1?o:o[0]}(t,...n)}}))}var p=r(2587);function m(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:{},m=arguments.length>2?arguments[2]:void 0,v=arguments.length>3?arguments[3]:void 0,{init:b,info:y,loader_config:w,runtime:x={loaderType:m},exposed:A=!0}=t;const E=(0,h.gG)();y||(b=E.init,y=E.info,w=E.loader_config),(0,i.Dg)(e,b||{}),(0,i.GE)(e,w||{}),(0,i.sU)(e,x),y.jsAttributes??={},d.v6&&(y.jsAttributes.isWorker=!0),(0,i.CX)(e,y),g();const T=function(e,t){t||(0,c.R)(e,"api");const h={};var g=a.ee.get(e),p=g.get("tracer"),m="api-",v=m+"ixn-";function b(t,r,n,o){const a=(0,i.C5)(e);return null===r?delete a.jsAttributes[t]:(0,i.CX)(e,{...a,jsAttributes:{...a.jsAttributes,[t]:r}}),x(m,n,!0,o||null===r?"session":void 0)(t,r)}function y(){}["setErrorHandler","finished","addToTrace","inlineHit","addRelease"].forEach((e=>h[e]=x(m,e,!0,"api"))),h.addPageAction=x(m,"addPageAction",!0,n.D.pageAction),h.setCurrentRouteName=x(m,"routeName",!0,n.D.spa),h.setPageViewName=function(t,r){if("string"==typeof t)return"/"!==t.charAt(0)&&(t="/"+t),(0,i.OP)(e).customTransaction=(r||"http://custom.transaction")+t,x(m,"setPageViewName",!0)()},h.setCustomAttribute=function(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2];if("string"==typeof e){if(["string","number"].includes(typeof t)||null===t)return b(e,t,"setCustomAttribute",r);(0,f.Z)("Failed to execute setCustomAttribute.\nNon-null value must be a string or number type, but a type of was provided."))}else(0,f.Z)("Failed to execute setCustomAttribute.\nName must be a string type, but a type of was provided."))},h.setUserId=function(e){if("string"==typeof e||null===e)return b("enduser.id",e,"setUserId",!0);(0,f.Z)("Failed to execute setUserId.\nNon-null value must be a string type, but a type of was provided."))},h.interaction=function(){return(new y).get()};var w=y.prototype={createTracer:function(e,t){var r={},i=this,a="function"==typeof t;return(0,o.p)(v+"tracer",[(0,s.z)(),e,r],i,n.D.spa,g),function(){if(p.emit((a?"":"no-")+"fn-start",[(0,s.z)(),i,a],r),a)try{return t.apply(this,arguments)}catch(e){throw p.emit("fn-err",[arguments,this,"string"==typeof e?new Error(e):e],r),e}finally{p.emit("fn-end",[(0,s.z)()],r)}}}};function x(e,t,r,i){return function(){return(0,o.p)(l.xS,["API/"+t+"/called"],void 0,n.D.metrics,g),i&&(0,o.p)(e+t,[(0,s.z)(),...arguments],r?null:this,i,g),r?void 0:this}}function A(){r.e(439).then(r.bind(r,7438)).then((t=>{let{setAPI:r}=t;r(e),(0,c.L)(e,"api")})).catch((()=>(0,f.Z)("Downloading runtime APIs failed...")))}return["actionText","setName","setAttribute","save","ignore","onEnd","getContext","end","get"].forEach((e=>{w[e]=x(v,e,void 0,n.D.spa)})),h.noticeError=function(e,t){"string"==typeof e&&(e=new Error(e)),(0,o.p)(l.xS,["API/noticeError/called"],void 0,n.D.metrics,g),(0,o.p)("err",[e,(0,s.z)(),!1,t],void 0,n.D.jserrors,g)},d.il?(0,u.b)((()=>A()),!0):A(),h}(e,v);return(0,h.Qy)(e,T,"api"),(0,h.Qy)(e,A,"exposed"),(0,h.EZ)("activatedFeatures",p.T),T}},3325:(e,t,r)=>{r.d(t,{D:()=>n,p:()=>i});const n={ajax:"ajax",jserrors:"jserrors",metrics:"metrics",pageAction:"page_action",pageViewEvent:"page_view_event",pageViewTiming:"page_view_timing",sessionReplay:"session_replay",sessionTrace:"session_trace",spa:"spa"},i={[n.pageViewEvent]:1,[n.pageViewTiming]:2,[n.metrics]:3,[n.jserrors]:4,[n.ajax]:5,[n.sessionTrace]:6,[n.pageAction]:7,[n.spa]:8,[n.sessionReplay]:9}}},n={};function i(e){var t=n[e];if(void 0!==t)return t.exports;var o=n[e]={exports:{}};return r[e](o,o.exports,i),o.exports}i.m=r,i.d=(e,t)=>{for(var r in t)i.o(t,r)&&!i.o(e,r)&&Object.defineProperty(e,r,{enumerable:!0,get:t[r]})},i.f={},i.e=e=>Promise.all(Object.keys(i.f).reduce(((t,r)=>(i.f[r](e,t),t)),[])),i.u=e=>(({78:"page_action-aggregate",147:"metrics-aggregate",242:"session-manager",317:"jserrors-aggregate",348:"page_view_timing-aggregate",412:"lazy-feature-loader",439:"async-api",538:"recorder",590:"session_replay-aggregate",675:"compressor",733:"session_trace-aggregate",786:"page_view_event-aggregate",873:"spa-aggregate",898:"ajax-aggregate"}[e]||e)+"."+{78:"ac76d497",147:"3dc53903",148:"1a20d5fe",242:"2a64278a",317:"49e41428",348:"bd6de33a",412:"2f55ce66",439:"30bd804e",538:"1b18459f",590:"cf0efb30",675:"ae9f91a8",733:"83105561",786:"06482edd",860:"03a8b7a5",873:"e6b09d52",898:"998ef92b"}[e]+"-1.236.0.min.js"),i.o=(e,t)=>Object.prototype.hasOwnProperty.call(e,t),e={},t="NRBA:",i.l=(r,n,o,a)=>{if(e[r])e[r].push(n);else{var s,c;if(void 0!==o)for(var u=document.getElementsByTagName("script"),d=0;d {s.onerror=s.onload=null,clearTimeout(h);var i=e[r];if(delete e[r],s.parentNode&&s.parentNode.removeChild(s),i&&i.forEach((e=>e(n))),t)return t(n)},h=setTimeout(l.bind(null,void 0,{type:"timeout",target:s}),12e4);s.onerror=l.bind(null,s.onerror),s.onload=l.bind(null,s.onload),c&&document.head.appendChild(s)}},i.r=e=>{"undefined"!=typeof Symbol&&Symbol.toStringTag&&Object.defineProperty(e,Symbol.toStringTag,{value:"Module"}),Object.defineProperty(e,"__esModule",{value:!0})},i.j=364,i.p="https://js-agent.newrelic.com/",(()=>{var e={364:0,953:0};i.f.j=(t,r)=>{var n=i.o(e,t)?e[t]:void 0;if(0!==n)if(n)r.push(n[2]);else{var o=new Promise(((r,i)=>n=e[t]=[r,i]));r.push(n[2]=o);var a=i.p+i.u(t),s=new Error;i.l(a,(r=>{if(i.o(e,t)&&(0!==(n=e[t])&&(e[t]=void 0),n)){var o=r&&("load"===r.type?"missing":r.type),a=r&&r.target&&r.target.src;s.message="Loading chunk "+t+" failed.\n("+o+": "+a+")",s.name="ChunkLoadError",s.type=o,s.request=a,n[1](s)}}),"chunk-"+t,t)}};var t=(t,r)=>{var n,o,[a,s,c]=r,u=0;if(a.some((t=>0!==e[t]))){for(n in s)i.o(s,n)&&(i.m[n]=s[n]);if(c)c(i)}for(t&&t(r);u {i.r(o);var e=i(3325),t=i(5763);const r=Object.values(e.D);function n(e){const n={};return r.forEach((r=>{n[r]=function(e,r){return!1!==(0,t.Mt)(r,"".concat(e,".enabled"))}(r,e)})),n}var a=i(9144);var s=i(5546),c=i(385),u=i(8e3),d=i(5938),f=i(3960),l=i(50);class h extends d.W{constructor(e,t,r){let n=!(arguments.length>3&&void 0!==arguments[3])||arguments[3];super(e,t,r),this.auto=n,this.abortHandler,this.featAggregate,this.onAggregateImported,n&&(0,u.R)(e,r)}importAggregator(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:{};if(this.featAggregate||!this.auto)return;const r=c.il&&!0===(0,t.Mt)(this.agentIdentifier,"privacy.cookies_enabled");let n;this.onAggregateImported=new Promise((e=>{n=e}));const o=async()=>{let t;try{if(r){const{setupAgentSession:e}=await Promise.all([i.e(860),i.e(242)]).then(i.bind(i,3228));t=e(this.agentIdentifier)}}catch(e){(0,l.Z)("A problem occurred when starting up session manager. This page will not start or extend any session.",e)}try{if(!this.shouldImportAgg(this.featureName,t))return void(0,u.L)(this.agentIdentifier,this.featureName);const{lazyFeatureLoader:r}=await i.e(412).then(i.bind(i,8582)),{Aggregate:o}=await r(this.featureName,"aggregate");this.featAggregate=new o(this.agentIdentifier,this.aggregator,e),n(!0)}catch(e){(0,l.Z)("Downloading and initializing ".concat(this.featureName," failed..."),e),this.abortHandler?.(),n(!1)}};c.il?(0,f.b)((()=>o()),!0):o()}shouldImportAgg(r,n){return r!==e.D.sessionReplay||!1!==(0,t.Mt)(this.agentIdentifier,"session_trace.enabled")&&(!!n?.isNew||!!n?.state.sessionReplay)}}var g=i(7633),p=i(7894);class m extends h{static featureName=g.t9;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];if(super(r,n,g.t9,i),("undefined"==typeof PerformanceNavigationTiming||c.Tt)&&"undefined"!=typeof PerformanceTiming){const n=(0,t.OP)(r);n[g.Dz]=Math.max(Date.now()-n.offset,0),(0,f.K)((()=>n[g.qw]=Math.max((0,p.z)()-n[g.Dz],0))),(0,f.b)((()=>{const t=(0,p.z)();n[g.OJ]=Math.max(t-n[g.Dz],0),(0,s.p)("timing",["load",t],void 0,e.D.pageViewTiming,this.ee)}))}this.importAggregator()}}var v=i(1117),b=i(1284);class y extends v.w{constructor(e){super(e),this.aggregatedData={}}store(e,t,r,n,i){var o=this.getBucket(e,t,r,i);return o.metrics=function(e,t){t||(t={count:0});return t.count+=1,(0,b.D)(e,(function(e,r){t[e]=w(r,t[e])})),t}(n,o.metrics),o}merge(e,t,r,n,i){var o=this.getBucket(e,t,n,i);if(o.metrics){var a=o.metrics;a.count+=r.count,(0,b.D)(r,(function(e,t){if("count"!==e){var n=a[e],i=r[e];i&&!i.c?a[e]=w(i.t,n):a[e]=function(e,t){if(!t)return e;t.c||(t=x(t.t));return t.min=Math.min(e.min,t.min),t.max=Math.max(e.max,t.max),t.t+=e.t,t.sos+=e.sos,t.c+=e.c,t}(i,a[e])}}))}else o.metrics=r}storeMetric(e,t,r,n){var i=this.getBucket(e,t,r);return i.stats=w(n,i.stats),i}getBucket(e,t,r,n){this.aggregatedData[e]||(this.aggregatedData[e]={});var i=this.aggregatedData[e][t];return i||(i=this.aggregatedData[e][t]={params:r||{}},n&&(i.custom=n)),i}get(e,t){return t?this.aggregatedData[e]&&this.aggregatedData[e][t]:this.aggregatedData[e]}take(e){for(var t={},r="",n=!1,i=0;i t.max&&(t.max=e),e 2&&void 0!==arguments[2])||arguments[2];super(e,r,j.t,n),c.il&&((0,t.OP)(e).initHidden=Boolean("hidden"===document.visibilityState),(0,N.N)((()=>(0,s.p)("docHidden",[(0,p.z)()],void 0,j.t,this.ee)),!0),(0,O.bP)("pagehide",(()=>(0,s.p)("winPagehide",[(0,p.z)()],void 0,j.t,this.ee))),this.importAggregator())}}var P=i(3081);class C extends h{static featureName=P.t9;constructor(e,t){let r=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(e,t,P.t9,r),this.importAggregator()}}var R,I=i(2210),k=i(1214),H=i(2177),L={};try{R=localStorage.getItem("__nr_flags").split(","),console&&"function"==typeof console.log&&(L.console=!0,-1!==R.indexOf("dev")&&(L.dev=!0),-1!==R.indexOf("nr_dev")&&(L.nrDev=!0))}catch(e){}function z(e){try{L.console&&z(e)}catch(e){}}L.nrDev&&H.ee.on("internal-error",(function(e){z(e.stack)})),L.dev&&H.ee.on("fn-err",(function(e,t,r){z(r.stack)})),L.dev&&(z("NR AGENT IN DEVELOPMENT MODE"),z("flags: "+(0,b.D)(L,(function(e,t){return e})).join(", ")));var M=i(6660);class B extends h{static featureName=M.t;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(r,n,M.t,i),this.skipNext=0;try{this.removeOnAbort=new AbortController}catch(e){}const o=this;o.ee.on("fn-start",(function(e,t,r){o.abortHandler&&(o.skipNext+=1)})),o.ee.on("fn-err",(function(t,r,n){o.abortHandler&&!n[M.A]&&((0,I.X)(n,M.A,(function(){return!0})),this.thrown=!0,(0,s.p)("err",[n,(0,p.z)()],void 0,e.D.jserrors,o.ee))})),o.ee.on("fn-end",(function(){o.abortHandler&&!this.thrown&&o.skipNext>0&&(o.skipNext-=1)})),o.ee.on("internal-error",(function(t){(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,o.ee)})),this.origOnerror=c._A.onerror,c._A.onerror=this.onerrorHandler.bind(this),c._A.addEventListener("unhandledrejection",(t=>{const r=function(e){let t="Unhandled Promise Rejection: ";if(e instanceof Error)try{return e.message=t+e.message,e}catch(t){return e}if(void 0===e)return new Error(t);try{return new Error(t+(0,D.P)(e))}catch(e){return new Error(t)}}(t.reason);(0,s.p)("err",[r,(0,p.z)(),!1,{unhandledPromiseRejection:1}],void 0,e.D.jserrors,this.ee)}),(0,O.m$)(!1,this.removeOnAbort?.signal)),(0,k.gy)(this.ee),(0,k.BV)(this.ee),(0,k.em)(this.ee),(0,t.OP)(r).xhrWrappable&&(0,k.Kf)(this.ee),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}onerrorHandler(t,r,n,i,o){"function"==typeof this.origOnerror&&this.origOnerror(...arguments);try{this.skipNext?this.skipNext-=1:(0,s.p)("err",[o||new F(t,r,n),(0,p.z)()],void 0,e.D.jserrors,this.ee)}catch(t){try{(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,this.ee)}catch(e){}}return!1}}function F(e,t,r){this.message=e||"Uncaught error with no additional information",this.sourceURL=t,this.line=r}let U=1;const q="nr@id";function G(e){const t=typeof e;return!e||"object"!==t&&"function"!==t?-1:e===c._A?0:(0,I.X)(e,q,(function(){return U++}))}function V(e){if("string"==typeof e&&e.length)return e.length;if("object"==typeof e){if("undefined"!=typeof ArrayBuffer&&e instanceof ArrayBuffer&&e.byteLength)return e.byteLength;if("undefined"!=typeof Blob&&e instanceof Blob&&e.size)return e.size;if(!("undefined"!=typeof FormData&&e instanceof FormData))try{return(0,D.P)(e).length}catch(e){return}}}var X=i(7243);class W{constructor(e){this.agentIdentifier=e,this.generateTracePayload=this.generateTracePayload.bind(this),this.shouldGenerateTrace=this.shouldGenerateTrace.bind(this)}generateTracePayload(e){if(!this.shouldGenerateTrace(e))return null;var r=(0,t.DL)(this.agentIdentifier);if(!r)return null;var n=(r.accountID||"").toString()||null,i=(r.agentID||"").toString()||null,o=(r.trustKey||"").toString()||null;if(!n||!i)return null;var a=(0,_.M)(),s=(0,_.Ht)(),c=Date.now(),u={spanId:a,traceId:s,timestamp:c};return(e.sameOrigin||this.isAllowedOrigin(e)&&this.useTraceContextHeadersForCors())&&(u.traceContextParentHeader=this.generateTraceContextParentHeader(a,s),u.traceContextStateHeader=this.generateTraceContextStateHeader(a,c,n,i,o)),(e.sameOrigin&&!this.excludeNewrelicHeader()||!e.sameOrigin&&this.isAllowedOrigin(e)&&this.useNewrelicHeaderForCors())&&(u.newrelicHeader=this.generateTraceHeader(a,s,c,n,i,o)),u}generateTraceContextParentHeader(e,t){return"00-"+t+"-"+e+"-01"}generateTraceContextStateHeader(e,t,r,n,i){return i+"@nr=0-1-"+r+"-"+n+"-"+e+"----"+t}generateTraceHeader(e,t,r,n,i,o){if(!