作 为举报人,我始终就只坚持一条：因为这是实验方法本身的问题,所以必须在对照上实验验证作者的实验方法,即 Y FISH ， Y/SPC 多重 标记。况且第一作者 3 月 6 日的 电邮中,她自已都说这两类实验她均不行,还得请我做几张 Y/SPC 多重 标记的片子。所以在台面上耶鲁作为世界名校无任何理由不去验证作者的实验方法,要么耶鲁能告诉我:作为名校他们的超级专家有更好,更确实,更简单明了的方法来获的这个非常简单的科学事实真相：作者的 Y FISH 方法 (protocol) 到底是一个如作者在其文章中所吹嘘那 样超高检测敏感度的方法 ( 文章中可使大于 99% 的公鼠骨髓 细胞被标记成 Y+ ，但无任何 图像 ) 还是只是一个检出率很低即漏检率很高的 Y FISH 方法。 遗憾的是耶鲁既不验证作者的实验方法,又不告诉我其超级专家的高招。耶鲁根本不提此案的关键点：作者实验方法的缺陷和由这种缺陷的实验方法所产生的别人无法在阳性对照上 ( 公鼠骨髓 细胞 ) 重复的, 魔 术般的, Y FISH 的神奇 实验数据 。 说其象魔术般神奇一点也不过分, 因为第二年这魔术在作者手中也玩不转了 - 请见我贴上的 3 月 6 日的 电邮第一作者本人对我所做的坦白！这耶鲁是否在刻意回避我所举报科研造假案的关键点？难道拥有如此雄厚的科学技术资源(包括生命科学领域的大专家学者)和学术界享有盛誉的世界名校接不起我这一在逻辑上,科学上和实验技术上如此简单的一招？如真是这样岂不贻笑天下？因为我只是一个无权,无势,无资源,无学术盛誉,但具有做为一名科研人员而应具备的科学良心和科研态度 - 事实求是的普通草民, 既四无一有的平民百姓,况且我所出的招非常简单明了, 就是从经济和时间角度上也是最佳获取我所举报的科研造假案真相的方法: 一步到位,简单明了( 在显微镜下只须看一眼由作者的高科技生产线 - Y FISH方法所能够生产的产品 - Y FISH标记的公鼠骨髓片子则真相大白 - 真可使各方无话可说。这也是在整个举报造假案过程中,我一直所要求的： 为何不能给科学和事实一次说话的机会？ )。 以下是我所 经历的耶鲁的所谓调查： 如我在事件回放中所述,由于作者和 中立方 的原因致使 内部方案 流 产（参见我的博文 : 事件回放）。我于 2008 年 4 月正式将此案 举报到耶鲁医学院 - 特 别办公室, 会见 了 Dr. Linda Mayes 并告 诉了她：我是如何发现老板的实验方法有问题,和抗体 - 抗 SPC （ Chemicon, AB3428) 的 问题。我明确说明这是实验方法本身的问题,即按作者的实验方法是无法得出其所发表文章中的相应 实验结果 的,而且 说明了 内部方案 (Action Plan) 是如何流 产的。她让我写出书面报告。 2008年5月我将书面报告交给 Dr. Linda Mayes (请见我的报告-英文版)。报告中我要求: 1.由中立方实验验证作者所有相关的实验方法(protocols); 2.由中立方重新检测作者3篇文章(FASEB 2007, Science 2004, Cell 2001)的实验组织样本 (组织的石蜡样本）。 2008年5月15日又会见 Dr.Linda Mayes,她以自已不懂技术(Y FISH,Y FISH和荧光免疫标记)为由,让我给她一点时间来找耶鲁的资深学者作为她的科技顾问来与我讨论技术问题。我告诉了她：作者的 Y FISH方法和Y/SPC双标记方法是不可能得出其相应实验结果的，我的相应方法比作者的好多了，但也得不出相应结果，这世上无人能得出那种实验结果- 这就是一个简单的技术瓶颈问题。作者方法最大的缺陷就是其Y FISH方法的低检测敏感度,即高漏检率,只需用对照实验验证其方法即可得真相,且这是唯一获得真相的方法。 2008年6月9日 Dr. Linda Mayes 和具有近50年经历的耶鲁资深学者 Dr. Michael Kashgarian 作为她的科技顾问与我讨论实验技术问题。此学者对我说: 他会看荧光免疫标记的片子,内部方案中的抗SPC片子是工作的。我只问了他一个问题: 那抗体-抗SPC (Chemicon, AB3428)是否从公司直接订购？他答: 不是,但他看了 装抗体的管子是 AB3428 。我说: 您打住,已没必要讨论这抗体的问题了(请见我的博文-事件回放)。现在我们来谈正事-Y FISH和Y/荧光免疫多重标记实验方法的问题。这位代表世界名校耶鲁资深学者的回答令我大跌眼镜,他说: 对于Y FISH实验技术除了这个名词以外,他一概不知 ,他需要时间上网去查相关信息。不是亲身经历,简直不敢相信此类黑色幽默,此人当然算资深学者, 但能否算这里所要讨论的 实验技术方面的专家？ Linda Mayes 说: 给他一个月时间去查Y FISH的资料。对于这种拖延战术,我又能说什么呢？那位专家离去后,我对 Linda Mayes 说:在这一小小块我所打击的实验技术领域, 我敢说我自已就是目前世上最好的专家之一, 因为我已建好的此类实验方法 (protocols)能产生最好的此类实验的真结果。所以我可以用自己的生命和人格担保: 作者文中公鼠骨髓细胞Y FISH 的实验数据只能是100%有意伪造的数字! 注： 自2008年4月份以后,有关各方就对我施压以让我承认所谓内部方案验证的抗SPC（AB3428）的结果,但绝口不提为何不从公司订购该抗体？为何不做 Y/SPC双标记验证？那位资深学者就是通过院办 (Dean's Office)安排读片的独立(independent) 专家！漏洞在那里,请看我的博文: 事件回放( 管子里的抗体是可以调包的 )! 2008年7月10日特别办的 Linda Mayes 又约我与 Dr. Kashgarian 谈技术问题。可此学者还大谈抗SPC的事。我说: 您还是先打住,该谈正事了。您Y FISH方法的技术资料查得如何？他居然告诉我: 他根本就没查Y FISH技术的资料,但他咨询了本 领域的超级专家(super experts), 专家认为在致死照射后骨髓移植, 受体鼠骨髓细胞替换(engraftment)可达90%,半致死照射后,可达50%的替换(engraftment),而且此文(FASEB 2007)是经过审稿的(peer-reviewed)所以他看不出文章有何问题。( 注 ：我不知道这位资深学者所指本领域为何领域,我只知道我们应该讨论的领域是Y FISH，Y/荧光免疫多重标记实验方法的技术问题) 我告诉他：作为一种专家学者的看法,我也赞成这看法,但这并不是我所挑战的问题。 我挑战的是作者文中那组公鼠骨髓细胞Y FISH实验数据： 小于1%的公鼠骨髓细胞为Y阴性,因为作者 所用实验方法具有大百份之几十的漏检率,也就是大百分之几十的公鼠细胞会被作者的方法所漏检成Y阴性而决非小于1%的公鼠细胞被漏检成Y阴性！ 这问题有何难懂吗？ 最后这位资深学者只得对我说： 步根, 实在抱歉我不知道你挑战是何问题 。这种幽默实在是不敢恭维, 一个代表世界名校的资深学者作为科技顾问与举报人来谈具体的实验技术问题, 而举报人举报此科研造假案的逻辑和科学实验依据就是 ：作者具体的 实验方法本身的缺陷 ,即按其方法是不可能得出其文章中相应实验结果的。先是说:对此类技术Y FISH除了名字以外,其余一概不知。给您一个月时间去查资料吧,再告诉你: 他根本就没查！只差没说公鼠细胞均含Y染色体了。最后再告诉你: 挑战的目标为何物,他也不知道！ 岂不象一场辩论完了后,对方对你说：这次辩论的主题我不知道！我应该有权质疑: 那您来干什么呢？ 这可不是儿戏。此学者离去后, Linda Mayes 居然还敢问我: 让他再去查有关资料,过段时间再和他一起讨论有关技术问题。我答: 已无此必要了,你为何不能请外校具有中立立场而又能听得懂该些技术的学者来讨论此技术问题呢？她又说:她会再找耶鲁其他的资深学者再与我讨论实验技术问题。并说:希望我第二天能将我的实验记录本(含有我未发表的Y FISH和Y/荧光免疫多重标记方法)交到她办公室以确保 其安全。第二天我去了特别办给她看了下我的记录本,并对她说:不劳您驾,我自已会将记录本锁好。 注: 自从问题上台面,举报人的核心目标就是打击造假者的死穴- 实验方法本身, 如内部方案中实验验证其方法,而当时所有各方均知道我手中拥有最好的此类实验方法(protocols)。所以老板使用各种借口想得到我的方法, 当然我是不会让她如愿的。当时我在这种无助的情况下也数次送电子邮件给官方求援, 并表明就是开除我,在我所举报问题解决之前谁也别想见到我的实验方法！在官方验证作者的实验方法结果出来之前, 我为何须死保自已的方法被漏出去。只须用常识就不难理解。因为: 耶鲁作为世界名校不会不知道一旦实验验证作者的Y FISH单标记和Y/SPC双标记方法的片子一出来, 在荧光显微镜下只需看一眼则真相大白: 只能是大百分之几十的公鼠细胞被作者超高检测敏感度的Y FSIH方法而漏检被标记成Y阴性细胞,而绝非小于1%的细胞缺乏Y染色体！此乃事实胜于雄辩, 耶鲁作为名校也无法将用眼睛就能看到的大百分之几十的Y阴性细胞硬说成小于1%的公鼠细胞为Y阴性细胞吧，因为任何一双正常的眼睛就能判别此种差异,无须具备高科技知识,更无须只有名校的超级专家才能判别这种差异 - 荧光显微镜下判别大百分之几十与小于百分之一 Y阴性细胞的差别吧！(耶鲁, 作者总不能说大比例的正常公鼠细胞都必发生了Y染色体丢失吧, 因为不管其做多少张片子,结果都会一样。这就是科学！）请见我的博文：事件回放, 实验系统和实验技术的解释, 图解中图6 到图10是我用作者的Y FISH方法所做的正常公鼠骨髓细胞片的Y标记 ,当然是大多数细胞被漏检而为Y阴性！这就是作者Y FISH方法本身所具有的高漏检率所决定。 如我博文中所讲,其方法本身的硬伤是：Y DNA变性条件实在太差,73度 5分钟 - 这不足以打开所有细胞核中Y DNA 的双链,而此乃Y探针杂交的先决条件！随着其Y FISH方法高漏检率的暴光,则作者有意伪造相应实验数据的多米诺骨牌较应马上见较(如其他组小鼠骨髓细胞Y FISH的数据：含Y染色体即Y阳性率均大于93%!)。 2008年7月29日 Dr. Linda Mayes 又约见我, 并有该特别办前主任 Dr. Larry Cohen。首先这位前主任就将我与老板的关系定义为: 两人之间的个人关系不和(因为此前我曾告诉官方: 老板有意给我穿小鞋,但并未难倒我,因为均为技术上的所谓难题, 而这正是我的强项), 所以耶鲁想解决此问题。耶鲁提供给我一个保护方案(protection plan)即由院长特别基金支付我的工资为一定时期(certain period) 而将我调出老板实验室, 但这基金只管工资(即我被挂起来, 无法再做实验)。我当即纠正他的说法: 我与老板无任何个人恩怨,只是她逼我做假而我不干且举报她做假而已。从7月30日到8月7日 Linda Mayes 一连给我送了4封电子邮件以催促我赶快进入保护方案我当然不会犯傻。很简单,我不接球,我让你玩你自已的单边游戏(我一个多年的美国朋友告诉我：这是典型的由耶鲁律师们为你量身订做而设的套,你不接球,则他们没招。并提醒我:千万别在任何文件上签字，因为你读不懂那些法律名词,就让他们双手抱球吧。并告诉我应该向美国政府ORI举报此案了。这哥们当时正在学法律)。我是可以将她的电子邮件暴光的。 注：因为与老板个人关系不和,学校出钱出力来解决,在名校作 bench scientist 能有此类好事吗？ 对我而言,这可成为潜在的陷井。在美国实验室干过的人均知,由老板科研基金所雇的人,只要与老板不和,可让你立马另找工作。但条例规定实名举报人须受到保护,所以老板无法开我。反观耶鲁的保护方案：一定时期的法律解释权均在耶鲁手中,即这可以是2个月也可为2年(基金可随时完了!),并且全为口说。 2008年8月我正式向美国政府监管机构ORI举报此案。我会另写博文讲ORI的作为。 2008年9月11日耶鲁收缴作者的原始记录时,作者无法交出与其文章 FASEB 2007有关的任何原始记录包括实验记录本。而只有一句话:所有的记录均找不到了。这篇文章可是篇规模不小的文章,用了60多只不同的小鼠,由多人所做的各种具体的实验， 难道大家的实验记录本均不约而同地一起消失？ 另一方面则以保护我为由强迫我交出相关的实验记录本( 含有我自已未发表的Y FISH和Y/荧光免疫多重标记的实验方法 )。 2008年10月22日耶鲁关闭此案,没给我任何科学事实,证据,依据可用来反驳我的依据和论据。只说无学术道德问题。院长的信:太完美的实验数据不一定是有意造假,别人无法重复可有多种原因。 关闭案例对我而言就是丧失实名举报人身份。 注： 这里院长大人玩的是偷梁换柱的把戏。因为我举报作者有意伪造实验结果数据的依据是: 其所用实验方法本身的缺陷, 即用作者的方法是无法得出其相应 实验结果的 - 方法学上的造假！就是按其所玩把戏:别人无法重复可有多种原因这其中一种最重要的原因就是:作者所伪造的结果数据远超过了该实验技术的瓶颈很远, 即用这种实验技术是无法得到此类完美的实验数据的 - 逻辑上的不可能事件 (这里是作者公鼠骨髓细胞Y FISH实验的结果数据：0.8 +/- 0.8 % 的公细胞缺乏Y染色体-这结果是说：其Y FISH方法标记公鼠骨髓细胞后,大于99%的细胞被标记为Y阳性- 这是一个技术上的天方夜谈 )所以无人能重复！有兴趣的网友可以上网查查看能否发现一张全景图像(含50-100个细胞): 公鼠骨髓细胞被Y FISH方法标记后只有少与10%的细胞为Y阴性-即漏检,我是找不到此类图像, 也没见过任何文章敢说其Y FISH方法能将90%或以上的公鼠骨髓细胞标记成Y阳性 - 当然世界名校耶鲁的专家学者我老板的团队例外！遗憾的是我老板团队从不显示她们完美的公鼠样本Y FISH标记全景图像, 作为其研究项目的继任研究人员工作了二年多,在内部我也无这眼福-看一看这完美Y FISH标记图像。倒是进入2008年可能是作者的倒霉年, 因为其刚用过不久的魔法般的实验方法：Y FISH和Y/SPC 双标记似乎失灵了。第一作者 Dr. Erica Herzog 在看完我用自已的Y/SPC多重标记方法所做的实物-片子后，于2008年3月6日送给我的电子邮件,此邮件中她明白无误地坦白： 她从未能将Y/SPC双标记搞工作过 ...连FISH看来都不工作了。 她怎敢送如此邮件给我,请见我的博文-事件回放。为了便于直观理解,在这里上传用我的方法所做的Y/SPC多重标记的野生型公鼠肺切片图像, 和用作者Y FISH方法所做的野生型公鼠骨髓细胞片子的Y标记 - 即实验验证作者的Y FISH方法(在我的博文: 图解中有更多的图像和图像解释)。此文中我也贴上作者2008年3月6日和2007年6月18, 19日的电子邮件看她两人在不同的时候是如何谈她们的同一 Y FISH方法的。 2008年12月2日老板给我一信：通知我90天后支付我职位的基金就到期了。2008年下半年老板先将我的位子从最大的基金转到一个将到期的小基金支付，我知道这把戏又能怎样呢。谁让我不合作呢？注：随着耶鲁10月22日关闭案例，我已不是举报人了！ 按理在举报人和被举报人之间耶鲁应该担当公正的裁判,作为世界名校其据有学术技术和逻辑的判断能力-如何得到我所举报案的真相。而获取真相这对举报方和被举报方均公平,如果我错了,则我为自己的行为承担所有后果包括面对作者,耶鲁，美国政府ORI的法律起诉因为我给各方找了麻烦，但如果我对了则作者须对她们自已的行为承担后果(在给官方邮件中我也写下了对自己的行为承担后果)。而正常用于这种涉及科研结果造假的程序是：只要举报人对发表的实验结果提出了科学技术上合理的质疑, 则自证清白的重担就转到作者的肩上了(proof of burden), 即作者必须拿出证据来证明其实验结果是真实的。也就是说这个程序与法庭上的无罪推论是相反的。耶鲁在这个程序中应该担当的是裁判或法官的角色,完全以证据本身作出判断和结论(可惜这里没有陪审员-在法庭上作有罪或无罪的决断)。 如果遵循程序, 即游戏规则(这也是美国最崇尚的基本价值观念之一),对双方的证据和证据的可靠度不采用双重标准,裁判还是裁判,则此案的解决太容易了。这是由于作者造假的方式所决定了的：在实验方法学上造假,即上演空城记且反复演此戏。其伪造的Y FISH实验结果不但远远超出其方法本身的检出率, 而且远超出此类实验技术的技术瓶颈 - 即这世上目前还没人拥有这种能达100%检出率的Y FISH方法 (protocol),况且也知道其方法检出率太低连包含几十个细胞均为Y阳性的假电子图像都无法做出,这么多年还敢这般玩法。真是无知才能无畏,连把戏不可久玩的道理都不懂！ 作为举报人在第一次举报时我就告诉特别办：抗SPC抗体(Chemicon, AB3428)在我手中从不工作,作者Y FISH方法检出率低,所以其文章 FASEB 2007中阳性对照组-公鼠骨髓Y FISH的实验数据只能是100%的伪造的数字,根本不可能为Y FISH的实验数据,还提供了作者自已所写的相关电子邮件包括第一作者 Erica 3月6日送给我的邮件。我还告诉 Dr. Linda Mayes：你不必相信我所说的,但用对照实验验证作者的Y FSIH方法则结果一目了然,而这也是得到作者Y FISH方法低检出率真相的唯一方法,而不是你说,我说,或她说。 注：当时我是可以将作者所有用于组织切片，细胞片的Y FISH方法,Y/荧光免疫多重标记方法全都实验验证,然后将片子放到校方的桌上的。我没那么做,因为我想给各方留足面子。 而作者方 则提供不出任何证据能说明其文章中那组公鼠骨髓 Y FISH 实验数据是真的实验数据，逻辑上至少你的方法要能够产生此类结果吧 ( 在公鼠骨髓片子上将 99% 以上的 细胞标记成 Y 阳性 ) 。 连是否做了此类 Y FISH 实验的证据也没提供。 唯一的证据就是作者在文章中所写下的阿拉伯数字 。我反复向官方要求让他们检查已拷贝的硬盘看是否有与作者神奇 Y FISH 数据相 对应的电子图像,可人家就是回避。作者连一丁点产生该文章的原始记录都无法提供：全找不到了 (2007 年 发表的文章 )! 在耶 鲁居然连这种借口也接受！ 出于无奈， 2009 年 2 月初我再 举报此案时得将物证作好：将我自已按作者 Y FISH 方法做的野生型公鼠骨髓 细胞片子,全景图像,作者的电子邮件,技术解释,书面报告等全交到院长办公室。也寄一份给美国政府 ORI 请他们 眼见为实！ 并请作者,中立方来挑战我所提供的物证：你们自已实验验证作者的Y FISH方法。这非常合理吧, 免得对方说: 举报人提供的物证无公信力 。我主动让你交互验证（cross-examination)总可以吧。作这种骨髓细胞Y FISH实验,第二天下午就能得到标记好的片子,即可在荧光显微镜下一显真相: 片子上到底是小于1% 的细胞被标记成Y阴性还是大百分之几十的细胞被标记成Y阴性则一目了然,任何各方均无话可说, 即让简单的科学事实来说话！ 从上一次举报到关案,我也学到不少东西。所以再举时报我全采用电子邮件方式,而避免上次那种闭门会谈: 被人一次次当猴耍了,还口说无凭。