("function"==typeof c._A?.btoa))return null;var a={v:[0,1],d:{ty:"Browser",ac:n,ap:i,id:e,tr:t,ti:r}};return o&&n!==o&&(a.d.tk=o),btoa((0,D.P)(a))}shouldGenerateTrace(e){return this.isDtEnabled()&&this.isAllowedOrigin(e)}isAllowedOrigin(e){var r=!1,n={};if((0,t.Mt)(this.agentIdentifier,"distributed_tracing")&&(n=(0,t.P_)(this.agentIdentifier).distributed_tracing),e.sameOrigin)r=!0;else if(n.allowed_origins instanceof Array)for(var i=0;i 2&&void 0!==arguments[2])||arguments[2];super(r,n,Z.t,i),(0,t.OP)(r).xhrWrappable&&(this.dt=new W(r),this.handler=(e,t,r,n)=>(0,s.p)(e,t,r,n,this.ee),(0,k.u5)(this.ee),(0,k.Kf)(this.ee),function(r,n,i,o){function a(e){var t=this;t.totalCbs=0,t.called=0,t.cbTime=0,t.end=E,t.ended=!1,t.xhrGuids={},t.lastSize=null,t.loadCaptureCalled=!1,t.params=this.params||{},t.metrics=this.metrics||{},e.addEventListener("load",(function(r){_(t,e)}),(0,O.m$)(!1)),c.IF||e.addEventListener("progress",(function(e){t.lastSize=e.loaded}),(0,O.m$)(!1))}function s(e){this.params={method:e[0]},T(this,e[1]),this.metrics={}}function u(e,n){var i=(0,t.DL)(r);i.xpid&&this.sameOrigin&&n.setRequestHeader("X-NewRelic-ID",i.xpid);var a=o.generateTracePayload(this.parsedOrigin);if(a){var s=!1;a.newrelicHeader&&(n.setRequestHeader("newrelic",a.newrelicHeader),s=!0),a.traceContextParentHeader&&(n.setRequestHeader("traceparent",a.traceContextParentHeader),a.traceContextStateHeader&&n.setRequestHeader("tracestate",a.traceContextStateHeader),s=!0),s&&(this.dt=a)}}function d(e,t){var r=this.metrics,i=e[0],o=this;if(r&&i){var a=V(i);a&&(r.txSize=a)}this.startTime=(0,p.z)(),this.listener=function(e){try{"abort"!==e.type||o.loadCaptureCalled||(o.params.aborted=!0),("load"!==e.type||o.called===o.totalCbs&&(o.onloadCalled||"function"!=typeof t.onload)&&"function"==typeof o.end)&&o.end(t)}catch(e){try{n.emit("internal-error",[e])}catch(e){}}};for(var s=0;s 1?e[1]=i:e.push(i)}else e[0]&&e[0].headers&&s(e[0].headers,n)&&(this.dt=n);function s(e,t){var r=!1;return t.newrelicHeader&&(e.set("newrelic",t.newrelicHeader),r=!0),t.traceContextParentHeader&&(e.set("traceparent",t.traceContextParentHeader),t.traceContextStateHeader&&e.set("tracestate",t.traceContextStateHeader),r=!0),r}}function x(e,t){this.params={},this.metrics={},this.startTime=(0,p.z)(),this.dt=t,e.length>=1&&(this.target=e[0]),e.length>=2&&(this.opts=e[1]);var r,n=this.opts||{},i=this.target;"string"==typeof i?r=i:"object"==typeof i&&i instanceof Y?r=i.url:c._A?.URL&&"object"==typeof i&&i instanceof URL&&(r=i.href),T(this,r);var o=(""+(i&&i instanceof Y&&i.method||n.method||"GET")).toUpperCase();this.params.method=o,this.txSize=V(n.body)||0}function A(t,r){var n;this.endTime=(0,p.z)(),this.params||(this.params={}),this.params.status=r?r.status:0,"string"==typeof this.rxSize&&this.rxSize.length>0&&(n=+this.rxSize);var o={txSize:this.txSize,rxSize:n,duration:(0,p.z)()-this.startTime};i("xhr",[this.params,o,this.startTime,this.endTime,"fetch"],this,e.D.ajax)}function E(t){var r=this.params,n=this.metrics;if(!this.ended){this.ended=!0;for(var o=0;o 2&&void 0!==arguments[2])||arguments[2];super(e,t,we.t,r),this.importAggregator()}}new class{constructor(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:(0,_.ky)(16);c._A?(this.agentIdentifier=t,this.sharedAggregator=new y({agentIdentifier:this.agentIdentifier}),this.features={},this.desiredFeatures=new Set(e.features||[]),this.desiredFeatures.add(m),Object.assign(this,(0,a.j)(this.agentIdentifier,e,e.loaderType||"agent")),this.start()):(0,l.Z)("Failed to initial the agent. Could not determine the runtime environment.")}get config(){return{info:(0,t.C5)(this.agentIdentifier),init:(0,t.P_)(this.agentIdentifier),loader_config:(0,t.DL)(this.agentIdentifier),runtime:(0,t.OP)(this.agentIdentifier)}}start(){const t="features";try{const r=n(this.agentIdentifier),i=[...this.desiredFeatures];i.sort(((t,r)=>e.p[t.featureName]-e.p[r.featureName])),i.forEach((t=>{if(r[t.featureName]||t.featureName===e.D.pageViewEvent){const n=function(t){switch(t){case e.D.ajax:return[e.D.jserrors];case e.D.sessionTrace:return[e.D.ajax,e.D.pageViewEvent];case e.D.sessionReplay:return[e.D.sessionTrace];case e.D.pageViewTiming:return[e.D.pageViewEvent];default:return[]}}(t.featureName);n.every((e=>r[e]))||(0,l.Z)("".concat(t.featureName," is enabled but one or more dependent features has been disabled (").concat((0,D.P)(n),"). This may cause unintended consequences or missing data...")),this.features[t.featureName]=new t(this.agentIdentifier,this.sharedAggregator)}})),(0,T.Qy)(this.agentIdentifier,this.features,t)}catch(e){(0,l.Z)("Failed to initialize all enabled instrument classes (agent aborted) -",e);for(const e in this.features)this.features[e].abortHandler?.();const r=(0,T.fP)();return delete r.initializedAgents[this.agentIdentifier]?.api,delete r.initializedAgents[this.agentIdentifier]?.[t],delete this.sharedAggregator,r.ee?.abort(),delete r.ee?.get(this.agentIdentifier),!1}}}({features:[J,m,S,class extends h{static featureName=oe;constructor(t,r){if(super(t,r,oe,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;const n=this.ee;let i;(0,k.QU)(n),this.eventsEE=(0,k.em)(n),this.eventsEE.on(se,(function(e,t){this.bstStart=(0,p.z)()})),this.eventsEE.on(ae,(function(t,r){(0,s.p)("bst",[t[0],r,this.bstStart,(0,p.z)()],void 0,e.D.sessionTrace,n)})),n.on(ce+ne,(function(e){this.time=(0,p.z)(),this.startPath=location.pathname+location.hash})),n.on(ce+ie,(function(t){(0,s.p)("bstHist",[location.pathname+location.hash,this.startPath,this.time],void 0,e.D.sessionTrace,n)}));try{i=new PerformanceObserver((t=>{const r=t.getEntries();(0,s.p)(te,[r],void 0,e.D.sessionTrace,n)})),i.observe({type:re,buffered:!0})}catch(e){}this.importAggregator({resourceObserver:i})}},C,xe,B,class extends h{static featureName=de;constructor(e,r){if(super(e,r,de,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;if(!(0,t.OP)(e).xhrWrappable)return;try{this.removeOnAbort=new AbortController}catch(e){}let n,i=0;const o=this.ee.get("tracer"),a=(0,k._L)(this.ee),s=(0,k.Lg)(this.ee),u=(0,k.BV)(this.ee),d=(0,k.Kf)(this.ee),f=this.ee.get("events"),l=(0,k.u5)(this.ee),h=(0,k.QU)(this.ee),g=(0,k.Gm)(this.ee);function m(e,t){h.emit("newURL",[""+window.location,t])}function v(){i++,n=window.location.hash,this[ve]=(0,p.z)()}function b(){i--,window.location.hash!==n&&m(0,!0);var e=(0,p.z)();this[pe]=~~this[pe]+e-this[ve],this[ye]=e}function y(e,t){e.on(t,(function(){this[t]=(0,p.z)()}))}this.ee.on(ve,v),s.on(be,v),a.on(be,v),this.ee.on(ye,b),s.on(ge,b),a.on(ge,b),this.ee.buffer([ve,ye,"xhr-resolved"],this.featureName),f.buffer([ve],this.featureName),u.buffer(["setTimeout"+le,"clearTimeout"+fe,ve],this.featureName),d.buffer([ve,"new-xhr","send-xhr"+fe],this.featureName),l.buffer([me+fe,me+"-done",me+he+fe,me+he+le],this.featureName),h.buffer(["newURL"],this.featureName),g.buffer([ve],this.featureName),s.buffer(["propagate",be,ge,"executor-err","resolve"+fe],this.featureName),o.buffer([ve,"no-"+ve],this.featureName),a.buffer(["new-jsonp","cb-start","jsonp-error","jsonp-end"],this.featureName),y(l,me+fe),y(l,me+"-done"),y(a,"new-jsonp"),y(a,"jsonp-end"),y(a,"cb-start"),h.on("pushState-end",m),h.on("replaceState-end",m),window.addEventListener("hashchange",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("load",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("popstate",(function(){m(0,i>1)}),(0,O.m$)(!0,this.removeOnAbort?.signal)),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}}],loaderType:"spa"})})(),window.NRBA=o})(); window.jQuery || document.write(' ') CKEDITOR_BASEPATH='https://f1000research.com/js/vendor/ckeditor/' window.reactTheme = 'research'; window.MathJax = { CommonHTML: { linebreaks: { automatic: true } }, 'HTML-CSS': { linebreaks: { automatic: true } }, SVG: { linebreaks: { automatic: true } }, AuthorInit: function() { MathJax.Hub.Register.MessageHook('End Process', function () { let timeout = false; // holder for timeout id const delay = 250; // delay after event is "complete" to run callback const reflowMath = function() { const dispFormulas = document.querySelectorAll('.disp-formula.panel'); if (!dispFormulas) { return; } for (const dispFormula of dispFormulas) { const child = dispFormula.querySelector('.MathJax_Preview').nextSibling.firstChild; const isMultiline = MathJax.Hub.getAllJax(dispFormula)[0].root.isMultiline; if (dispFormula.offsetWidth < child.offsetWidth || isMultiline) { MathJax.Hub.Queue(['Rerender', MathJax.Hub, dispFormula]); } } }; window.addEventListener('resize', function() { clearTimeout(timeout); // clear the timeout timeout = setTimeout(reflowMath, delay); // start timing for event "completion" }); }); }, }; if (window.location.hash == '#_=_'){ window.location = window.location.href.split('#')[0] } !function(f,b,e,v,n,t,s){if(f.fbq)return;n=f.fbq=function() {n.callMethod? n.callMethod.apply(n,arguments):n.queue.push(arguments)} ;if(!f._fbq)f._fbq=n; n.push=n;n.loaded=!0;n.version='2.0';n.queue=[];t=b.createElement(e);t.async=!0; t.src=v;s=b.getElementsByTagName(e)[0];s.parentNode.insertBefore(t,s)}(window, document,'script','https://connect.facebook.net/en_US/fbevents.js'); fbq('init', '1641728616063202'); fbq('track', "PixelInitialized", {}); (function(h,o,t,j,a,r){ h.hj=h.hj||function(){(h.hj.q=h.hj.q||[]).push(arguments)}; h._hjSettings={hjid:2318163,hjsv:6}; a=o.getElementsByTagName('head')[0]; r=o.createElement('script');r.async=1; r.src=t+h._hjSettings.hjid+j+h._hjSettings.hjsv; a.appendChild(r); })(window,document,'https://static.hotjar.com/c/hotjar-','.js?sv='); search file_upload Submit your research search menu close search Browse Gateways & Collections How to Publish Submit your Research My Submissions Article Guidelines Article Guidelines (New Versions) Open Data, Software and Code Guidelines Open Data and Accessible Source Materials Guidelines (HSS) Open Data, Software and Code Guidelines (PSE) Prepublication Checks Production Process Posters and Slides Guidelines Document Guidelines Article Processing Charges Peer Review Finding Article Reviewers About How it Works For Reviewers Our Advisors Policies Glossary FAQs For Developers Newsroom Contact My Research Submissions Content and Tracking Alerts My Details Sign In file_upload Submit your research { "@context": "https://schema.org", "@type": "ScholarlyArticle", "mainEntityOfPage": { "@type": "WebPage", "@id": "https://f1000research.com/articles/12-257" }, "headline": "Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in...", "datePublished": "2023-03-09T15:40:30", "dateModified": "2023-03-09T15:40:30", "author": [ { "@type": "Person", "name": "I Nyoman Bayu Mahendra" }, { "@type": "Person", "name": "I Nyoman Gede Budiana" }, { "@type": "Person", "name": "I Gede Mega Putra" }, { "@type": "Person", "name": "Anom Suardika" }, { "@type": "Person", "name": "Anak Agung Gede Putra Wiradnyana" }, { "@type": "Person", "name": "Alisza Novrita Sari" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": "Background: Incidence of cervical cancer is higher in low- and middle-income countries, including Indonesia. In Asia, Indonesia ranks second both in mortality and incidence of cervical cancer. Infection by Human Papilloma Virus (HPV) type 16 is a known risk factor, especially with mutations of the E6 or E7 oncogene that lead to chronic infection and malignancy. Increase in cyclin D1 expression is found on cervical cancer cells, including those resistant to chemotherapy. The role of cyclic D1 in increasing cellular proliferation and decreasing apoptosis leads to cervical cancer. Methods: This study was a cross-sectional observational study carried out between August 2020 until August 2021 at Prof. Dr. I.G.N.G. Ngoerah Hospital, Denpasar, Bali, Indonesia. Samples were collected by consecutive sampling of eligible patients. Polyclinic patients newly diagnosed with cervical cancer and who gave informed consent were further examined. We determined their oncogene E6 and E7 HPV type 16 mutation patterns and their association with cyclin D1 expression using contingency coefficient correlation test. Other outcomes including age distribution, parturition history, BMI, pathological type, clinical stage, and E6/E7 mutation characteristics was also recorded. Results: We included 31 eligible subjects with cervical cancer and HPV type 16-positive, divided into mutant E6/E7 (n=12/31; 38.7%) and wild type (n=19/31;61.3%). Primary outcome of association between mutant oncogene and cyclin D1 expression was weak (c=0.283; p-value=0.1). Secondary outcome showed majority of age on 50 years old or over; normal or overweight BMI; squamous cell carcinoma was the most commonly found (n=28/31; 90%); and clinical stage II (n=18/31; 38%) in both groups. Conclusions: This study concluded that there is no association between mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/12-257/v1", "name": "Association between mutant E6 and E7 Human Papilloma Virus Type 16..." } } ] } Home Browse Association between mutant E6 and E7 Human Papilloma Virus Type 16... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Mahendra INB, Budiana ING, Putra IGM et al. Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.12688/f1000research.128537.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] I Nyoman Bayu Mahendra 1 , I Nyoman Gede Budiana 1 , I Gede Mega Putra 1 , Anom Suardika 1 , Anak Agung Gede Putra Wiradnyana 1 , Alisza Novrita Sari https://orcid.org/0000-0003-1714-242X 1 I Nyoman Bayu Mahendra 1 , I Nyoman Gede Budiana 1 , [...] I Gede Mega Putra 1 , Anom Suardika 1 , Anak Agung Gede Putra Wiradnyana 1 , Alisza Novrita Sari https://orcid.org/0000-0003-1714-242X 1 PUBLISHED 09 Mar 2023 Author details Author details 1 Obstetrics and Gynecology Department, Prof. Dr. I.G.N.G. Ngoerah Hospital/Medical Faculty of Udayana University, Denpasar, Bali, 80113, Indonesia I Nyoman Bayu Mahendra Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization I Nyoman Gede Budiana Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization I Gede Mega Putra Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Anom Suardika Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Anak Agung Gede Putra Wiradnyana Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Alisza Novrita Sari Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Cell & Molecular Biology gateway. Abstract Background: Incidence of cervical cancer is higher in low- and middle-income countries, including Indonesia. In Asia, Indonesia ranks second both in mortality and incidence of cervical cancer. Infection by Human Papilloma Virus (HPV) type 16 is a known risk factor, especially with mutations of the E6 or E7 oncogene that lead to chronic infection and malignancy. Increase in cyclin D1 expression is found on cervical cancer cells, including those resistant to chemotherapy. The role of cyclic D1 in increasing cellular proliferation and decreasing apoptosis leads to cervical cancer. Methods: This study was a cross-sectional observational study carried out between August 2020 until August 2021 at Prof. Dr. I.G.N.G. Ngoerah Hospital, Denpasar, Bali, Indonesia. Samples were collected by consecutive sampling of eligible patients. Polyclinic patients newly diagnosed with cervical cancer and who gave informed consent were further examined. We determined their oncogene E6 and E7 HPV type 16 mutation patterns and their association with cyclin D1 expression using contingency coefficient correlation test. Other outcomes including age distribution, parturition history, BMI, pathological type, clinical stage, and E6/E7 mutation characteristics was also recorded. Results: We included 31 eligible subjects with cervical cancer and HPV type 16-positive, divided into mutant E6/E7 (n=12/31; 38.7%) and wild type (n=19/31;61.3%). Primary outcome of association between mutant oncogene and cyclin D1 expression was weak (c=0.283; p-value=0.1). Secondary outcome showed majority of age on 50 years old or over; normal or overweight BMI; squamous cell carcinoma was the most commonly found (n=28/31; 90%); and clinical stage II (n=18/31; 38%) in both groups. Conclusions: This study concluded that there is no association between mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. READ ALL READ LESS Keywords cervical cancer, oncogene, cyclin expression, HPV type 16, observational study, gynecological cancer Corresponding Author(s) I Nyoman Bayu Mahendra ( [email protected] ) Close Corresponding author: I Nyoman Bayu Mahendra Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2023 Mahendra INB et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Mahendra INB, Budiana ING, Putra IGM et al. Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.12688/f1000research.128537.1 ) First published: 09 Mar 2023, 12 :257 ( https://doi.org/10.12688/f1000research.128537.1 ) Latest published: 09 Mar 2023, 12 :257 ( https://doi.org/10.12688/f1000research.128537.1 ) Introduction The estimated incidence of cervical cancer in the world is 13.1 every 100,000 women. The incidence of cervical cancer is found to be higher in lower- and middle-income countries like Indonesia. In 2018, Indonesia ranked second in the incidence and mortality rate of cervical cancer in Asia 1 , 2 One of the known risk factors of cervical cancer is the Human Papilloma Virus (HPV). HPV types 16 and 18 are the most common types found in cases of cervical squamous cell carcinoma. The HPV oncoproteins that play a role in the development of cervical cancer are E6 and E7. 3 – 5 E6 and E7 initiate a series of processes leading to chronic infection and transformation into cervical cancer. E6 causes degradation of p53, and p21 inhibition leads to a decrease in cellular apoptosis. E7 inactivates pRb so that infected cells experience increased cell proliferation, causes an aberrant synthesis phase of the cell cycle, and increases transcription factor E2F. The sequences carried by E6 and E7 lead to an increase in cyclin D1 expression. 5 – 7 Increase in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy. 8 – 10 Cyclin D1 overexpression can augment cell cycle and increase cellular proliferation. The expression of Cyclin D1 also affects the function of apoptosis, hypertrophy and differentiation. Cyclin D1 is thought to have mitogenic activity so as to stimulate hypertrophic growth. 11 Determination of E6 and E7 mutations for HPV type 16 can be useful in the development of HPV molecular diagnostics, prevention, and therapeutic efforts to control or eliminate cervical cancer. Moreover, the development of agents to target cyclin D1 activity, directly or indirectly, by targeting cyclin-dependent kinase (CDK) has also been studied. These agents are used concurrently with chemotherapy and have been shown to reduce resistance to chemotherapy and increase patient survival rates 12 – 14 when patients are referred for chemotherapy or radiotherapy. 15 , 16 Given their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out. Methods An observational analytic, cross-sectional study was conducted from August 2020 until August 2021 at the Gynecology polyclinic of RSUP Prof. Dr I.G.N.G. Ngoerah, Denpasar laboratory of microbiology, faculty of medicine Universitas Udayana (FK UNUD), Denpasar and laboratory of histology FK UNUD, Denpasar. This study was approved by the Ethical Board for Research of FK UNUD, Denpasar on 21 December 2021 with protocol number 2021.02.1.1247. The sample size was calculated with the following formula: N = Z α 2 PQ d 2 = 1.96 2 × 0.70 × 0.30 0.18 2 = 24.89 sample (rounded to 25) N=Minimum sample size Zα=Confidence interval with α score of 5% and Zα is 1.960 P=Proportion of oncogene E6 and E7 mutation of HPV 16 was around 70% based on a study by Zhe et al. (2019) 17 Q=1-p (proportion of oncogene E6 and E7 of HPV 16 which have compability with the prototype), around 30% d=Error limitation or absolute precision, 18% Data was collected from cervical cancer patients who visited the polyclinic between August 2020 until August 2021. The sampling method used was consecutive sampling of the population and included patients who fulfilled the eligibility criteria. Patient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1 . Patient eligibility was assessed with inclusion criteria such as first-time cervical cancer diagnosis during the study setting; not having received surgery, chemotherapy, or radiotherapy. The exclusion criteria were cervical cancer that was not caused by HPV type-16 and if the cervical cancer tissue samples were damaged or not representative for DNA PCR or immunohistochemistry examination. Eligible patients were asked for and provided written informed consent. Table 1. Eligibility criteria. Eligibility criteria Explanation Patient provided informed consent. Inclusion criteria First time diagnosed with cervical cancer during the study setting and location. Have not received surgery, chemotherapy, and radiotherapy. Exclusion criteria Cervical cancer not caused by Human Papilloma Virus (HPV) – HR 16. Cervical cancer tissue samples were damaged or not representative for DNA PCR or immunohistochemistry examination. Eligible patients were asked for demographic characteristics, clinical history, received physical examination, and pathological examination of cervical cancer cells. Patient characteristics such as age, parturition history, body mass index (BMI), cervical cancer clinical stage, and pathological findings were recorded. Cervical cancer tissue specimens were tested for oncogene E6 or E7 mutation by HPV DNA PCR The PCR analyses were conducted at our institution laboratory, DNA isolation was carried out with Roche DNA extraction kit following the product standard protocol. After extraction, a PCR was performed to determine positive and negative HPV with primers My09 (5′-CGT CCM ARR GGA WAC TGA TC-3′) and My11 (5′-GCM CAG GGW CAT AAY AAT GG-3′). Meanwhile, DNA PCR for HR-HPV type-16 were performed with primers (Forward: 5′-GAC CCA GAA AGT TAC CAC AG-3′ and reverse: 5′-CAC AAC GGT TTG TTG TAT TG-3′). The HPV HR Type 16 PCR program is the same as the PCR program for universal HPV. To determine the quality of the extracted DNA, a PCR was performed for the betaglobin gene with the primers (Forward 5-CAA CTT CAT CCA CGT TCA CC-3 and reverse: 5-GAA GAG CCA AGG ACA GGT AC-3); The PCR results were then compared with the wild type gene on GenBank according to sequence K02718/HPV16R. Amplification of E6 and E7 gene was done with primer sequences as follow: upstream , 5′AAG GGC GTA ACC GAA AT3′; downstream , 5′TCC ATT ACA TCC CGT ACC CTC3′ (Primer 2 OD; 1 OD=33 μg). The primer was dissolved up to 100 mmol/L at a concentration of 10 μM (M=mol/L). The PCR reaction mixture of E6 and E7 genes (25 μL) wasas follows: 1 μL DNA template (1:20), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 12.5 μL of 2× Taq PCR MasterMix, and double distilled water. The PCR cycle conditions were as follows: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds; and final extension at 72°C for 5 minutes. After PCR was completed, 5 mL of sample were analyzed by electrophoresis using 1.5% agarose gel; The MEGA6 software was utilized for the sequencing of the E6 and E7 genes. BLAST software was used to compare the nucleotide sequences of the E6 and E7 genes to the HPV 16 prototype (HPv16.P, GenBank access code: NC 001526) from the European variation; cyclin D1 expression was measured by immunohistochemistry with rabbit primary ERβ polyclonal antibodies (BY-02101, Yueyan Biotechnology, Shanghai, China) and rabbit secondary polyclonal antibodies tagged with horseradish peroxidase (K500711, Gene Biotechnology, Shanghai, China) and categorized by Immunoreactive Scoring System (IRS); diagnosis of cervical cancer was made based on histopathological findings with histological classification such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma. After the patient was diagnosed with cervical cancer, they were grouped according to E6 or E7 oncogene mutation and each group was tested for cyclin D1 expression. The data was analyzed with IBM SPSS for Windows version 22.0. The correlation between oncogene E6/E7 mutation and cyclic D1 expression was analyzed with a contingency coefficient correlation test. The result ranges from 0 until 1, 0 indicating no correlation and 1 a very strong correlation. Results From August 2020 until August 2021, a total of 100 cervical cancer patients was tested for HPV 16. 