但写下来的东西总得认帐吧。再则就是使案例最简单化：只打击一个实验方法：用于骨髓细胞片子的Y FISH方法; 和一组该方法所产生的Y FISH实验数据: 阳性对照组-野生型公鼠骨髓细胞的数据: 0.8 +/- 0.8 % 的细胞缺乏Y然色体这结果是说: 在整组此公鼠的所有骨髓细胞片子上, 作者用其Y FISH方法标记Y染色体DNA, 其结果是平均只有0.8% 的细胞被标记为Y阴性,标准差或标准误也只有0.8% (作者还有意不将这0.8% 的Y阴性说成漏检,而说成缺乏Y染色体; 其潜台词是 : 她的方法本身的检出率还是 100%，这0.8% 的细胞是由于没有Y然色体,所以作者没测到。这潜台词是给审稿者看的, 表示作者的Y FISH方法确实具有100%的检出率而可作为其文章中的逻辑推理前提，所以在实验鼠样本中(含有公鼠细胞和母鼠细胞 - 供，受体鼠性别相反模型)对其Y FISH实验结果作者可以作如下反推理: 在所谓的公鼠细胞中如作者未能用其Y FISH方法检测到Y就等于Y染色体丢失! 对于她们自已用了多年的看家本事-Y FISH方法具有很低的检出率作者自已当然知道, 她们所蒙的是外人并不知此真相而信以为真。因为此领域使用该方法的实验室并不多。有关祥情请见我的博文：实验系统和实验技术解释。我怎么能知道真相呢？我是她们研究相目的继任者，即这方法学上所演的空城记的城中之人, 我当然得用她们的实验方法！)。 即再次举报造假案非常简单明了：次案的命运只系于一组公鼠骨髓细胞Y FISH的实验数据。 我举报的立足点或依据: 作者的Y FISH方法具有很低的检出率而绝非其文章中所称超高的检出率。用其方法 (protocol)标记任何正常公鼠骨髓细胞片子只能产生具有大百分之几十的细胞被标记成Y阴性的公鼠骨髓细胞片子；这就决定了在获取这组Y FISH实验数据时 (data acquisition),作者的双眼在荧光显微镜下所能看到的每一个视野只能是百分之几十的细胞被标记成Y阴性, 而绝非小于1% 的细胞被标记为Y阴性; 并且任何一双诚实的眼睛是无法将百分之几十的Y阴性细胞重复地判别,记数成小于1%的Y阴性细胞的；所以文章中那组Y FISH的所谓实验数据只能是100%的有意伪造的数字,而根本不可能为次类实验的数据。这就是我的证据链。证明完毕。在举报时我已将这些证据放上了台面,如果作者方, 耶鲁方想在科学上推翻我的结论，也必须用以科学技术为基础的证据链来打段我的证据链。逻辑上这组数据只能是真实验数据或伪造的数字，总不能亦真亦假吧？我证的是这只能为作者有意伪造的数字, 因为其所用方法(protocol)无法产生此神奇结果,而作者文章的发表又需要此神奇数字 - 则只能伪造,或者别发这文章！遗憾的是在整个所谓的调查过程中,我看不到耶鲁对作者的这个Y FISH实验方法(protocol)作出任何的科学评估即用这方法作者能否有可能得出其文章中的相应结果数据；更没看到耶鲁对那组神奇数据作出任何的科学评估即这神奇数据是否有可能是真的Y FISH实验的结果。 而事实上我再次举报的造假案就是这两个目标！ 难到作为拥有如此强大的科研和学术实力的世界名校真接不起由我这个平民百姓所出的在科学技术上如此简单的招或不愿意接这招可又不便说出？作为纳税人我有权知道真相，因为那篇文章是由纳税人的钱-NIH基金资助的。而且我个人认为：科学面前人人平等；科学的基本精神就是事实求是；在实验科学上实践是检验真理的唯一标准也应该是普世真理！我深信这一点：科学就是自然法则(这也是我从小学到研究生所受的教育), 谁也无法将自然法则象面团一样任意揉捏。这也就是为何我敢叫板的根本原因。我还真不信有人能象舞台上变戏法一样用作者的Y FISH实验方法 (protocol)在任何公鼠骨髓细胞片子上将99%的细胞标记成Y阳性。就这个非常简单的科学技术问题我敢跟你名校耶鲁,美国政府的监管机构ORI打擂台,从理论上,实验原理上,实践上,为何作者的Y FISH方法只能是一个低检出率的方法,我所打击的数据只能是作者有意伪造的数字。如我输了,则承担所有后果,如我赢了,也只要求你名校,你政府机构回到你们自已所订的游戏规则和公平,公正的价值观上来。我所举报的科研造假案不是靠权力和所谓的公信力就能改变的，因为权力与所谓的公信力无法改变这样一个非常简单的科学事实：作者的Y FISH方法只能是低检出率的实验方法这是已被自然法则 (DNA 分子原位杂交的实验原理)所决定了的。在此我也呼吁世界上任何具有公信力的科学实验室: 愿意出面来实验验证作者用于小鼠骨髓细胞的Y FISH方法,我可以提供所有的技术支持。 更无法理解的是我反复送电子邮件给耶鲁，问其一个简单的问题：我是否为此案的实名举报人？对方回复我的邮件，但不回答我的问题。耶鲁怎么回答，如说我是，则老板精心安排想将我踢出的方案则会前功尽弃，如说不是吧，则太不合逻辑，所以还是回避! 这就是他们所谓非常严肃的调查。耶鲁的所谓结论我7个月以后才从ORI索取到，有多莫名奇妙我会在下一篇博文写出。 我并不是一个职业打假员，在极困难的环境下我仍在默默的干科研。不经意间科学给了我丰厚的回报。我在按照自已的想法所做的模型中偶然发现了整个成年干细胞发育可塑性研究领域谁也没见过的实验现像 (该领域从2004年以后基本处于仃摆状态)，因为这是一株老板团队没做出任何名堂而准备杀掉的小鼠,也不属于她的科研基金项目。所以对我没雷池。我的发现是在同一个体的同一靶器官小鼠骨髓中的干细胞可以两种完全不同的途径变成特化的组织细胞：1.与宿主的细胞融合而变成该组织细胞（在 2004 年以前有 许多大文章证明此现像); 2.直接分化成为该组织的细胞 (此现象未被证明过)。由于靶器官的原因,如果第二种途径能被确证,则意味着我在不经意 间证明了一个当年这领域中许多大实验室均想证明而未能证明的一个非常重要基本概念：转分化。我的模型证明: 属于中胚层的骨髓细胞中含有成年多能干细胞其可以突破现存理论规定的胚胎种子层的限制而直接转分化成为只能来源于内胚层的上皮细胞。当我刚看到此现像时,我已有策略和技术方法去确证该现像。当我告诉老板这事时,她反而不让我去完成此证明。所以,我只得利用晚上和周末来做证明转分化的实验。2007年11月我将一些主要全景图像给干细胞中心主任看了,他也劝我以此为机会让老板自我纠错- 即送她一篇大文章而又指出她一，二篇文章中方法学上的缺陷,而让她自己体面的撤下这文章。我说: 早已试过此招,没用。 以下为附上的电子邮件，第一次的举报书面报告，两张图像(第一张为用我自已的Y/SPC多重标记的正常野生型公鼠肺切片以显示肺II型细胞在肺泡上的生理分布 - 即Y/SPC双阳性细胞，SPC+为胞浆很纯的绿荧光，第二张为实验验证作者的Y FISH方法 - 野生型公鼠骨髓细胞的Y标记：兰色为细胞核，核中很强的红色点为Y信号，即Y+，没红点的细胞核即为Y-，其它荧光均为自发荧光）： ----- Forwarded message from Erica Herzog erica.lyndrup@yale.edu ----- Date: Thu, 06 Mar 2008 14:51:55 -0500 From: Erica Herzog erica.lyndrup@yale.edu Reply-To: Erica Herzog erica.lyndrup@yale.edu Subject: slides To: bugen.hu@yale.edu hi bugen i left the slides and results for you on the bench. were they in any way correct? i never was able to get pro-spc to work with fish because of the autofluor but if you are able to that's great. i always did prospc first and detected that way so the signal was really bright and then did fish after doing confocal. your control looked nice and like real signal. the few experimental cells that i saw that were possibly spc+ didn't look exactly like the control. anyway let me know how this compared with your results also since my lab cannot seem to get fish to work diane said you guys would be willing to collaborate if i gave you some slides for staining - if so can i bring you the slides sometime soon? best, erica Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注：她问我她的判片结果怎样（看了我的实验样本片)？ 她不能将Y/SPC双标记方法搞工作的原因是其Y FISH方法本身和抗SPC抗体 (Chemicon, AB3428) 本身不工 作。对于这种Y/荧光免疫多重标记方法的有关详情，请见我的博文：实验系统和实验技术的解释。autofluor （自发荧光）总是存在的， 这根本就不是她的双标记方法 不工作的原因！在实验方法学上此人一贯狡辩。其 Y/SPC 双 标记的方法都不工作, 但敢在文章 FASEB 2007 中将此双 标记结果写成 1000 分之 1000 的双阳性（ 8 只野生型公鼠肺石蜡切片,表 -2 p.2596). 她所 说其 lab 不能将 FISH 搞工作, 应该是指很 高的漏检率。当时她已不敢再对我说她们的 Y FISH 是 100% 的 检 出率了 , 因为她 还等着我帮她一把。 请见她 2007 年 6 月 18,19 日 电邮, 看她们当时是怎样说她们的 同一Y FISH方法的。 当时她应该还不知道我老板已将我逼到死角, 我已别无选择, 准备打假了, 否则她不 会给我送这电子邮件。 ----- Forwarded message from Diane Krause diane.krause@yale.edu ----- Date: Tue, 19 Jun 2007 06:32:54 -0400 From: Diane Krause diane.krause@yale.edu Reply-To: Diane Krause diane.krause@yale.edu Subject: Re: Y loss in males To: Erica Herzog erica.lyndrup@yale.edu What % of the male into male transplants were Y-? At this point Bugen is using cytospins. So far, he's only done older transplanted and untransplanted SPC-/- and untransplanted WT males. So far, with cytospins, we never see 20% without a Y. Diane On Jun 18, 2007, at 9:29 PM, Erica Herzog wrote: age matched but not irradiated truly less than 1% were Y neg unlike the male into male transplants how old are his mice - are they the old ones for the Y loss project also depending on fixation, size of probe, length of permeablization etc there can be issues.depending on the PFA strength, age of reagents there can be big issues. how is his X staining? Hi Erica In rereading the FASEB paper, I see that we don't have the % of SPC+ cells in males that are Y- by confocal. Did you do this with any males? If so, were they age-matched or younger? I am concerned that we don't know the sensitivity of the assay to detect for Y loss after cell fusion . Bugen is seeing epithelial cells have no Y on lung cytospins from male mice. Thanks for any info that you have. Diane -- Diane Krause MD, PhD Yale University School of Medicine Associate Professor, Department of Laboratory Medicine PO Box 208035 New Haven, CT 06520-8035 Phone: (203) 688-4829 Fax: (203) 688-2748 Office: BML 462 Administrative Associate, Pat Sember: (203) 688-3265 Pager: (203) 412-0805 Krauselab website: http://info.med.yale.edu/labmed/faculty/labs/krauselab/index.html Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注： 这是老板看了我用自己的 Y/CK 多重 标记方法所做的公小鼠肺细胞片子后, 她与 Erica 之 间就所谓 Y FISH 方法的 电子邮件 , 但有意转给我。她两人在 Y FISH 方法漏 检 率的 问题 上 给 我演双簧 , 其实就是想逼我将我所做的公小鼠肺细胞上的 Y漏检 , 当 Y 丢失的结论。 老板已知道我当时刚建好的多重标记方法已 突破了她 们文章 FASEB 2007 中方法的技 术 瓶 颈 , 想让我用自己的方法做出 Y 丢失 的结果去 掩盖文章中谎谬的 Y 丢失结果和结论。两人装着一副好象不知道她们的 Y FISH 方法有漏 检 似的 (因她们总是说其 Y FISH 方法具有 100% 的 检出率)。 我当然是装 糊涂, 对这种双簧毫无反应, 所以老板也拿我没办法。请见 Erica 于 2008 年 3 月 6 日 给我的电子邮件, 看她是如何说她们的 Y FISH 方法。 老板说: 步根在公鼠肺细胞片子上已看到 CK 阳性但 Y 阴性的细胞（其实就是 Y 漏 检) 也应该是警示 Erica : 其 实我已知道她们的秘密只是不说而已。 这里 Erica 的所 谓解释在技术上均是一派胡言。 这就是老板 2008 年 3 月 12 日在收到我的 邮件后,立马给我的回复。在上午一对一的会谈中我根本就没主动提过 Erica, 更没 说过 Erica 造假, 而只是 给老板看了她以前的 fellow-Erica 3 月 6 日送 给我的电子邮件,看完后她即失态。我还告诉她了一句有关抗 SPC 抗体（ Chemicon, AB3428) 的 实话。尔后她即将我大骂一通,让我走人。此回复中居然请我别辞职,详情请见我的博文：事件回放。 ----- Forwarded message from Diane Krause diane.krause@yale.edu ----- Date: Wed, 12 Mar 2008 14:25:30 -0400 From: Diane Krause diane.krause@yale.edu Reply-To: Diane Krause diane.krause@yale.edu Subject: Re: Request for your comment on me. To: bugen.hu@yale.edu Bugen Please don't quit. I would really like to work this out. I have no issues with your scientific conduct, and I am very pleased with all that you have done in the laboratory. In fact, I think that you're terrific in the lab, and I couldn't ask for someone who works more diligently, and with more care. I am also completely content with negative data in your studies using SPC-/- recipients. Negative data, if it is definitively negative, is still excellent data. That is why I needed to see the RT-PCR data. What concerned me today, and it has been building, is your repetition of concern that Erica is dishonest. I have heard you, and I have neither agreed nor disagreed with you - I have remained neutral on the subject without data to support your claims. Let's try to work this out. I am sorry that I got angry this morning. Let's just talk about your data and leave concerns regarding Erica out of our discussions. Let me know what you think, Diane Dear Diane: First of all thank you very much for offering me the position in your lab in Oct., 2006. I really appreciated that opportunity by my research performance. Now I determined that enough is enough. Please write down what you said to me including your comments on me about my personality (including that I was fired from my previous job because I pissed off some person, and you have evidence for this claim), my attitude to science, and my performance of research, then you concluded that I am not suited for working in your lab. Please give me this form letter with your signature. I hope that you will give me two weeks grace period. Thank you for your attention. Sincerely, Bugen -- Diane Krause MD, PhD Yale University School of Medicine Professor, Department of Laboratory Medicine PO Box 208073 New Haven, CT 06520 Phone: (203) 737-1678 Fax: (203) 785-7095 Office: Amistad 237b Administrative Associate, Pat Sember: (203) 737-1685 Pager: (203) 412-0805 Krauselab website: http://info.med.yale.edu/labmed/faculty/labs/krauselab/index.html ----- End forwarded message ----- May 7, 2008 Dear Dr. Mayes Here is the report you requested after our discussion of April 16, 2008. 1. FASEB 2007 At page 2594, in the section Results Bone marrow engraftment: These Y chromosome counts differed from untransplanted male WT and Sp-C null controls, which contained occasional cells that lacked the Y chromosome (0.8+/- 0.8%, P0.001, 0.92+/- 1.03%, P0.001). As a reader, my understanding of this sentence is that on the BM cytospins the authors detected average more than 98% BM cells containing Y by interphase Y FISH for the male WT and Sp-C null (16 mice per group), in other words, their Y FISH detection sensitivity for mouse BM cytospin is more than 98%. I do not dare to say that the detection sensitivity of my interphase Y FISH for mouse BM cytospins is such high. I do not know of anyone other than Dr. Herzog and Dr. Krause who have claimed this sensitivity for interphase Y FISH on mouse BM cytospin. I am at a loss to explain how this number could have been produced by experimental data, my conclusion is that this number is fabricated number instead of experimental data. I did not see any evidence that the authors really did wt female bone marrow transplantation to spcko male recipients. The authors misinterpret experimental phenomenon of interphase Y FISH. When I raised this issue clearly during my one-to-one meeting, Dr. Krause became emotional. 2. Science 2004 The authors misinterpret experimental phenomenon of interphase Y FISH and Cre-Lox experimental system. Issue of the commercial anti-pro-spc antibody that was used for immunofluorescent staining on recipient lung cytospin to identify donor bone marrow stem cell derived type II pneumocyte. 3. Cell 2001 During our lab meeting around Oct. 2007, the major agenda was to ask everyones suggestion about research projects ( Cell, 2001). Dr. Krause presented some information about this report. After the publication of this paper, no one including Dr. Krause's lab could reproduce the results by even using 1000 stem cells. In 2002, Dr. Weissmans lab at Stanford did a very similar experimental project and published their results in Science (Little Evidence for Developmental Plasticity of Adult Hematopoietic Stem Cells, 27 September 2002 Vol. 297, Page 2256). There was a difference between two teams in the isolation of single hematopoietic stem cell. Dr. Krause said that Dr. Weissman admitted this minor difference. Dr. Weissman's team replied with a challenge: his team will give their cells to Dr. Krause's team, and her team would give his team their cells, then both teams will carry out the same two experiments by using the two kinds of cells. Dr. Krause said that for five years her team did not take Dr. Weissmans challenge because Saul Sharkis (co-author) refused to do so. Finally, she said that she received a small grant for this project years ago, and now she doesnt know whether her team should continue to do this kind project or stop. She asked for suggestions from the lab. There was no evidence that would convince me that Dr. Krause was sincerely willing to resolve this scientific discrepancy, otherwise I would have offered two proposals to resolve this scientific discrepancy: Plan 1. Provided the paraffin blocks of sample (that generated the data for the 2001 paper in Cell ), I could use my detection methods to reexamine the tissue samples because my detection methods are much better and more accurate than the methods used for identifying bone marrow derived tissue type cells in multiple organs in the paper. Based on the results, we could draw appropriate conclusions. Plan 2. Step1: Verify all the experimental protocols used in that paper in our own lab by using positive and negative controls to see if all work. If all protocols pass, go to the Step 2: Re-evaluate the protocols used for identifying bone marrow stem cell derived tissue type cells in multiple organs to see if these methods can really identify such cells based on commonly accepted criteria in this research area. If pass, then go to the Step 3: Use these protocols to reexamine the paraffin tissue samples. If such cells are identified in multiple organs, publish the results. Failure at any step would require Dr. Krause to decide what is the right thing to do. The paper (Cell 2001) is one of the most controversial papers in this research area; so far no one has been able to reproduce the results. Even an attempt by the authors could not reproduce the results using 1000 such stem cells. In 2003, the authors published a one page comment in Science (Comment on Little Evidence for Developmental Plasticity of Adult Hematopoietic Stem Cells Science vol 299 28 February 2003). In this paper the authors used unpublished data to argue that they used Y FISH, and its detection sensitivity is much higher than the GFP used by Dr. Weissmans team. In the paper the authors state for example, following transplantation of male Rosa marrow cells into lethally irradiated female wild type mice, the spleen showed 90% engraftment by Y chromosome analysis, As a reader, my understanding of this statement is: 1. in their female recipient, more than 90% spleen mass is replaced by donor bone marrow derived spleen cells. 2. the detection sensitivity of the authors Y FISH protocol (before Feb. 2003, I supposed the protocol they used in the paper in Cell 2001) is 90%. Detection sensitivity of the authors Y FISH can be easily verified by a neutral third party. I request that: all experimental protocols published in the papers related to stem cell derived tissue type cell by Dr. Diane Krause or Dr. Erica Herzog be verified by a neutral third party. all the samples involved in papers (Cell 2001, Science 2004, FASEB 2007) be reexamined by a neutral third party. I have decided to leave it to Yale University School of Medicine to draw conclusions. Thank you for your attention. Sincerely, Bugen Hu