31 samples tested positive for HPV 16 with oncogene E6/E7 mutation distribution of wild type of n=19; 61%, and mutated oncogene n=12; 39%. Table 2 summarizes the demographics and characteristics of sample including age, parturition history, BMI, cervical cancer histological classification, and cancer stage which were distributed normally. Mean age for wildtype and mutant type were 50 and 54.5 years old, respectively. Mean body mass index for wildtype was 23.7 and 22.1 for the mutant. Squamous cell carcinoma was the most common histological findings, and cancer clinical stage II was the most common in both types. Table 2. Demographics and characteristics of sample. Mutation Wild-type p-value Age 54.5 (21-58) 50 (33-61) 0.72 Parturition history 0.25 1. ≤2 6 (50) 14 (73.7) 2. >2 6 (50) 5 (26.3) BMI (kg/m 2 ) 22.1 (19.5-28.2) 23.7 (18.4-36.1) 0.34 Histological classification 0.63 1. Squamous cell carcinoma 11 (91.7) 17 (89.4) 2. Adenocarcinoma 1 (8.3) 1 (5.3) 3. Neuroendocrine carcinoma 0 1 (5.3) Cancer stage 0.87 Stage I 1 (8.3) 2 (10.5) IA1 0 1 (5.3) IB1 1 (5.3) 1 (5.3) Stage II 7 (58.3) 11 (47.9) IIA 0 1 (5.3) IIA1 0 1 (5.3) IIA2 1 (5.3) 0 IIB 6 (50) 9 (47.4) Stage III 4 (33.3) 6 (31.6) IIIB 4 (33.3) 6 (31.6) Table 3 summarize the characteristic and proportion of E6 and E7 mutation on this study. The proportion of E6 mutants in this study group was 25.8% (8/31), while in the E7 group it was 12.9% (4/31). The population characteristics of the E6 mutation group were dominated by the nucleotide position of 27 prototype T variant C (n=5; 16.1%) followed by position 360 prototype A variant G (n=2; 6.4%) and position 371 prototype G variant A (n=1; 3.2%). The mutation in strain E6 is a synonymous mutation and G371A/R124K the only non-synonymous mutation. In the E7 group, the same proportion was obtained, namely one sample (3.2%) for each nucleotide position, prototype and variant. At E7 the majority of mutations were non-synonymous 9.6% (3/31) in N29T; N29S; R77C. Table 3. E6 and E7 Mutation characteristics and proportion. Nucleotide position Prototype Variant N (%) Amino acid position Prototype Variant N (%) E6 27 T C 5 (16.1) 9 F F - 360 A G 2 (6.4) 120 E E - 371 G A 1 (3.2) 124 R K 1 (3.2) E7 86 A C 1 (3.2) 29 N T 1 (3.2) 86 A G 1 (3.2) 29 N S 1 (3.2) 229 C T 1 (3.2) 77 R C 1 (3.2) 285 T C 1 (3.2) 95 S S - The association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer is summarized in Table 4 . There was no association between mutations in the E6/E7 HPV type 16 oncogene with cyclin D1 expression (c=0.283; p value=0.1). The correlation coefficient between the mutations of the E6 and E7 HPV type 16 oncogenes with Cyclin D1 expression was weak, as seen in Table 4 . Table 4. Association of mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. Cyclin D1 expression Weak Strong C score p-value Mutant E6 or E7 5 6 0.283 0.1 Wild type E6 or E7 15 5 Discussion In this study, we described the association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. Our study included 31 patients with cervical cancer and HPV type 16 infection based on pathological examination and HPV DNA PCR. The samples median age was 54.5 (21-58) years old, in accordance with a study in Denmark and Sweden with a higher incidence of cervical cancer in patients aged 50 years or older. 18 , 19 Women aged 50 years and over are usually in the perimenopausal or postmenopausal phase; therefore, their physiological and pathological characteristics differ significantly from women of childbearing age. 20 The mean BMI was 22.1 kg/m 2 in cervical cancer patients with mutant oncogenes E6 and E7 with a BMI range of 19.5-28.2 kg/m 2 . In this study, patients were found to be in the ideal BMI and overweight categories. The effect of body mass index (BMI) on precancerous cervical lesions (PCL) and cervical cancer is not clear. There are claims that underweight women may have an increased risk of cervical lesions while overweight women have a reduced risk of PCL compared to women with a normal BMI. 21 One of the identified risk factors for PCL is being underweight (BMI<18.5 kg/m 2 ), so the probability of being positive for PCL was fourfold higher among underweight women than women with a normal BMI. This relationship can be explained by the possible association between low BMI, poor nutritional status and low immunity. Therefore, participants with a low BMI tend to be malnourished, which in turn can compromise their immunity. 22 Proportion of oncogene E6 and E7 mutation The HPV genome consists of small, conserved double-stranded DNA with an approximate size of 8000 base pairs, and consists of three regions. The molecular biology of this tiny DNA molecule is complex. There are six starting proteins, three regulatory proteins (E1, E2, and E4) and three oncoproteins (E5, E6, and E7) encoded in 4000 base pairs that participate in viral replication and cell transformation. 23 In HPV16-positive cells, it was found that viral genes E6 and E7 remained integrated into the host genome and expressed, although in some HPV16-infected cells, E6/E7 overexpression could be absent. In addition, E6/E7 overexpression was also found in cells infected with other HPV types. E6 and E7 are small proteins composed of 100 to 150 amino acids with no known enzymatic activity; they can affect host cell activity when they bind to cell receptor proteins. E6, for example, binds to E6-associated protein (E6AP), a ubiquitin ligase that causes structural changes in E6 that allow it to bind to p53, a tumor-inhibiting protein in a cell cycle control manner to form the E6/E6AP/p53 complex. Therefore, E6 and E7 are important factors in the occurrence of cervical cancer in HPV16-positive cells. 24 The HPV genome may integrate with the host genome or remain in an episomal form, with 83% of cases of HPV-positive cervical cancer showing evidence of integration of the HPV genome into host cells. In the case of integration of the viral genome with the host genome, it often leads to disruption of the E2 gene site. The E2 gene is responsible for suppressing E6 and E7, thereby causing E6 and E7 to be activated upon integration of the viral genome into the host genome. Throughout the course of infection, the activities of E6 and E7 are responsible for the doubling of the viral genome with the help of cellular machinery. They can trick cells into becoming oncogenic in the process of viral replication. Therefore, HPV-mediated tumor development can be defined as collateral damage from viral infection. 25 The E6 protein is thought to increase cell proliferation by stimulating the degradation of the tumor suppressor protein p53 through the formation of a complex consisting of E6, p53 and the cellular ubiquitination enzyme E6-AP. E6-stimulated degradation interferes with the biological function of p53, thereby interfering with the control of cell cycle progression, which ultimately leads to increased tumor cell growth. Although it is generally accepted that the ability of high-risk HPV types E6 to target p53 for degradation contributes to virus-induced cellular transformation, it is also clear that the E6 protein has oncogenic activity independent of p53. 26 The E7 protein encoded by high-risk HPV types, such as HPV 16 and HPV 18, binds to the Rb protein with much higher affinity than that encoded by low-risk HPV types, such as HPV type 6 and HPV type 11. E7 binds to the Rb protein with the region being called ‘pocket domain’. The ‘pocket domain’ sequence of Rb is critical for its tumor suppressor function, with many naturally occurring Rb loss-of-function mutations appearing clustered within this ‘pocket domain’. One of the major biochemical functions of Rb is to bind to transcription factors of the E2F family and suppress the gene expression of the replication enzymes. As a result of mutations in E7 resulting in loss of function of Rb resulting in loss of replication suppression so that cells continue to replicate and cause cancer. 27 To ensure continued cell proliferation, HPV-infected cells also need to pass the mitotic test, which is also carried out by E6 and E7 simultaneously. E6 is dependent on a p53-dependent pathway, while E7 avoids the spindle checkpoint in a p53-independent manner, with the help of pRb. As a result of alterations in several cell cycle regulators, the cyclin-CDK complex is a major player of a dramatically altered cell cycle in HPV-infected cervical cancer cells. Cyclin D1-CDK4 and cyclin D1-CDK2 associations is reduced in E6-expressing cells and completely abolished in E6 and E7-expressing cells. 28 Association of mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression There was not statistically significant difference between the mutant and wild-type groups. This indicates that the mutant and wild-type have a weak correlation with the emergence of cyclin D1 expression and mutation or lack thereof does not determine whether the mutant or wild-type has a higher or less cyclin D1 value. Cancer development and progression involve activation of oncogenes, inactivation of tumor suppressor genes, and imbalance of immune system regulatory mechanisms, a combination which causes cellular variability and leads to tissue transformation into cancer. Cyclin, and especially cyclin D1, functions as a cell cycle regulator and promotescell proliferation. Mutation or over expression of Cyclin D1 may alter the cell cycle in various cancers. 27 Many studies have focused on the expression of cyclin D1 and its junction variants cyclin D1a and cyclin D1b, which arise from single nucleotide polymorphisms (SNPs) in the CCND1 gene in cervical cancer. While cyclin D1 has been associated with different clinical and pathological stages of cervical cancer, few studies have focused on its correlation with cervical cancer prognosis. Currently, the accepted view is that Cyclin D1 and its isoforms play an important role in the development and progression of cervical cancer. During the normal cell cycle, cyclin D1 forms a complex with CDK4, which promotes phosphorylation of the tumor suppressor protein. The transcription factor E2F is then initiated to promote DNA synthesis, allowing the completion of cell division by moving from the G1 phase to the S phase. cyclin D1 can thus be seen as a shortening of the G1 phase of the cell cycle. When the control of the cyclin D1 protein is abnormal and several cancer-associated genes result in an increase in its expression, the time the cell spends in the G1 phase of the cell cycle is significantly reduced, causing the cell to enter the S phase earlier, which in turn results in uncontrolled cell proliferation and transformation leading to carcinogenesis. Currently, cyclin D1 is recognized as a proto-oncogene, and its overexpression can alter progression through the cell cycle, leading to uncontrolled cell proliferation and malignancy. Unlike the findings of this study, in a study to detect Cyclin D1 expression, it was found that there was a fourfold increase in Cyclin D1 expression in cancer conditions. But in this study, the comparison of the cancer group was normal cells (no malignancy activity at all) and the research focused on uterine cancer. 28 Research by Park et al. (2016) supports the result of our study. Their study stated that there is a role for glycogen synthase kinase 3β (GSK3β), a pluripotent protein kinase that is involved in cancer development through the regulation of various oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of the target proteins that GSK3β regulates. Squamous cell carcinoma has an increased expression of GSK3β which plays a role in cervical carcinogenesis and has an inverse correlation with cyclin D1 expression in this process. 29 Another study by Bae et al. (2001) found that decreased cyclin D1 is regulated at the transcriptional level in cervical cancer. Cyclin D1 is less expressed in cervical neoplasia, and is more frequently expressed in malignant lesions. This may be because cyclin D1 is no longer required for G1 progression in HPV-transformed cells due to the binding of HPV E7 to Rb leading to the release of the transcription factor E2F. 30 Pyeon et al. (2007) found that there was no significant difference between the increase in cyclin D1 and cervical cancer. In HPV-positive cancers, p16 is expressed at high levels and cyclin D1 at low levels, in contrast to HPV-negative cancers. 