Bugen Hu August 7, 2009 Legend for the lung images by using my unpublished protocols of Y FISH and immunofluorescent staining contained in the CD All the images are coming from the original images when I examined the slides under fluorescent microscope, I did not do any art work or edit on any of such images. Amplification: 40X Wild type male mouse lung paraffin section labeled with Y FISH, anti-SPC (in-house guinea pig anti-spc serum from Jeffs lab), and anti-CD45/F4/80. Signals labeled as the following: Rhodamine for Y (intense pink dots in the DAPI counter stained blue nucleus), Alexafluor 488 for SPC (green in cytoplasmic part), Texas-Red for CD45 and F4/80 (orange red on cell membrane). If the authors could really get experimental protocols of Y FISH and immunofluorescent staining (anti-SPC, and anti-CD45) work, this image should be positive control image for the FASEB paper Fig 2A. This image can show the people, including the peer-reviewers, and readers how well the experimental methods (protocols) are, and what should be the positive markers for each labeling (Y/SPC/CD45), and these positive markers right locations on the slide and in the cell. The most important information of this image should show is that after all labeling is finished (Y FISH, and immunofluorescent staining), as a whole picture what really looks like (Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells (on the alveoli) how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive). In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative (detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image). c. At right physiological locations of type II cells there are a lot of SPC and Y doubt positive cells (definitely certain percentage of type II cells must be stained as SPC negative because I dont think that my protocols can reach 100% detection sensitivity for anti-SPC immunofluorescent staining). d. There is no cell being SPC and CD45 double positive (in this field there is no obvious CD45 positive cell). spcko male mouse lung paraffin section labeled with Y FISH, anti-spc, anti-CD45, and anti-F4/80. Compared with the image of wt male control, overall spcko has much stronger autofluorescence (the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section). For Formalin or PFA fixed tissues, lung is one of the tissues with the strongest autofluorescence, another one is GI tissue), and the alveoli have more fibers than the alveoli of wt male mouse. In this image: a. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. b. No single cell is labeled as SPC positive, no single cell even really looks like the SPC positive cell in the wt male control image (even there are some cells with green in their cytosplasmic part look-like positive, but this so-called green signal is the same as that of alveoli fibers but different from the real SPC positive staining in the image of wt male slide, it is very easy to tell difference between signals of SPC specific staining and green autofluorescence when compared with the real positive cells in the wt male control). c. In this field there is obvious CD45 positive cell. If the authors protocols for Y FISH and anti-SPC really work, at least double-labeling image should be presented as the image of spcko male control Fig 2B for the paper FASEB 2007. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues (whether or not Y can achieve similar detection sensitivity in both tissues), and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung. Paraffin section from the lung of spcko male recipient of wt female bone marrow transplantation (model mouse ID: spc-14) labeled with Y FISH, anti-SPC, anti-CD45, and anti-F4/80. This image shows chimera characteristics of such model. In this image: a. Whole structure of alveoli is clearly shown up (as green autofluorescence of thick fibers). b. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative. c. There are several obvious CD45 positive and Y negative cells (leukocytes derived from donor bone marrow cells, that is, so-called engraftment). d. There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse. If the authors actually did such model, and their published protocols of Y FISH and immunofluorescent staining really work, by using such model to identify and prove that in vivo donor bone marrow cell can derive into type II cell by the mechanism of fusion with recipient cell, this is the image the authors should present as the Fig 2C in the paper FASEB 2007 (at least to show people that the image is really coming from lung of such model, their image Fig 2C logically can come from any mouse lung, and so-called SPC positive could be anything including autofluorescence of cytoplasmic part). This is the image (spc-14) containing the two look-like positive cells with double positive Dr. Diane Krause really wanted and put huge pressure on me trying to force me change my own judgment call on the two cells from negative to positive cells (Y and spc double positive). When I put the images of wt male and spc-14 side by side, and asked her: Even when you compare image of the two cells with image of positive control (wt male), the color and staining pattern is same or not? She said: not same, but stem cell derived type II cell is not as same as real type II cell. And I repeated my principle by saying: As PI you can make your own judgment call on my slides any way you want, and it is perfectly fine with me, but I only hold responsibility for my own judgment call. Then she forced me to trust and accept the results form the paper (FASEB 2007), after I firmly refused she became so emotional and angry with me. Finally she sent an email to Dr. Erica Herzog to ask her as blinder to examine my slides under microscope and ask Dr. Erica Herzog to show us electronic images of anti-spB the authors used for the paper FASEB 2007 (the facts are: a. Erica never showed us any images of anti-spB she only said that she needs to find such images of anti-spB from the computer., but she never showed us any anti-spB image (I dont think that she has such anti-spB images). b. No single image of anti-spB was presented in the paper. c. In the inventory boxes for immunofluorescent reagents in Dr. Krauses lab I had never seen any vials of anti-spB antibody before February, 2008. I ordered my anti-spB from Chemicon in February 2008. I have the email to prove). On March 6, 2008 after looking at my slides under fluorescent microscope, Dr. Erica Herzog sent me that famous email. Normal wt male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few cells being Y negative, for example, one cell at the bottom showing most part of its nucleus. b. There are three cells being labeled as Y and SPC double positive (one is located at left of the image, two are located in the middle). c. At the left of the image adjacent to the Y SPC double positive cell there are several cells being labeled as Y positive and with yellow-green autofluorescence in their cytosplamic parts (such Y positive cells with yellow-green autofluorescence in cytoplasmic parts are common on the male mouse lung cytospins slides labeled with just Y FISH without any other immunofluorescent staining, such cells with yellow-green autofluorescence in their cytoplasmic parts are common on the mouse lung cytospins slides without any labeling, that is, blank control for immunofluorescent staining if the samples are fixed with Formalin or PFA). d. There is a cell labeled as Y positive and look-like CD45 positive located in up-right area of the image. e. There is no single cell being labeled as SPC and CD45 double positive. This image can show real clear-cut difference between signal of real cytoplasmic marker (here is SPC) immunofluorescent staining and signal of cytoplasmic autofluorescence on the mouse lung cytospins. If the images of Fig 4A, 4B (paper FASEB 2007) are really coming from the lung of the model spcko female recipient of male marrow, and the authors published protocols of Y FISH and anti-CK staining really work, for Fig 4A, and 4B, the authors should present the images like this one showing Y and cytoplasmic marker positive cells plus information of signal of X FISH in nuclei, and cells adjacent to the identified positive cells. Normal spcko male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few nuclei are labeled as Y negative, located at the bottom of the image. b. No single cell is stained as SPC positive, but there are a lot of cells with strong green autofluorescence in their cytoplasmic parts. c. There are several cells containing multiple dots of Y signal in its nucleus. And scientific fact is that every cell on the slide can only contain one molecule of Y chromosomal DNA, so this image can clearly demonstrate this scientific fact that one molecule of Y DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal in the nucleus by interphase Y FISH. Summary: These five images of mouse lung samples (paraffin sections and cytospins slides) labeled with Y FISH and immunofluorescent staining show: For Formalin or PFA fixed mouse lung samples, cytoplasmic autofluorescence is common and strong on both paraffin