31 In this study, there was also no relationship between cyclin D1 and the occurrence of mutations in HPV-positive cancer, and cyclin D1 levels were not associated with changes in lesions from pre-cancerous to cancerous. The limitation of this study was a limited number of samples we found in a one-year, single centre study. Further study with a greater sample population can provide more accurate result such as on national level. Conclusions This study concluded that there is no association of mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. Data availability Figshare: Data of Cervical Cancer HPV 16 oncogen E6 or E7 with Cyclin D1 expression.xlsx, https://doi.org/10.6084/m9.figshare.21786644.v1 . 32 Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). References 1. Bray F, Ferlay J, Soerjomataram I, et al. : Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018; 68 (6): 394–424. PubMed Abstract | Publisher Full Text 2. World Health Organization: Indonesia Source GLOBOCAN 2018. International Agency for Research on Cancer. 2019; 256 : 1–2. 3. Wipperman J, Neil T, dan Williams T: Cervical Cancer: Evaluation and Management. Am. Fam. Physician. 2018; 15 (7): 43–47. Publisher Full Text 4. Tadlaoui KA, Hassou N, Bennani B, et al. : Emergence of oncogenic high-risk human papillomavirus types and cervical cancer , Emerging and Reemerging Viral Pathogens: Volume 1: Fundamental and Basic Virology Aspects of Human, Animal and Plant Pathogens. Elsevier Inc.; 2019. Publisher Full Text 5. Vonsky M, Shabaeva M, Runov A, et al. : Carcinogenesis Associated with Human Papillomavirus Infection. Mechanisms and Potential for Immunotherapy. Biochem. Mosc. 2019; 84 (7): 782–799. PubMed Abstract | Publisher Full Text 6. Doorbar J: Molecular biology of human papillomavirus infection and cervical cancer. Clin. Sci. 2006; 110 (5): 525–541. Publisher Full Text 7. Hoppe-Seyler K, Bossler F, Braun JA, et al. : The HPV E6/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets. Trends Microbiol. 2018; 26 (2): 158–168. Elsevier Ltd. PubMed Abstract | Publisher Full Text 8. Van De Putte G, Kristensen GB, Lie AK, et al. : Cyclins and proliferation markers in early squamous cervical carcinoma. Gynecol. Oncol. 2004; 92 (1): 40–46. PubMed Abstract | Publisher Full Text 9. Bahnassy AA, Zekri A-R, El-din HA, et al. : The role of cyclins and cyclins inhibitors in the multistep process of HPV-associated cervical carcinoma. J. Egypt. Natl. Canc. Inst. 2006; 18 (4): 292–302. PubMed Abstract 10. Leal-Esteban LC, dan Fajas L: Cell cycle regulators in cancer cell metabolism. Biochim. Biophys. Acta Mol. Basis Dis. 2020; 1866 (5): 165715. Elsevier B. V. Publisher Full Text 11. Haybar H, Shahrouzian M, Gatavizadeh Z, et al. : Cyclin D1: A Golden Gene in Cancer, Cardiotoxicity, and Cardioprotection. Jundishapur J. Chronic Dis. Care. 2021; 10 (3): e112413. Publisher Full Text 12. Whittaker SR, Mallinger A, Workman P, et al. : Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacol. Ther. 2017; 173 : pp. 83–105. Elsevier Inc. PubMed Abstract | Publisher Full Text | Free Full Text 13. Almeida AM, Queiroz JA, Sousa F, et al. : Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins. Drug Discov. Today. 2019; 24 (10): 2044–2057. PubMed Abstract | Publisher Full Text 14. Roskoski R: Cyclin-dependent protein serine/threonine kinase inhibitors as anticancer drugs. Pharmacol. Res. 2019; 139 : pp. 471–488. Elsevier Ltd. Publisher Full Text 15. Winata IGS, Hidayat YM, Winarno GN, et al. : Pulsatility Index and Hypoxia Inducible Factor-1α Expression Predict the Clinical Response after External Radiation in Patients with Stage IIB to IVA Cervical Cancer. Asian Pac. J. Cancer Prev. 2019; 20 (7): 2073–2078. PubMed Abstract | Publisher Full Text | Free Full Text 16. Winata IGS, Budiana ING, Jawi IM, et al. : Neoadjuvant Chemotherapy in Stadium IB3, IIA2 and IIB Cervical Cancer a Narrative Review. Biomed. Pharmacol. J. 2022; 15 (2): 901–910. Publisher Full Text 17. Zhe X, Xin H, Pan Z, et al. : Genetic variations in E6, E7 and the long control region of human papillomavirus type 16 among patients with cervical lesions in Xinjiang, China. Cancer Cell Int. 2019; 19 (1): 1–11. Publisher Full Text 18. Moore KN, Java JJ, Slaughter KN, et al. : Is age a prognostic biomarker for survival among women with locally advanced cervical cancer treated with chemoradiation? An NRG Oncology/Gynecologic Oncology Group ancillary data analysis. Gynecol. Oncol. 2016; 143 (2): 294–301. PubMed Abstract | Publisher Full Text | Free Full Text 19. Lynge E, Lönnberg S, Törnberg S: Cervical cancer incidence in elderly women-biology or screening history? Eur. J. Cancer. 2017; 74 : 82–88. Publisher Full Text 20. Yan J, He Y, Wang M, et al. : Prognostic Nomogram for Overall Survival of Patients Aged 50 Years or Older with Cervical Cancer. Int. J. Gen. Med. 2021; 14 : 7741–7754. PubMed Abstract | Publisher Full Text | Free Full Text 21. Feng YH: The association between obesity and gynecological cancer. Gynecol. Minim. Invasive Ther. 2015; 4 (4): 102–105. Publisher Full Text 22. Taye BT, Mihret MS, Muche HA: Risk factors of precancerous cervical lesions: The role of women’s socio-demographic, sexual behavior and body mass index in Amhara region referral hospitals; case-control study. PLoS One. 2021; 16 (3 March): e0249218–e0249215. PubMed Abstract | Publisher Full Text | Free Full Text 23. Jing Y, Wang T, Chen Z, et al. : Phylogeny and polymorphism in the long control regions E6, E7, and L1 of HPV Type 56 in women from southwest China. Mol. Med. Rep. 2018; 17 (5): 7131–7141. PubMed Abstract | Publisher Full Text | Free Full Text 24. Chan CK, Aimagambetova G, Ukybassova T, et al. : Human Papillomavirus Infection and Cervical Cancer: Epidemiology, Screening, and Vaccination - Review of Current Perspectives. J. Oncol. 2019; 2019 : 1–11. PubMed Abstract | Publisher Full Text | Free Full Text 25. Pal A, dan Kundu, R.: Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy. Front. Microbiol. 2020; 10 (January). PubMed Abstract | Publisher Full Text | Free Full Text 26. Yim E-K, Park J-S: The Role of HPV E6 and E7 Oncoproteins in HPV-associated Cervical Carcinogenesis. Cancer Res. Treat. 2005; 37 (6): 319–324. PubMed Abstract | Publisher Full Text | Free Full Text 27. Casimiro MC, Di Sante G, Di Rocco A, et al. : Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis. Cancer Res. 2017; 77 (13): 3391–3405. PubMed Abstract | Publisher Full Text | Free Full Text 28. Tripathi R, Rath G, Jawanjal P, et al. : Cyclin D1 protein affecting global women’s health by regulating HPV mediated adenocarcinoma of the uterine cervix. Sci. Rep. 2019; 9 (1): 1–6. Publisher Full Text 29. Park H, et al. : Glycogen synthase kinase 3β and cyclin D1 expression in cervical carcinogenesis. Obstet. Gynecol. Sci. 2016; 59 (6): 470–478. PubMed Abstract | Publisher Full Text | Free Full Text 30. Bae DS, Cho SB, Kim YJ, et al. : Aberrant expression of cyclin D1 is associated with poor prognosis in early stage cervical cancer of the uterus. Gynecol. Oncol. 2001; 81 (3): 341–347. PubMed Abstract | Publisher Full Text 31. Pyeon D, Newton MA, Lambert PF, et al. : Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus-Positive and Human Papillomavirus-Negative Head/Neck and Cervical Cancers. Cancer Res. 2007; 67 (10): 4605–4619. PubMed Abstract | Publisher Full Text | Free Full Text 32. Mahendra INB, Budiana ING, Putra IGM, et al. : Data of Cervical Cancer HPV 16 oncogen E6 or E7 with Cyclin D1 expression.xlsx. Dataset. figshare. 2022. Publisher Full Text Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 09 Mar 2023 ADD YOUR COMMENT Comment Author details Author details 1 Obstetrics and Gynecology Department, Prof. Dr. I.G.N.G. Ngoerah Hospital/Medical Faculty of Udayana University, Denpasar, Bali, 80113, Indonesia I Nyoman Bayu Mahendra Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization I Nyoman Gede Budiana Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization I Gede Mega Putra Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Anom Suardika Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Anak Agung Gede Putra Wiradnyana Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization Alisza Novrita Sari Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (1) version 1 Published: 09 Mar 2023, 12:257 https://doi.org/10.12688/f1000research.128537.1 Copyright © 2023 Mahendra INB et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Mahendra INB, Budiana ING, Putra IGM et al. Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.12688/f1000research.128537.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 09 Mar 2023 Views 0 Cite How to cite this report: Kushwah AS. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r266381 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-266381 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 17 Sep 2024 Atar Singh Kushwah , Oncological Sciences, Mount Sinai Health System, New York, New York, USA; Department of Zoology, University of Lucknow, Lucknow, Uttar Pradesh, India; Department of Zoology, Banaras Hindu University, Banaras, Uttar Pradesh, India; Biological Sciences, Alabama State University, Montgomery, Alabama, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.141136.r266381 Mahendra et al, demonstrated the relationship between mutations in the E6 and E7 oncogenes of HPV Type 16 and Cyclin D1 expression in cervical cancer patients. The study included 31 cervical cancer patients, 38.7% of whom had mutations in the ... Continue reading READ ALL Mahendra et al, demonstrated the relationship between mutations in the E6 and E7 oncogenes of HPV Type 16 and Cyclin D1 expression in cervical cancer patients. The study included 31 cervical cancer patients, 38.7% of whom had mutations in the E6/E7 oncogenes. Despite the known role of these oncogenes in promoting cancer through disruption of cellular processes like p53 degradation and pRb inactivation, the study found no significant association between these mutations and Cyclin D1 expression. Cyclin D1 is commonly overexpressed in various cancers, but this study suggests that its expression in cervical cancer may not be directly influenced by E6/E7 mutations. The findings indicate that other molecular mechanisms may be involved in Cyclin D1 regulation, warranting further research with larger sample sizes to clarify these pathways. While reviewing the manuscript, I found the following limitations that need to be rectified for further consideration: The statistical tests used in the study are not clearly described. It appears that t-tests, Fisher's exact tests, and chi-square tests were used, but this is not explicitly stated. Furthermore, the chi-square test in Table 4 is questionable due to low cell counts, potentially making it unreliable. The sample size formula used is incorrect for the study's objectives. The formula calculates the sample size for a single sample proportion test, but the study involves comparing two groups, requiring a different calculation method. Table 4 contains inconsistencies with the rest of the manuscript, showing 11 cases with mutant E6/E7 and 20 cases with wild-type E6/E7, which do not match earlier data. Key details about the DNA extraction kit, PCR conditions, and immunohistochemistry procedures are missing, making replication difficult. Some references cited to support claims about Cyclin D1 expression are not appropriate or do not back the statements made. The authors concluded no association between E6/E7 mutations and Cyclin D1 expression based solely on a p-value greater than 0.05. However, the c-score (0.283) suggests a medium correlation, indicating that the study may be underpowered rather than the relationship being non-existent. The potential functional consequences of non-synonymous mutations in the E6/E7 oncogenes are not thoroughly discussed. The effects of synonymous mutations on protein structure and function are also not addressed. The study does not offer significant new findings or insights into the relationship between E6/E7 mutations and Cyclin D1 expression. Previous research has explored Cyclin D1's role in cervical cancer, and the study does not significantly advance the current understanding. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: PharmacoEpiGenetics, Clinical Oncology and Biotherapeutics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Kushwah AS. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r266381 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-266381 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Siegel E. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r307750 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-307750 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Aug 2024 Eric Siegel , University of Arkansas for Medical Sciences, Little Rock, AR, USA Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.141136.r307750 This cross-sectional study enrolled cervical-cancer patients that were positive for HPV type 16. Its two purposes were (1) to characterize the patients' HPV genomes for whether they carried mutations in either the E6 or E7 oncoprotein, and (2) to test ... Continue reading READ ALL This cross-sectional study enrolled cervical-cancer patients that were positive for HPV type 16. Its two purposes were (1) to characterize the patients' HPV genomes for whether they carried mutations in either the E6 or E7 oncoprotein, and (2) to test for whether the presence of such mutations are associated with Cyclin D1 expression. They obtained 31 subjects that were positive for HPV16, of which 19 had wildtype E6/E7 and 12 had mutated E6/E7. They classified Cyclin D1 expression as Weak or Strong, and tested for association with E6/E7 mutation status using a 2x2 contingency table with an unidentified statistical test, and used the contingency coefficient (c-score) as the association's effect-size measure. They obtained c-score=0.283 with p-value = 0.1 from the unidentified statistical test. From this result, they concluded that there is no association of mutation in the E6/E7 HPV-16 oncogenes with Cyclin D1 expression in patients with cervical cancer. The authors' conclusion is false for two reasons. One, it is a fallacy to conclude that no association exists simply because the p-value is greater than 0.05. The fact that the authors obtained a c-score greater than 0 means that some level of association did exist, but that the study was underpowered to detect it. Furthermore, the fact that the authors obtained a c-score of 0.283 from Table 4 means that the authors also obtained a phi coefficient of ±0.295 from the same Table 4. Phi coefficients of magnitude 0.1, 0.3, and 0.5 were classified respectively as small , medium , and large by Cohen J (1992) Ref 1. By this classification, phi=±0.295 represents a medium association, and therefore, the authors' c-score=0.283 represents an association that is medium, not weak as the authors claimed. Because a medium association, if it exists, could have relevance to public health, it is dangerous to pretend that a medium association equals no association simply because the p-value was greater than 0.05. Two, the authors have a mistake somewhere in their Table 4. The top row of Table 4 shows only 11 cases with mutant E6 or E7, while the bottom row of Table 4 shows 20 cases with wildtype E6 or E7. These numbers are not consistent with the numbers in Table 2, the numbers in the first paragraph of the Results section, or the numbers in the Results section of the abstract. Correcting Table 4 to be consistent with the rest of the manuscript has a 50% chance of pushing the p-value down below 0.05, but also a 50% chance of pulling it up to 0.15 Two other statistical issues. The first issue is, the authors never say what statistical test procedures they used. It appears that they used t-tests and Fisher's exact tests in Table 2, and used the chi-square test in Table 4, but because they never say, the reader cannot tell for sure. The authors need to say what tests they used and what variables they used them on. The authors probably also ought to stop using the chi-square test in Table 4 because of the warnings about expected cell counts being too small. The second statistical issue is this. The authors correctly calculated a "minimum" sample size from the formula that they used (page 3 of manuscript), but they used the wrong formula. The sample-size formula they used is the formula for testing a single sample for whether its observed proportion P-hat of mutant E6/E7 is significantly different from the hypothesized constant P0=70% based on Zhe et al . 2019. Moreover, the formula they used treats "d=error limitation or absolute precision" as the ±95% margin of error on P-hat given P0, which is the same as saying that the minimum sample size calculated by their formula is the minimum sample size needed to obtain 50% power (not 80% power) to detect the difference between P-hat and P0. But what the authors actually did in this study was, they split their n=31 cases into two sample, determined the number and proportion of strong Cyclin D1 in each sample, and used the chi-square test to compare the two samples to each other for the difference in their strong Cyclin-D1 proportions. To calculate the minimum sample size for that two-sample comparison of Cyclin-D1 proportions will require a different formula from the one the authors used. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No References 1. Cohen J: A power primer. Psychol Bull . 1992; 112 (1): 155-9 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: I am a biostatistician with 25 years of expertise practicing statistics. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Siegel E. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r307750 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-307750 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Macedo J. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r170486 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-170486 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 May 2023 Jacyara Macedo , Department of Biochemistry, Institute of Biology Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, State of Rio de Janeiro, Brazil Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.141136.r170486 The work was developed in order to investigate possible association of mutated HPV E6 and E7 oncgenes with cyclin D1 expression in cervical cancer cells. Introduction section should be improved. The importance of E6 and E7 oncogenes ... Continue reading READ ALL The work was developed in order to investigate possible association of mutated HPV E6 and E7 oncgenes with cyclin D1 expression in cervical cancer cells. Introduction section should be improved. The importance of E6 and E7 oncogenes and the cyclin D1 expression should have been better explored to support the study. References for the sentence “The sequences carried by E6 and E7 lead to an increase in cyclin D1 expression.” were not adequate. The sentence “Increase in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy.” is not supported by the references 8 and 10. I could not check the reference 9. The sentence below should be rewritten: “Given their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out.” Methodology was not properly described. The sentence “Patient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1” could be excluded. “Table 1” should be included at the end of the paragraph. Some parts were not presented or were partially presented. For example, there are different Roche DNA extraction kits. No information about the kit used for DNA extraction in this work was given. References of primers used in this work were not mentioned. PCR conditions used in the amplification of different regions of the viral genome were not properly described. Details of the immunohistochemistry and DNA sequencing were not described. The results should be better explored. What are the effects of non-synonymous mutations in the HPV E6 and E7 oncogenes in the protein structure/function? In mutated E6 protein, arginine was replaced by lysine. In mutated E7 proteins, asparagine at the position 29 was replaced by hydroxylated aminoacids, threonine or serine, and arginine at the position 77 was replaced by cysteine. Do synonymous mutations in the HPV E6 and E7 genes cause any change in protein expression, conformation or function? Is it correct to group all mutations to assess possible associations of E6/E7 mutations with cyclin D1 expression? These issues were not properly discussed. In fact, the limited number of samples did not allow for a deeper discussion. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly References 1. Sauna ZE, Kimchi-Sarfaty C: Understanding the contribution of synonymous mutations to human disease. Nat Rev Genet . 2011; 12 (10): 683-91 PubMed Abstract | Publisher Full Text 2. Xiong Y, Wang Y, Li T, Yu X, et al.: A novel function for cyclin D1 as a transcriptional role in oncogenesis and tumor development by ChIP-Seq and RNA-Seq. J Cancer . 2021; 12 (17): 5181-5192 PubMed Abstract | Publisher Full Text 3. Gu J, Zhang X, Yang Z, Wang N: Expression Of Cyclin D1 Protein Isoforms And Its Prognostic Significance In Cervical Cancer. Cancer Manag Res . 2019; 11 : 9073-9083 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Molecular biology of cancer, Molecular epidemiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Macedo J. Reviewer Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r170486 ) The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-170486 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 09 Mar 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 1 09 Mar 23 read read read Jacyara Macedo , Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil Eric Siegel , University of Arkansas for Medical Sciences, Little Rock, USA Atar Singh Kushwah , Mount Sinai Health System, New York, USA; University of Lucknow, Lucknow, India; Banaras Hindu University, Banaras, India; Alabama State University, Montgomery, USA Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Kushwah A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 17 Sep 2024 | for Version 1 Atar Singh Kushwah , Oncological Sciences, Mount Sinai Health System, New York, New York, USA; Department of Zoology, University of Lucknow, Lucknow, Uttar Pradesh, India; Department of Zoology, Banaras Hindu University, Banaras, Uttar Pradesh, India; Biological Sciences, Alabama State University, Montgomery, Alabama, USA 0 Views copyright © 2024 Kushwah A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Mahendra et al, demonstrated the relationship between mutations in the E6 and E7 oncogenes of HPV Type 16 and Cyclin D1 expression in cervical cancer patients. The study included 31 cervical cancer patients, 38.7% of whom had mutations in the E6/E7 oncogenes. Despite the known role of these oncogenes in promoting cancer through disruption of cellular processes like p53 degradation and pRb inactivation, the study found no significant association between these mutations and Cyclin D1 expression. Cyclin D1 is commonly overexpressed in various cancers, but this study suggests that its expression in cervical cancer may not be directly influenced by E6/E7 mutations. The findings indicate that other molecular mechanisms may be involved in Cyclin D1 regulation, warranting further research with larger sample sizes to clarify these pathways. While reviewing the manuscript, I found the following limitations that need to be rectified for further consideration: The statistical tests used in the study are not clearly described. It appears that t-tests, Fisher's exact tests, and chi-square tests were used, but this is not explicitly stated. Furthermore, the chi-square test in Table 4 is questionable due to low cell counts, potentially making it unreliable. The sample size formula used is incorrect for the study's objectives. The formula calculates the sample size for a single sample proportion test, but the study involves comparing two groups, requiring a different calculation method. Table 4 contains inconsistencies with the rest of the manuscript, showing 11 cases with mutant E6/E7 and 20 cases with wild-type E6/E7, which do not match earlier data. Key details about the DNA extraction kit, PCR conditions, and immunohistochemistry procedures are missing, making replication difficult. Some references cited to support claims about Cyclin D1 expression are not appropriate or do not back the statements made. The authors concluded no association between E6/E7 mutations and Cyclin D1 expression based solely on a p-value greater than 0.05. However, the c-score (0.283) suggests a medium correlation, indicating that the study may be underpowered rather than the relationship being non-existent. The potential functional consequences of non-synonymous mutations in the E6/E7 oncogenes are not thoroughly discussed. The effects of synonymous mutations on protein structure and function are also not addressed. The study does not offer significant new findings or insights into the relationship between E6/E7 mutations and Cyclin D1 expression. Previous research has explored Cyclin D1's role in cervical cancer, and the study does not significantly advance the current understanding. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise PharmacoEpiGenetics, Clinical Oncology and Biotherapeutics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Kushwah AS. Peer Review Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r266381) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-266381 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Siegel E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Aug 2024 | for Version 1 Eric Siegel , University of Arkansas for Medical Sciences, Little Rock, AR, USA 0 Views copyright © 2024 Siegel E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This cross-sectional study enrolled cervical-cancer patients that were positive for HPV type 16. Its two purposes were (1) to characterize the patients' HPV genomes for whether they carried mutations in either the E6 or E7 oncoprotein, and (2) to test for whether the presence of such mutations are associated with Cyclin D1 expression. They obtained 31 subjects that were positive for HPV16, of which 19 had wildtype E6/E7 and 12 had mutated E6/E7. They classified Cyclin D1 expression as Weak or Strong, and tested for association with E6/E7 mutation status using a 2x2 contingency table with an unidentified statistical test, and used the contingency coefficient (c-score) as the association's effect-size measure. They obtained c-score=0.283 with p-value = 0.1 from the unidentified statistical test. From this result, they concluded that there is no association of mutation in the E6/E7 HPV-16 oncogenes with Cyclin D1 expression in patients with cervical cancer. The authors' conclusion is false for two reasons. One, it is a fallacy to conclude that no association exists simply because the p-value is greater than 0.05. The fact that the authors obtained a c-score greater than 0 means that some level of association did exist, but that the study was underpowered to detect it. Furthermore, the fact that the authors obtained a c-score of 0.283 from Table 4 means that the authors also obtained a phi coefficient of ±0.295 from the same Table 4. Phi coefficients of magnitude 0.1, 0.3, and 0.5 were classified respectively as small , medium , and large by Cohen J (1992) Ref 1. By this classification, phi=±0.295 represents a medium association, and therefore, the authors' c-score=0.283 represents an association that is medium, not weak as the authors claimed. Because a medium association, if it exists, could have relevance to public health, it is dangerous to pretend that a medium association equals no association simply because the p-value was greater than 0.05. Two, the authors have a mistake somewhere in their Table 4. The top row of Table 4 shows only 11 cases with mutant E6 or E7, while the bottom row of Table 4 shows 20 cases with wildtype E6 or E7. These numbers are not consistent with the numbers in Table 2, the numbers in the first paragraph of the Results section, or the numbers in the Results section of the abstract. Correcting Table 4 to be consistent with the rest of the manuscript has a 50% chance of pushing the p-value down below 0.05, but also a 50% chance of pulling it up to 0.15 Two other statistical issues. The first issue is, the authors never say what statistical test procedures they used. It appears that they used t-tests and Fisher's exact tests in Table 2, and used the chi-square test in Table 4, but because they never say, the reader cannot tell for sure. The authors need to say what tests they used and what variables they used them on. The authors probably also ought to stop using the chi-square test in Table 4 because of the warnings about expected cell counts being too small. The second statistical issue is this. The authors correctly calculated a "minimum" sample size from the formula that they used (page 3 of manuscript), but they used the wrong formula. The sample-size formula they used is the formula for testing a single sample for whether its observed proportion P-hat of mutant E6/E7 is significantly different from the hypothesized constant P0=70% based on Zhe et al . 2019. Moreover, the formula they used treats "d=error limitation or absolute precision" as the ±95% margin of error on P-hat given P0, which is the same as saying that the minimum sample size calculated by their formula is the minimum sample size needed to obtain 50% power (not 80% power) to detect the difference between P-hat and P0. But what the authors actually did in this study was, they split their n=31 cases into two sample, determined the number and proportion of strong Cyclin D1 in each sample, and used the chi-square test to compare the two samples to each other for the difference in their strong Cyclin-D1 proportions. To calculate the minimum sample size for that two-sample comparison of Cyclin-D1 proportions will require a different formula from the one the authors used. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No References 1. Cohen J: A power primer. Psychol Bull . 1992; 112 (1): 155-9 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise I am a biostatistician with 25 years of expertise practicing statistics. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Siegel E. Peer Review Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r307750) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-307750 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Macedo J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 May 2023 | for Version 1 Jacyara Macedo , Department of Biochemistry, Institute of Biology Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, State of Rio de Janeiro, Brazil 0 Views copyright © 2023 Macedo J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The work was developed in order to investigate possible association of mutated HPV E6 and E7 oncgenes with cyclin D1 expression in cervical cancer cells. Introduction section should be improved. The importance of E6 and E7 oncogenes and the cyclin D1 expression should have been better explored to support the study. References for the sentence “The sequences carried by E6 and E7 lead to an increase in cyclin D1 expression.” were not adequate. The sentence “Increase in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy.” is not supported by the references 8 and 10. I could not check the reference 9. The sentence below should be rewritten: “Given their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out.” Methodology was not properly described. The sentence “Patient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1” could be excluded. “Table 1” should be included at the end of the paragraph. Some parts were not presented or were partially presented. For example, there are different Roche DNA extraction kits. No information about the kit used for DNA extraction in this work was given. References of primers used in this work were not mentioned. PCR conditions used in the amplification of different regions of the viral genome were not properly described. Details of the immunohistochemistry and DNA sequencing were not described. The results should be better explored. What are the effects of non-synonymous mutations in the HPV E6 and E7 oncogenes in the protein structure/function? In mutated E6 protein, arginine was replaced by lysine. In mutated E7 proteins, asparagine at the position 29 was replaced by hydroxylated aminoacids, threonine or serine, and arginine at the position 77 was replaced by cysteine. Do synonymous mutations in the HPV E6 and E7 genes cause any change in protein expression, conformation or function? Is it correct to group all mutations to assess possible associations of E6/E7 mutations with cyclin D1 expression? These issues were not properly discussed. In fact, the limited number of samples did not allow for a deeper discussion. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly References 1. Sauna ZE, Kimchi-Sarfaty C: Understanding the contribution of synonymous mutations to human disease. Nat Rev Genet . 2011; 12 (10): 683-91 PubMed Abstract | Publisher Full Text 2. Xiong Y, Wang Y, Li T, Yu X, et al.: A novel function for cyclin D1 as a transcriptional role in oncogenesis and tumor development by ChIP-Seq and RNA-Seq. J Cancer . 2021; 12 (17): 5181-5192 PubMed Abstract | Publisher Full Text 3. Gu J, Zhang X, Yang Z, Wang N: Expression Of Cyclin D1 Protein Isoforms And Its Prognostic Significance In Cervical Cancer. Cancer Manag Res . 2019; 11 : 9073-9083 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Molecular biology of cancer, Molecular epidemiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Macedo J. Peer Review Report For: Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali [version 1; peer review: 1 approved with reservations, 2 not approved] . F1000Research 2023, 12 :257 ( https://doi.org/10.5256/f1000research.141136.r170486) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-257/v1#referee-response-170486 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Stay Updated Sign up for content alerts and receive a weekly or monthly email with all newly published articles Register with F1000Research Already registered? Sign in Not now, thanks close PLEASE NOTE If you are an AUTHOR of this article, please check that you signed in with the account associated with this article otherwise we cannot automatically identify your role as an author and your comment will be labelled as a “User Comment”. If you are a REVIEWER of this article, please check that you have signed in with the account associated with this article and then go to your account to submit your report, please do not post your review here. If you do not have access to your original account, please contact us . All commenters must hold a formal affiliation as per our Policies . The information that you give us will be displayed next to your comment. User comments must be in English, comprehensible and relevant to the article under discussion. We reserve the right to remove any comments that we consider to be inappropriate, offensive or otherwise in breach of the User Comment Terms and Conditions . Commenters must not use a comment for personal attacks. When criticisms of the article are based on unpublished data, the data should be made available. I accept the User Comment Terms and Conditions Please confirm that you accept the User Comment Terms and Conditions. Affiliation ✕ refresh Please enter your institution. Note: To add your institution or organisation, start typing the name and then select the correct name from the list. Where applicable, the name will appear in both the original language and in English. Do not paste in the name. If the name does not appear in the drop-down list, we will display the information you have entered. ✕ refresh Country/Region * USA UK Canada China France Germany Afghanistan Aland Islands Albania Algeria American Samoa Andorra Angola Anguilla Antarctica Antigua and Barbuda Argentina Armenia Aruba Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia and Herzegovina Botswana Bouvet Island Brazil British Indian Ocean Territory British Virgin Islands Brunei Bulgaria Burkina Faso Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Christmas Island Cocos (Keeling) Islands Colombia Comoros Congo Cook Islands Costa Rica Cote d'Ivoire Croatia Cuba Cyprus Czech Republic Democratic Republic of the Congo Denmark Djibouti Dominica Dominican Republic Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Ethiopia