section and cytospins slide, but there is clear-cut difference or manifestation between specific immunofluorescent staining signal of cytosplasmic marker and cytosplasmic autofluorescence. Without showing the real image of real positively labeled control, people not having direct experience in this technique can be easily fooled by cytoplasmic autofluorescence especially when the authors just present images with a single cell or a few cells without reference system. For both paraffin section and cytospins slide, my unpublished Y FISH protocols can achieve much higher detection sensitivity. But I can say here that even using my own protocols I have never achieved 99% detection sensitivity for either paraffin or cytospins slide, even not close to 99% positive (my standard is whole slide, not just one chosen image). By using my protocols or protocols that can achieve similar detection sensitivity of Y, dishonest people can easily make fake image (containing 20, 30, or 50 cells) showing 100% cells being labeled as Y positive by just cutting off single cell or a few cells that are labeled as Y negative from the image. For example, by using my protocols of Y FISH and immunofluorescent staining (edited image), for male sample I can generate the double-labeled images (at 40x amplification, containing 20, 30, 50, or even 100 cells) showing 100% Y detection sensitivity, and beautiful immunofluorescent staining with preserved tissue and cell morphology by just cutting off a cell or a few cells labeled as Y negative from the image. If I was dishonest, and I need such logic premise: 100% detection sensitivity of Y FISH, I submit such images as results and claim that my Y FISH protocol can reach 100% detection sensitivity, definitely based on manifestation of the edited image this statement is true, but scientifically this image does not truthfully reflect the real experimental phenomenon of the slide because the Y negative cell or cells in the real image or whole picture were intentionally cutting off from the image presented (edited image). What the real images of mouse lung sample should look like after multiple labeling of Y FISH and immunofluorescent staining. In the nucleus of one normal male cell, one molecule of Y chromosomal DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal. Physiological distribution of type II cells labeled as Y and SPC double positive on alveoli of the normal male mouse lung paraffin section. For any people if their protocols of Y FISH and anti-SPC immunofluorescent staining really work, and the primary antibody of anti-SPC really works on Formalin or PFA fixed samples, they should be able to produce the similar images from the normal male mouse lung paraffin section as real wt positive control image if they want to use Y and anti-SPC dual markers to identify donor stem cell derived type II cell from the tissue of spc knockout sex-mismatched model. How important it is to have both positive and negative control slides and reference system on the same slide when experimental methods of interphase Y FISH and immunofluorescent staining are used to identify donor stem cell derived tissue type cells in the recipient tissues, that is, without the controls or reference system to compare with, the so-called identified positive cell in the electronic image could be anything but real positive cell. Because in this situation two different kinds of experimental methods, in situ DNA hybridization and antigen- antibody interaction (immune staining) are used, and none of the methods can achieve 100% detection sensitivity and none of immunofluorescent staining can achieve 100% detection accuracy. The authors, Dr. Diane Krause, and Dr. Erica Herzog should know this principle pretty well because they have been using such methods to identify donor stem cell derived tissue type cells from the tissues of sex-mismatched models for years, and published multiple papers. Feb. 4, 2009 Technical Information and Explanation of Erica Herzogs Experimental Protocol for Y FISH on Bone Marrow Cytospins Published in the FASEB Paper (2007) Here the most important step is missing: Denature the target DNA, i.e. Y chromosomal DNA (the size of this DNA is 97.5Mb, for C57 background mouse) to open the double stranded target DNA. The larger the size of the target DNA, the harsher condition is required to open the target DNA. To open the double stranded target DNA is precondition for any DNA hybridization experiment, otherwise the specific probe can not bind to the complement sequence on the target. So-called Denature step in the authors protocol is 73C for 5 mins. Actually the major function of this step is not to denature the target DNA, but the probe, just like for normal DNA hybridization or classic FISH some people do denature the probe 1 st at 70-80C for about 10 mins before apply the probe to the target. Under this denature condition (73C, 5 mins) there is only certain percentage of cells their Y chromosomal DNA getting partial denature, i.e. double stranded DNA opened in some sections of the Y chromosomal DNA. That is, sacrifice the detection sensitivity for retaining the cells on the slide and cell morphology. Here the probe we used for Y FISH is a mixture of DNA, sizes from 100 to 500 nucleotides, its complement sequences on the target will cover the whole Y chromosomal DNA except for super high repeated sequences that will also appear in other chromosomes (the other name of Y FISH is whole Y chromosome painting). When utilize Y FISH (identify the origin of donor) combined with immunofluorescent staining (identify the specific tissue marker) technique to identify the donor stem cell derived specific tissue cell in sex-mismatched models, the authors encounter technical dilemma: Y FISH requires very harsh condition which will destroy the tissue physiological structure and cells morphology including cytoplasmic and cell membrane component (like CD45) , while immunofluorescent staining is just antigen antibody interaction (here is protein and protein interaction) which requires mild condition. If just add these two different kinds of experimental methods together it will not work, regardless doing immunofluorescent staining 1 st or Y FISH 1 st because in order to identify donor stem cell (Y as marker of origin for donor or recipient) derived specific tissue type cell, after both experimental procedures the both signals need to be present on the same slide otherwise the mission is impossible. If the authors use the classic denature condition for Y FISH on tissue paraffin sections as they published, 1M sodium thiocyanate at 80C for 20 mins, then neutralized with 0.2N HCl at room temperature for 12 mins, after Y FISH for most cells only nuclei left. If the authors use such denature condition on cytospins, after Y FISH probably only a few nuclei left on the slide, or nothing left on the slide. And imagine even there is such biological event- bone marrow stem cell derives into specific tissue type cell (here is from BM to type II pneumocyte) happening in vivo, the frequency of such event is very low even based on the authors calculation (less than 1 per 1000). Since no immunofluorescent staining is 100% specific and accurate, the universal accepted standard to identify donor stem cell derived specific tissue cell is triple labeling: Y, tissue specific marker and CD45 in sex-mismatched models to exclude the leukocytes that can be present in any tissue. How to solve this technical dilemma, it took me about 6 months to solve the technical dilemma above mentioned: interphase Y FISH combined with immunofluorescent staining while keeping the tissue and cell morphology normal. The strategy I used is to find the common window of experimental conditions that will fit for both Y FISH and immunofluorescent staining, then hard working of error and try. Even I say that I solved the technical dilemma, but for Y FISH my protocols cannot get 99% detection sensitivity on either paraffin section or cytospin slide. Definition of detection sensitivity needs to be