Falkland Islands Faroe Islands Federated States of Micronesia Fiji Finland France French Guiana French Polynesia French Southern Territories Gabon Georgia Germany Ghana Gibraltar Greece Greenland Grenada Guadeloupe Guam Guatemala Guernsey Guinea Guinea-Bissau Guyana Haiti Heard Island and Mcdonald Islands Holy See (Vatican City State) Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Israel Italy Jamaica Japan Jersey Jordan Kazakhstan Kenya Kiribati Kosovo (Serbia and Montenegro) Kuwait Kyrgyzstan Lao People's Democratic Republic Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macao Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Martinique Mauritania Mauritius Mayotte Mexico Minor Outlying Islands of the United States Moldova Monaco Mongolia Montenegro Montserrat Morocco Mozambique Myanmar Namibia Nauru Nepal Netherlands Antilles New Caledonia New Zealand Nicaragua Niger Nigeria Niue Norfolk Island North Korea North Macedonia Northern Mariana Islands Norway Oman Pakistan Palau Palestinian Territory Panama Papua New Guinea Paraguay Peru Philippines Pitcairn Poland Portugal Puerto Rico Qatar Reunion Romania Russian Federation Rwanda Saint Helena Saint Kitts and Nevis Saint Lucia Saint Pierre and Miquelon Saint Vincent and the Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa South Georgia and the South Sandwich Is South Korea South Sudan Spain Sri Lanka Sudan Suriname Svalbard and Jan Mayen Swaziland Sweden Switzerland Syria Taiwan Tajikistan Tanzania Thailand The Gambia The Netherlands Timor-Leste Togo Tokelau Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Turks and Caicos Islands Tuvalu UK USA Uganda Ukraine United Arab Emirates United States Virgin Islands Uruguay Uzbekistan Vanuatu Venezuela Vietnam Wallis and Futuna West Bank and Gaza Strip Western Sahara Yemen Zambia Zimbabwe Please select your country/region. You must enter a comment. Competing Interests Please disclose any competing interests that might be construed to influence your judgment of the article's or peer review report's validity or importance. Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Please state your competing interests The comment has been saved. An error has occurred. Please try again. Cancel Post var lTitle = "Association between mutant E6 and E7 Human...".replace("'", ''); var linkedInUrl = "http://www.linkedin.com/shareArticle?url=https://f1000research.com/articles/12-257/v1" + "&title=" + encodeURIComponent(lTitle) + "&summary=" + encodeURIComponent('Read the article by '); var deliciousUrl = "https://del.icio.us/post?url=https://f1000research.com/articles/12-257/v1&title=" + encodeURIComponent(lTitle); var redditUrl = "http://reddit.com/submit?url=https://f1000research.com/articles/12-257/v1" + "&title=" + encodeURIComponent(lTitle); linkedInUrl += encodeURIComponent('Mahendra INB et al.'); var offsetTop = /chrome/i.test( navigator.userAgent ) ? 4 : -10; var addthis_config = { ui_offset_top: offsetTop, services_compact : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_expanded : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_custom : [ { name: "LinkedIn", url: linkedInUrl, icon:"/img/icon/at_linkedin.svg" }, { name: "Mendeley", url: "http://www.mendeley.com/import/?url=https://f1000research.com/articles/12-257/v1/mendeley", icon:"/img/icon/at_mendeley.svg" }, { name: "Reddit", url: redditUrl, icon:"/img/icon/at_reddit.svg" }, ] }; var addthis_share = { url: "https://f1000research.com/articles/12-257", templates : { twitter : "Association between mutant E6 and E7 Human Papilloma Virus Type.... Mahendra INB et al., published by " + "@F1000Research" + ", https://f1000research.com/articles/12-257/v1" } }; if (typeof(addthis) != "undefined"){ addthis.addEventListener('addthis.ready', checkCount); addthis.addEventListener('addthis.menu.share', checkCount); } $(".f1r-shares-twitter").attr("href", "https://twitter.com/intent/tweet?text=" + addthis_share.templates.twitter); $(".f1r-shares-facebook").attr("href", "https://www.facebook.com/sharer/sharer.php?u=" + addthis_share.url); $(".f1r-shares-linkedin").attr("href", addthis_config.services_custom[0].url); $(".f1r-shares-reddit").attr("href", addthis_config.services_custom[2].url); $(".f1r-shares-mendelay").attr("href", addthis_config.services_custom[1].url); function checkCount(){ setTimeout(function(){ $(".addthis_button_expanded").each(function(){ var count = $(this).text(); if (count !== "" && count != "0") $(this).removeClass("is-hidden"); else $(this).addClass("is-hidden"); }); }, 1000); } close How to cite this report {{reportCitation}} Cancel Copy Citation Details $(function(){R.ui.buttonDropdowns('.dropdown-for-downloads');}); $(function(){R.ui.toolbarDropdowns('.toolbar-dropdown-for-downloads');}); $.get("/articles/acj/128537/141136") new F1000.Clipboard(); new F1000.ThesaurusTermsDisplay("articles", "article", "141136"); $(document).ready(function() { $( "#frame1" ).on('load', function() { var mydiv = $(this).contents().find("div"); var h = mydiv.height(); console.log(h) }); var tooltipLivingFigure = jQuery(".interactive-living-figure-label .icon-more-info"), titleLivingFigure = tooltipLivingFigure.attr("title"); tooltipLivingFigure.simpletip({ fixed: true, position: ["-115", "30"], baseClass: 'small-tooltip', content:titleLivingFigure + " " }); tooltipLivingFigure.removeAttr("title"); $("body").on("click", ".cite-living-figure", function(e) { e.preventDefault(); var ref = $(this).attr("data-ref"); $(this).closest(".living-figure-list-container").find("#" + ref).fadeIn(200); }); $("body").on("click", ".close-cite-living-figure", function(e) { e.preventDefault(); $(this).closest(".popup-window-wrapper").fadeOut(200); }); $(document).on("mouseup", function(e) { var metricsContainer = $(".article-metrics-popover-wrapper"); if (!metricsContainer.is(e.target) && metricsContainer.has(e.target).length === 0) { $(".article-metrics-close-button").click(); } }); var articleId = $('#articleId').val(); if($("#main-article-count-box").attachArticleMetrics) { $("#main-article-count-box").attachArticleMetrics(articleId, { articleMetricsView: true }); } }); var figshareWidget = $(".new_figshare_widget"); if (figshareWidget.length > 0) { window.figshare.load("f1000", function(Widget) { // Select a tag/tags defined in your page. In this tag we will place the widget. _.map(figshareWidget, function(el){ var widget = new Widget({ articleId: $(el).attr("figshare_articleId") //height:300 // this is the height of the viewer part. [Default: 550] }); widget.initialize(); // initialize the widget widget.mount(el); // mount it in a tag that's on your page // this will save the widget on the global scope for later use from // your JS scripts. This line is optional. //window.widget = widget; }); }); } close Error Close Add Reset F1000.MICROSERVICES.AFFILIATION = ''; $(document).ready(function () { $('.js-affiliations-form').each((index, form) => { new AffiliationForm({ formId: form.id, institutionErrorSelector: '.comment-enter-institution', departmentErrorSelector: '.comment-enter-department', placeSelector: '.js-add-comment-place', stateSelector: '.js-add-comment-state', zipCodeSelector: '.js-add-comment-zipcode', countrySelector: '.js-add-comment-country', countryErrorSelector: '.comment-enter-country', }); }); }); $(document).ready(function () { var reportIds = { "294148": 0, "259842": 0, "294149": 0, "294150": 0, "294151": 0, "294144": 0, "259846": 0, "294145": 0, "294146": 0, "259844": 0, "294147": 0, "259850": 0, "259849": 0, "259855": 0, "259854": 0, "259853": 0, "259857": 0, "259856": 0, "203799": 0, "203803": 0, "165914": 0, "203802": 0, "165915": 0, "203801": 0, "203800": 0, "165918": 0, "165919": 0, "165916": 0, "203804": 0, "165917": 0, "307749": 0, "165920": 0, "307750": 8, "307756": 0, "307757": 0, "307758": 0, "307759": 0, "307752": 0, "307754": 0, "307755": 0, "307760": 0, "168774": 0, "168775": 0, "168773": 0, "224331": 0, "224330": 0, "224329": 0, "224335": 0, "224334": 0, "224333": 0, "224332": 0, "224338": 0, "224337": 0, "224336": 0, "276076": 0, "276077": 0, "276078": 0, "276079": 0, "276073": 0, "276074": 0, "276075": 0, "276080": 0, "276081": 0, "276082": 0, "266380": 0, "266381": 7, "266382": 0, "266383": 0, "266376": 0, "266377": 0, "266378": 0, "266379": 0, "266384": 0, "266385": 0, "230819": 0, "230818": 0, "230817": 0, "230823": 0, "230822": 0, "230821": 0, "230820": 0, "230826": 0, "230825": 0, "230824": 0, "289213": 0, "289214": 0, "289215": 0, "289216": 0, "289217": 0, "289218": 0, "289229": 0, "289230": 0, "170486": 22, "170487": 0, "170490": 0, "170488": 0, "174584": 0, "294142": 0, "170489": 0, "294143": 0, }; $(".referee-response-container,.js-referee-report").each(function(index, el) { var reportId = $(el).attr("data-reportid"), reportCount = reportIds[reportId] || 0; $(el).find(".comments-count-container,.js-referee-report-views").html(reportCount); }); var uuidInput = $("#article_uuid"), oldUUId = uuidInput.val(), newUUId = "e4bb6d23-6ac2-46da-ba01-4d9ad32b37a2"; uuidInput.val(newUUId); $("a[href*='article_uuid=']").each(function(index, el) { var newHref = $(el).attr("href").replace(oldUUId, newUUId); $(el).attr("href", newHref); }); }); An innovative open access publishing platform offering rapid publication and open peer review, whilst supporting data deposition and sharing. Browse Gateways Collections How it Works Contact For Developers Cookie Notice Privacy Notice RSS Submit Your Research Follow us © 2012-2026 F1000 Research Ltd. ISSN 2046-1402 | Legal | Partner of Research4Life • CrossRef • ORCID • FAIRSharing R.templateTests.simpleTemplate = R.template(' $text $text $text $text $text '); R.templateTests.runTests(); var F1000platform = new F1000.Platform({ name: "f1000research", displayName: "F1000Research", hostName: "f1000research.com", id: "1", editorialEmail: "[email protected]", infoEmail: "[email protected]", usePmcStats: true }); $(function(){R.ui.dropdowns('.dropdown-for-authors, .dropdown-for-about, .dropdown-for-myresearch');}); // $(function(){R.ui.dropdowns('.dropdown-for-referees');}); $(document).ready(function () { if ($(".cookie-warning").is(":visible")) { $(".sticky").css("margin-bottom", "35px"); $(".devices").addClass("devices-and-cookie-warning"); } $(".cookie-warning .close-button").click(function (e) { $(".devices").removeClass("devices-and-cookie-warning"); $(".sticky").css("margin-bottom", "0"); }); $("#tweeter-feed .tweet-message").each(function (i, message) { var self = $(message); self.html(linkify(self.html())); }); $(".partner").on("mouseenter mouseleave", function() { $(this).find(".gray-scale, .colour").toggleClass("is-hidden"); }); }); Sign In Remember me Forgotten your password? Sign In Cancel Email or password not correct. Please try again Please wait... $(function(){ // Note: All the setup needs to run against a name attribute and *not* the id due the clonish // nature of facebox... $("a[id=googleSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("GOOGLE"); $("form[id=oAuthForm]").submit(); }); $("a[id=facebookSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("FACEBOOK"); $("form[id=oAuthForm]").submit(); }); $("a[id=orcidSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("ORCID"); $("form[id=oAuthForm]").submit(); }); }); If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password. The email address should be the one you originally registered with F1000. Email address not valid, please try again You registered with F1000 via Google, so we cannot reset your password. To sign in, please click here . If you still need help with your Google account password, please click here . You registered with F1000 via Facebook, so we cannot reset your password. To sign in, please click here . If you still need help with your Facebook account password, please click here . Code not correct, please try again Reset password Cancel Email us for further assistance. Server error, please try again. If your email address is registered with us, we will email you instructions to reset your password. If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance. Please wait... Register $(document).ready(function () { signIn.createSignInAsRow($("#sign-in-form-gfb-popup")); $(".target-field").each(function () { var uris = $(this).val().split("/"); if (uris.pop() === "login") { $(this).val(uris.toString().replace(",","/")); } }); });