defined by thousands of cells on the same slide, but not by a few cells of electronic image of very small field. In order to test the detection sensitivity of Y FISH on bone marrow cytospins, and verify the authors experimental results generated by using such protocol, I just did Y FISH on bone marrow cytospins from the perfectly normal young wild type mouse by strictly following Dr Erica Herzog and Dr Diane Krause published protocol in their paper, FASEB vol.21 August 2007, p2592-2601. I will send the slide and a CD that contains the electronic images of such slides to the Deans Office, Yale School of Medicine. Here are technical parameters: Channel 1: DAPI to stain for DNA, blue color. Channel 2: FITC, green color. Channel 3: Rhodamine for Y signal, pink color (intense dot in nuclei). Merged image: besides the Y signal, some yellow, green signals are coming from autofluorescence. Amplification: 40x. Rhodamine-conjugated anti-digoxigenin antibody (Roche), the same as Dianes lab always used. Digoxigenin labeled Y probe, the same as Dianes lab always used. IPLAB software is used to capture images. After examine the whole slide under fluorescent microscope, my conclusion is: there is no way for the authors to obtain their experimental results as they claimed in their paper (FASEB 2007) because the Y FISH detection sensitivity is too low. So their results of Y FISH on bone marrow are just intentionally fabricated numbers, not scientific experimental results at all. Bugen Hu
所有 图像均为 40 倍物 镜下所拍摄。 图 1 到 图 5 (Fig.1-Fig.5) 是用我自己的 Y/SPC/CD45 多重免疫 标记所作的野生型和 SPCKO 公鼠肺切片和 细胞片的全景图像。 图 6 到 图 10 (Fig.6-Fig.10) 是我 2009 年 2 月份 实验验证作者文章 FASEB 2007 中 Y FISH 方法在正常野生型公鼠骨髓 细胞片子的全景图像。 信号或 标记: SPC 为 Alexafluor 所 标记 - 胞 浆中绿色荧光。 Y 为 罗丹明所标记 - 在 DAPI 所染成的 兰色胞核中的芝麻大小的很强的红点。 CD45 为 Texas-Red 所 标 记 - 胞膜上桔 红色。 图 1. 野生型公鼠, 可 见完整的肺泡结构, 大部分细胞被标记为 Y 阳性, 但也有很少 细胞为 Y 阴性 - 漏 检, 许多 Y/SPC 双阳性 细胞 - 肺 II 型 细胞相隔分布于肺泡上。如果作者的 Y/SPC 双 标记方法真能工作, 则其文章 FASEB 2007 中 图 2A 应该为此图像, 作 为双阳性对照。 图 2. SPCKO 公鼠, 可 见完整的肺泡结构, 但整个结构有更强的绿色自发荧光, 大部分细胞被标记为 Y 阳性, 但也有很少 细胞为 Y 阴性 - 漏 检, 无 SPC 阳性 细胞, 但有不少细胞胞浆中有很强的绿色自发荧光 - 与真正的 SPC 所 标记的绿色荧光并不相同。如果作者 Y/SPC 双 标记真能工作, 则其文章 FASEB 2007 中 图 2B 因 该为此图像。 图 3. 模型 - 接受了野生型母鼠骨髓移植的 SPCKO 公鼠的肺, 有不少 Y 阴性但 CD45 阳性的白 细胞 - 来自于供体 ( 母鼠)的干 细胞变成即所谓的 engraftment 。其他方面与 图 2 相似。如果作者的 Y/SPC 双 标记方法真能工作, 则其文章 FASEB 2007 图 2C 因 该为此图。这就是我的模型鼠14号的图像, 老板非要两个阳性细胞以证明她的融 合机理。 因 为作者的技术瓶颈: Y FISH 方法的高漏 检率, 用于石蜡切片的 Y FISH 方法 对组织和细胞结构的破坏, 抗体 - 抗 SPC (Chemicon, Ab3428) 不工作,只要具有任何一条所指缺陷, 则作者无法显示我所显示的以上图像。况且作者的方法具有以上所有的缺陷。事实上作者根本无法显示任何公鼠Y FISH的全景图像, 因为那将暴露其最大的秘密-其YFISH 方法的高漏检率。 图4. 野生型公鼠肺细胞片子, 大多数细胞为Y阳性, 但也有少量漏检。有3个Y/SPC双阳性细胞, 不少细胞的胞浆中有黄到绿的自发荧光。如果作者的用于细胞片 (cytospins) 的Y/荧光免疫多重标记方法真能工作, 则其文章 FASEB 2007 中图4A, 4B 应该显示此类全景多重标记图像。还是因为其方法上的缺陷, 所以作者只能显 点 而 无法显面, 因为只要显面则露陷。文中的图像是假图像, 即并非作者在图解中所说 (Figure legend)。更祥细的说明请看英文版的图解说明。 图5. SPCKO公鼠肺细胞片子, 大多数细胞为Y阳性, 但也有少量漏检。许多细胞胞浆中有很强的绿色自法荧光, 但无真正的SPC阳性细胞。此图最重要之点是显示有些细胞胞核中含有多点Y信号, 这就明确无误的说明: 任何一个公鼠细胞在Y FISH以后, 或为Y阴性-漏检, 或为含一点Y, 或为含多点Y信号。而这是由其实验原理所决定了的。 图6到图10均为正常野生型公鼠骨髓细胞片子, 严格按照文章 FASEB 2007 中作者所发表的Y FISH方法所做的Y FISH实验-即实验验证作者的Y FISH方法。这里只有兰色胞核中的红色点-芝麻大小为Y信号, 其他颜色的均为自发荧光。核中有芝麻 大小红点的即为Y阳性, 无此Y信号的则为Y阴性-漏检(在这里每一个细胞核均含有 一条Y染色体, 只是该Y FISH方法未能检测出而已)! 所有的图像中大部分公鼠骨髓细胞核被标记成Y阴性-即漏检, 这就证明作者的Y FISH方法具有很高的漏检率, 也就是很低的检测敏感度！决非象作者长期以来所吹嘘的超高检测敏感度。这就在逻辑上实验技术上证明作者无法得出其文章中公鼠骨髓细胞Y FISH实验结果的数据。此数据只能是有意伪造的数字。 Bugen Hu August 7, 2009 Legend for the lung images by using my unpublished protocols of Y FISH and immunofluorescent staining contained in the CD All the images are coming from the original images when I examined the slides under fluorescent microscope, I did not do any art work or edit on any of such images. Amplification: 40X Wild type male mouse lung paraffin section labeled with Y FISH, anti-SPC (in-house guinea pig anti-spc serum from Jeffs lab), and anti-CD45/F4/80. Signals labeled as the following: Rhodamine for Y (intense pink dots in the DAPI counter stained blue nucleus), Alexafluor 488 for SPC (green in cytoplasmic part), Texas-Red for CD45 and F4/80 (orange red on cell membrane). If the authors could really get experimental protocols of Y FISH and immunofluorescent staining (anti-SPC, and anti-CD45) work, this image should be positive control image for the FASEB paper Fig 2A. This image can show the people, including the peer-reviewers, and readers how well the experimental methods (protocols) are, and what should be the positive markers for each labeling (Y/SPC/CD45), and these positive markers right locations on the slide and in the cell. The most important information of this image should show is that after all labeling is finished (Y FISH, and immunofluorescent staining), as a whole picture what really looks like (Y signal: what percentage of cells is labeled as Y positive, SPC signal: in the physiological locations of lung type II cells (on the alveoli) how well the type II cells are labeled as SPC positive, CD45 signal: whether or not membrane of leukocyte can be labeled as CD45 positive). In this image: a. The whole structure of alveoli is clearly shown up at normal exposure time due to autofluorescence of the tissue itself, b. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative (detection sensitivity of Y FISH protocol, but this image is still extremely good image in terms of Y FISH and SPC staining detection sensitivity if you have doubt about it, you can do literature search on internet to see if there is any image of lung paraffin section labeled with Y FISH and immunofluorescent staining in any published papers is better than this image). c. At right physiological locations of type II cells there are a lot of SPC and Y doubt positive cells (definitely certain percentage of type II cells must be stained as SPC negative because I dont think that my protocols can reach 100% detection sensitivity for anti-SPC immunofluorescent staining). d. There is no cell being SPC and CD45 double positive (in this field there is no obvious CD45 positive cell). spcko male mouse lung paraffin section labeled with Y FISH, anti-spc, anti-CD45, and anti-F4/80. Compared with the image of wt male control, overall spcko has much stronger autofluorescence (the whole alveoli structure is clearly shown up as very strong green autofluorescence even exposure conditions I used is the same as I used for wt male control section). For Formalin or PFA fixed tissues, lung is one of the tissues with the strongest autofluorescence, another one is GI tissue), and the alveoli have more fibers than the alveoli of wt male mouse. In this image: a. Most of nuclei are labeled as Y positive, but there is still a small percentage of nuclei being labeled as Y negative. b. No single cell is labeled as SPC positive, no single cell even really looks like the SPC positive cell in the wt male control image (even there are some cells with green in their cytosplasmic part look-like positive, but this so-called green signal is the same as that of alveoli fibers but different from the real SPC positive staining in the image of wt male slide, it is very easy to tell difference between signals of SPC specific staining and green autofluorescence when compared with the real positive cells in the wt male control). c. In this field there is obvious CD45 positive cell. If the authors protocols for Y FISH and anti-SPC really work, at least double-labeling image should be presented as the image of spcko male control Fig 2B for the paper FASEB 2007. Only using above mentioned wt male and spcko male multiple labeled sections can really function as controls to show: how well Y FISH protocol works in both wt male and spcko male tissues (whether or not Y can achieve similar detection sensitivity in both tissues), and clear-cut difference between wt male and spcko male sections in terms of SPC staining, and physiological distribution of SPC positive labeled type II cells in the lung. Paraffin section from the lung of spcko male recipient of wt female bone marrow transplantation (model mouse ID: spc-14) labeled with Y FISH, anti-SPC, anti-CD45, and anti-F4/80. This image shows chimera characteristics of such model. In this image: a. Whole structure of alveoli is clearly shown up (as green autofluorescence of thick fibers). b. Most nuclei of CD45 negative cells are labeled as Y positive, but there is still a small percentage of such cells being labeled as Y negative. c. There are several obvious CD45 positive and Y negative cells (leukocytes derived from donor bone marrow cells, that is, so-called engraftment). d. There is no single cell being SPC positive, in terms of SPC specific staining the situation is the same as I mentioned for the image of spcko male control mouse. If the authors actually did such model, and their published protocols of Y FISH and immunofluorescent staining really work, by using such model to identify and prove that in vivo donor bone marrow cell can derive into type II cell by the mechanism of fusion with recipient cell, this is the image the authors should present as the Fig 2C in the paper FASEB 2007 (at least to show people that the image is really coming from lung of such model, their image Fig 2C logically can come from any mouse lung, and so-called SPC positive could be anything including autofluorescence of cytoplasmic part). This is the image (spc-14) containing the two look-like positive cells with double positive Dr. Diane Krause really wanted and put huge pressure on me trying to force me change my own judgment call on the two cells from negative to positive cells (Y and spc double positive). When I put the images of wt male and spc-14 side by side, and asked her: Even when you compare image of the two cells with image of positive control (wt male), the color and staining pattern is same or not? She said: not same, but stem cell derived type II cell is not as same as real type II cell. And I repeated my principle by saying: As PI you can make your own judgment call on my slides any way you want, and it is perfectly fine with me, but I only hold responsibility for my own judgment call. Then she forced me to trust and accept the results form the paper (FASEB 2007), after I firmly refused she became so emotional and angry with me. Finally she sent an email to Dr. Erica Herzog to ask her as blinder to examine my slides under microscope and ask Dr. Erica Herzog to show us electronic images of anti-spB the authors used for the paper FASEB 2007 (the facts are: a. Erica never showed us any images of anti-spB she only said that she needs to find such images of anti-spB from the computer., but she never showed us any anti-spB image (I dont think that she has such anti-spB images). b. No single image of anti-spB was presented in the paper. c. In the inventory boxes for immunofluorescent reagents in Dr. Krauses lab I had never seen any vials of anti-spB antibody before February, 2008. I ordered my anti-spB from Chemicon in February 2008. I have the email to prove). On March 6, 2008 after looking at my slides under fluorescent microscope, Dr. Erica Herzog sent me that famous email. Normal wt male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few cells being Y negative, for example, one cell at the bottom showing most part of its nucleus. b. There are three cells being labeled as Y and SPC double positive (one is located at left of the image, two are located in the middle). c. At the left of the image adjacent to the Y SPC double positive cell there are several cells being labeled as Y positive and with yellow-green autofluorescence in their cytosplamic parts (such Y positive cells with yellow-green autofluorescence in cytoplasmic parts are common on the male mouse lung cytospins slides labeled with just Y FISH without any other immunofluorescent staining, such cells with yellow-green autofluorescence in their cytoplasmic parts are common on the mouse lung cytospins slides without any labeling, that is, blank control for immunofluorescent staining if the samples are fixed with Formalin or PFA). d. There is a cell labeled as Y positive and look-like CD45 positive located in up-right area of the image. e. There is no single cell being labeled as SPC and CD45 double positive. This image can show real clear-cut difference between signal of real cytoplasmic marker (here is SPC) immunofluorescent staining and signal of cytoplasmic autofluorescence on the mouse lung cytospins. If the images of Fig 4A, 4B (paper FASEB 2007) are really coming from the lung of the model spcko female recipient of male marrow, and the authors published protocols of Y FISH and anti-CK staining really work, for Fig 4A, and 4B, the authors should present the images like this one showing Y and cytoplasmic marker positive cells plus information of signal of X FISH in nuclei, and cells adjacent to the identified positive cells. Normal spcko male mouse lung cytospins slide labeled with Y FISH, anti-SPC, and anti-CD45. In this image: a. Most of nuclei of cells are labeled as Y positive, but a few nuclei are labeled as Y negative, located at the bottom of the image. b. No single cell is stained as SPC positive, but there are a lot of cells with strong green autofluorescence in their cytoplasmic parts. c. There are several cells containing multiple dots of Y signal in its nucleus. And scientific fact is that every cell on the slide can only contain one molecule of Y chromosomal DNA, so this image can clearly demonstrate this scientific fact that one molecule of Y DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal in the nucleus by interphase Y FISH. Summary: These five images of mouse lung samples (paraffin sections and cytospins slides) labeled with Y FISH and immunofluorescent staining show: For Formalin or PFA fixed mouse lung samples, cytoplasmic autofluorescence is common and strong on both paraffin section and cytospins slide, but there is clear-cut difference or manifestation between specific immunofluorescent staining signal of cytosplasmic marker and cytosplasmic autofluorescence. Without showing the real image of real positively labeled control, people not having direct experience in this technique can be easily fooled by cytoplasmic autofluorescence especially when the authors just present images with a single cell or a few cells without reference system. For both paraffin section and cytospins slide, my unpublished Y FISH protocols can achieve much higher detection sensitivity. But I can say here that even using my own protocols I have never achieved 99% detection sensitivity for either paraffin or cytospins slide, even not close to 99% positive (my standard is whole slide, not just one chosen image). By using my protocols or protocols that can achieve similar detection sensitivity of Y, dishonest people can easily make fake image (containing 20, 30, or 50 cells) showing 100% cells being labeled as Y positive by just cutting off single cell or a few cells that are labeled as Y negative from the image. For example, by using my protocols of Y FISH and immunofluorescent staining (edited image), for male sample I can generate the double-labeled images (at 40x amplification, containing 20, 30, 50, or even 100 cells) showing 100% Y detection sensitivity, and beautiful immunofluorescent staining with preserved tissue and cell morphology by just cutting off a cell or a few cells labeled as Y negative from the image. If I was dishonest, and I need such logic premise: 100% detection sensitivity of Y FISH, I submit such images as results and claim that my Y FISH protocol can reach 100% detection sensitivity, definitely based on manifestation of the edited image this statement is true, but scientifically this image does not truthfully reflect the real experimental phenomenon of the slide because the Y negative cell or cells in the real image or whole picture were intentionally cutting off from the image presented (edited image). What the real images of mouse lung sample should look like after multiple labeling of Y FISH and immunofluorescent staining. In the nucleus of one normal male cell, one molecule of Y chromosomal DNA can be detected as Y negative, one dot of Y signal, or multiple dots of Y signal. Physiological distribution of type II cells labeled as Y and SPC double positive on alveoli of the normal male mouse lung paraffin section. For any people if their protocols of Y FISH and anti-SPC immunofluorescent staining really work, and the primary antibody of anti-SPC really works on Formalin or PFA fixed samples, they should be able to produce the similar images from the normal male mouse lung paraffin section as real wt positive control image if they want to use Y and anti-SPC dual markers to identify donor stem cell derived type II cell from the tissue of spc knockout sex-mismatched model. How important it is to have both positive and negative control slides and reference system on the same slide when experimental methods of interphase Y FISH and immunofluorescent staining are used to identify donor stem cell derived tissue type cells in the recipient tissues, that is, without the controls or reference system to compare with, the so-called identified positive cell in the electronic image could be anything but real positive cell. Because in this situation two different kinds of experimental methods, in situ DNA hybridization and antigen- antibody interaction (immune staining) are used, and none of the methods can achieve 100% detection sensitivity and none of immunofluorescent staining can achieve 100% detection accuracy. The authors, Dr. Diane Krause, and Dr. Erica Herzog should know this principle pretty well because they have been using such methods to identify donor stem cell derived tissue type cells from the tissues of sex-mismatched models for years, and publi shed multiple papers.
这篇博文专门解释与实验系统和实验方法相关的技术细节问题, 尤其是为何作者 Y FISH 方法漏 检率高就一定是作者有意伪造由该方法所得的实验数据？为何其 Y FISH 方法的高漏 检率的秘密对我老板团队如此重要, 一旦暴光就会象多米诺骨牌较 应等等问题？这里有一个事实: 在成年干细胞可塑性研究领域中使用 Y FISH 方法和 荧光免疫多重标记来鉴定由供体干细胞在受体鼠体内变成的组织特化细胞的实验 室并不多（其实是一个纯技术的原因, 请见我以下的技术解释), 我老板的实验室是 这方面的大老级实验室。 其 实整个成年干细胞发育可塑性研究的大命题就是一个: 体内是否还存在此类干细 胞？如有此类干细胞, 则此类干细胞能否在体内分化成某些组织特化细胞, 如有的话, 这个生物学事件也是一个很低概率事件。所以此领域研究的核心就是如何用实 验 的方法来证明这个生物学过程: 成年干细胞在体内分化为某种组织 特化细胞。 我在博文-事件回放中所说, 老板团队一直所用的实验系统就是: 供, 受体鼠性别相 反的模型(sex mismatched model) 即以公鼠作为骨髓干细胞的来源, 受体鼠则为致死 剂量照射的母鼠。即在宿主(母鼠) 组织中以Y染色体作为供体 (公鼠)干细胞来源的 识别标志。实验方法或手段就是: Y FISH 以标记Y作为来源于供体干细胞的证据, 和荧光免疫标记组织细胞特化蛋白, 如胞浆中的角质蛋白-CK就代表上皮细胞, 包 膜上的CD45代表白细胞。逻辑上和理论上这种实验系统非常简单明了, 是典型的 反推证明模式: 以Y/荧光免疫多重标记方式证明受体 (母鼠) 组织中此阳性细胞-Y阳性和细胞标志蛋白阳性但CD45阴性只能由供体 (公鼠) 细胞变成, 即中间过 程无需也无法证明。但这就要求在技术上多重标记的每个标志的真实性得以确证-在阳性和阴性甚至空白对照上。理论上Y FISH外加荧光免疫的多重标记是最直接和确实的鉴定方法。但当时世界上大多数本领域有名的实验室并不用此种Y /荧光 免疫多重标记方法以鉴定受体鼠组织中来源于供体鼠干细胞变成的组织特化细胞, 而最多使用的是绿荧光蛋白(GFP)作为供体干细胞来源的标志。其实原因 非常简 单, 纯技术上的原因。Y FISH其实就是DNA原位杂交, 而荧光 免疫标记就是抗原-抗体的反应, 这里是蛋白质-蛋白质的相互作用, 如CK和其相应抗体的结合- 这种结合力并不强 (与DNA分子杂交相比), 一旦遇上剧烈的实验条件如Y FISH 实验中的 DNA变性条件（DNA denature, Y DNA is 97.5Mbp, it requires very harsh conditions to open its double strands, which is precondition for the Y probes to bind to its complementary sequences on the target- Y DNA) 既使已结合在目标蛋白上的抗体也会解离 (dissociate), 即达不到双标记的目地。如果按传统的Y FSIH方法 (用于石蜡 切片的方法-硫氰酸 钠 80度处理, 再用盐酸室温下处理) 先作Y标记, Y FISH后则造成了组织和细胞结构 的很大破坏而使胞浆, 胞膜目标蛋白甚至不存在于片子上。即还是 达不到双标记目地。也就是这是两类实验条件不兼容的实验方法, 即技术上的两难 (dilemma)。 况且Y FISH的检测敏感度也不见得很高, 一般而论, 在公小鼠 组织石蜡切片上传统 的Y FISH方法标记后可达40%的细胞漏检而被标记成Y阴性。 如用更温和的条件下来作Y FISH以换取对组织和细胞结构的较轻破坏, 则Y FISH的检测敏感度会进一步 降低。所以即使有人应用Y FISH方法在母鼠组织中鉴定出了 Y 阳性细胞, 也只说该细胞来源于公鼠的供体细胞。没人会作如此反推理: 在公鼠样本中或公鼠细胞中未检测到Y就等于Y染色体丢式, 因为所有应用Y FISH方法的人均知道该方法有百分之几十的漏检率而将公鼠细胞标记成Y阴性 (除非他的Y FISH方法具有100% 的检测敏感度或检出率, 他才能作以上反推理。这在技术上是不可能的-技术瓶颈)。 所以老板的团队在其文章FASEB 2007 中得有意伪造一组Y FISH阳性对照组的完美的实验结果或数据以向审稿人证明作者在该文章中所用的逻辑推理前提是真实的: 即她们用于标记小鼠骨髓细胞片子 (bone marrow cytospins ) 的Y FISH方法 (protocol) 具有100%的检测敏感度-也就是漏检率为零, 所以在该文章中对于她们在实验样本中 (含公, 母鼠细胞-sex mismatched model) 的Y FISH结果作者可以做以上所讲的反 推理。该文章中作者所用的Y FISH阳性对照组就是正常的野生型公鼠骨髓细胞的Y FISH结果: 0.8 +/- 0.8 %的细胞缺乏Y染色体。这里作者还对这个假数据进行了偷梁 换柱的解读 (这 0.8 +/- 0.8% 的正常公鼠骨髓细胞是缺乏Y染色体-即作者所言的 Y 染色体丢失而不是其Y FISH方法的漏检) 以给审稿人送去信息: 即作者的Y FISH方法的检出率本身仍为100%, 而那 0.8 +/- 0.8 % 的正常公鼠骨髓细胞是由于Y染色体丢失导致作者未能测到。这可是实验方法学上一场彻头彻尾的骗局, 因为作者完全知道她们用了多年的Y FISH方法（protocol) 在公鼠细胞片子 (肺细胞, 骨髓细胞)上具有很低的检出率也就是很高的漏检率, 即Y FSIH标记完后, 片子上仍会有很大比例的公鼠细胞因漏检而被标记成Y 阴性。这就是为何作者从来不在其文章中显示 Y FISH实验阳性对照组全景图像 (包含有几十个公鼠 细胞在Y FISH以后的图像) 的根本原因。 作者Y FISH方法的实验原理如下: 这种方法的全称是间期细胞Y FISH（间期细胞中所谓染色体是成染色质状态-chromatin), Y探针是去与Y DNA上的互补序列杂交。探针的制备是以正常公鼠的Y DNA 为模板用 DOR-PCR先制备此探针的材料: 为500bp到几个Kb大小的DNA片段 , 涵盖几乎整个Y DNA序列 (极高重复序列除外-有可能出现在其他染色体DNA序列中), 尔后用商品试剂合 Dig-Nick(其实就是 nick-translation) 反应将 Dig (digoxigenin) 标记到 DOR-PCR 所制备的 DNA 片段上, Dig 标记完后其产物就为100-500bp（Dig 标记的Y探针) 大小的混合物, 即Y探针混合物分段杂交到Y DNA 上的相应互补序列, 信号则来自于罗丹明标记的抗-Dig(Roche公司）。所以其实验原理已决定: 任何一个公鼠细胞在Y FISH标记以后, 或为Y阴性 (漏检, 因为无人的Y FISH 能达 100%的检出率且相差甚远）或含一个Y信号 (核中芝麻大小的粉红点) 或含多个 Y 信号, 即根本就不存在胞核中一个Y信号就代表一条Y染色体的对应关系-这是其实验原理已决定了！这也正是任何做Y FISH实验的人会在荧光显微镜下看到的现象。 那怕就是用同样的Y探针和同样的抗-Dig, 不同的Y FISH方法 (protocol) 当然会在同样的阳性对照样本上 (正常公鼠组织细胞片或切片) 产生不同的检出率。因为不同 的方法(protocol) 对实验步骤规定了不同的实验条件（温度, 时间, pH 等等), 也就是说每一个不同的Y FISH方法 (protocol) 所具有的检出率已被其本身所决定, 例如我的Y FISH方法的检出率就远高于作者的Y FISH方法（因为我的 protocol 所规定的实验条件不同于作者的 protocol), 这就是科学技术。作者用于小鼠细胞片子 (cytospins) Y FISH 方法的硬伤是: 其 Y DNA 变性条件太差, 73度 5分钟, 此条件不足以打开所有细胞核中的 Y DNA 双链, 而这正是 Y 探针杂交的先决条件！而按照任何一个已建立的Y FISH方法 (protocol) 去做Y FISH实验则很简单, 即操作过程 并不复杂。 作者 Y FISH 方法 (protocol) 的高漏 检率与由这方法所得出的实验结果的关系: 仍以作者文章 FASEB 2007 该方法的阳性对照组为例: 正常野生型公鼠骨髓细胞片子 (bone marrow cytospins)Y FISH 结果 ; 平均 值正负标准差或标准误为: 0.8 +/- 0.8 % 的骨髓 细胞缺乏 Y 染色体 。 这里只要知道两个任何人也无法改变和否认的 事实, 则这组阳性对照组的 Y FISH 实验数据的命运就已被决定了。 1. 作者 Y FISH 方法本身所决定的高漏 检率, 而得到这个证据或事实的最确切和唯一的办法就是实验验证其方法本身, 即严格按照作者的方法在正常公鼠骨髓细胞片子上作 Y FISH 实验, 标记完片子后, 在荧光显微镜下则真相一目了然: 所看到的只能是大百分几十 的细胞因漏检而被标记成 Y 阴性细胞 - 即所称的假 阴性, 因为在片子上每一个细胞均含有一条 Y 染色体, 而只是作者的 Y FISH 方法检测不出而已。这正是由其方法 (protocol) 本身所具有的高漏 检率所决定的, 不管何人包括作者本人, 何时用这方法 (protocol) 在阳性 对照上都会得到同样的结果: 即大百分之几十的公鼠骨髓细胞因漏检而被标记成 Y 阴性细胞 , 即作者的 Y FISH 方法只能 产生高 比例的假 阴性而决非 小于 1% 的公鼠 细胞为 Y 阴性。此仍实践是检验真理的唯一标准, 而不是作者说或某 人说其 Y FISH 方法 (protocol) 能达 100% 的 检出率, 这方法就能真达 100% 检出 率！ 这 里所遵循的是实验科学的一个基本原则：可重复性, 即任何实验方法必须在同 样的对照上产生同一趋式的结果。这里还有一个特点: 被检测目标为 Y 染色体 DNA, 这是所有正常公鼠细胞中的常态恒定成分 ( 这也是遗传学的基本原则, 所以 作者不能说其 Y FISH 方法在 2007 年以前能在公鼠 细胞上测出 100% 检出率, 现在不行了, 因被测目标 - Y DNA 与以前不同了）。2. 获 得 这种 Y FISH 实验结 果数据的唯一方式(data acquisition)就是作者必 须 用自已的双眼在 荧 光 显 微 镜 下去看由她 们 自已的 Y FISH方法所 标记 的正常公鼠骨髓 细 胞片子, 根据一个非常 简单 和明 显 的依据- 在被DAPI染成 兰 色的胞核中有或无Y信号(粉 红 色点)而 对每 一个 细 胞判 别为 Y+ 或 Y- 并分 类计 数, 一个 视 野接着一个 视 野(一 张 骨髓 细 胞片子包含几十个 视 野),而 获 得那 组 阳性 对 照的Y FISH 实验 数据(0.8 +/- 0.8 % 细 胞缺乏Y染色体)作者必 须 看,判 别 , 计 数几百个 这样 的 视 野。整个 过 程无高科技,而只需要正确的科研精神-即 诚实 或事 实 求是: 将自已所看到的真 实记录 下来以采集到真正的 Y FISH 实验结 果的数据。作者Y FISH方法的高漏 检 率就决定了在此Y FISH 实验结 果的数据采集 过 程中, 每 个 视 野她 们 只能看到大百分之几十的公鼠骨髓 细 胞因漏 检 而被 标记为 Y 阴 性 细 胞而决非小于1%的 细 胞 为 Y 阴 性,而任何一双 诚实 的眼睛是无法将 每 个 视 野下大百分之几十的 Y 阴 性 细 胞判 别 , 计 数成小于1%的Y 阴 性 细 胞的。 证 明完 毕 : 作者的 这组 Y FISH 实验 数据只能是有意 伪 造的一 组 数字。因 为 文章需要此完美的数据, 否 则 无法通 过审 稿(文章中的 逻辑 推理前提), 即作者以欺 诈 的方式而 获 得 这组 完美的Y FISH 实验 数据: 一 则 可能根本就没做此 实验 , 因 为 她 们 知道其Y FISH方法的高漏 检 率是无法真正得到那 种 她 们 所需的完美数据的,有 谁 会有意去做无用功呢？其二作者有意将 每 个 视 野下所看到的大百分之几十的Y 阴 性 细 胞写成 为 小于1% 的Y 阴 性 细 胞。不管是那 种 情况,作者都是有意 伪 造 实验 数据,在本案中 连 诚实 的 错误 (honest error)也失 较 ! 就象我再次 举报此案时反复对官方强调的那样, 此案可算史无前例。这是由作者的造假方式所决定的, 作者胆大妄为, 其有意伪造的 Y FISH 实验数据不但远远 超出其方法的检测敏感度, 而且远超出该种实验技术的技术瓶颈。这种实验方法学上的 造假就决定了, 这种造假极易用逻辑上的反证法将其一步证死。作为举报人我可以 用对照样本证明在逻辑上和科学技术上用作者的实验方法是不可能得出其文章中相应的实验数据的并相差几十倍: 这里是公鼠骨髓细胞 Y FISH 方法的漏 检率只能是大百分之几十, 但文章中作者白纸黑字写下了 0.8 +/- 0.8 % 细 胞缺乏 Y 染色体！ 所以此案的 关键点就只有一个且非常简单: 到底作者 Y FISH 方法的漏 检率是零还 是大百分之几十？ 此真相一出则此案大白。所以我于 2009 年 2 月再次 举报此案时, 只打击一个实验方法和一组阳性对照组的 Y FISH 实验结果数据 0.8 +/- 0.8 % 公 鼠骨髓 细胞缺乏 Y 染色体 。并将自已 实验验证的片子本身 (物证), 所照的全景图像, 技术解释等等给了耶鲁和 ORI 让官方眼见为实。而官方对我提供的铁证只能刻意回避, 因为对方无法面对这两难处境。官方可以说我作为举报人所提供的物证没公信力 (credential) ，但你可以 对 我所提供的最直接的物 证进行交互验证吧 (cross-examination), 你耶 鲁, ORI ,作者自己去 验证总可以吧。这正是我一直所要求的, 但 ORI 告 诉我: 他 们看了也没用, 因为他们没裁决权 ! 对方拿着我的物证是两难: 如说他们已看了我所做的片子, 则必须承认大多数 (majority) 公鼠骨髓 细胞漏检而被 标记成 Y 阴性, 即假阴性 , 这就必须承认作者有意伪造 Y FISH 实验数据。如对方想挑战我的实验验证, 则他们须实验证明作者的方法可以产生漏检率为零或小于 1% 的公鼠骨髓片子。我也明确无 误 地告 诉 官方, 尽管我只打 击 一个 实验 方法和一 组 数据, 但只要其Y FISH方法的高漏 检 率暴光 则为 多米 诺 骨牌 较应 。 作者 Y FISH 方法的高漏 检率和多米诺较应: 一旦其方法的高漏 检率暴光, 例如漏检率为 50% ,只 须用同理可得的道理则作者 FASEB 2007 文章中的下列 结果的命运会如何: SPCKO 公鼠 组骨髓细胞片子 Y FISH 的 结果数据 : 0.92 +/- 1.03 % 细胞缺乏 Y 染色体, 即 Y 阴性 p.2594 ；接受了野生型公鼠骨髓移植的 SPCKO 母鼠 组骨髓细胞片子 Y FISH 的 结果数据: 94.18 +/- 2.48 % 细胞含有 Y 染色体 , 即 Y 阳性; 接受了 SPCKO 公鼠骨髓移植的 SPCKO 母鼠 组骨髓细胞片子的 Y FISH 的 结果数据: 93.28 +/- 1.78 % 细胞含有 Y 染色体,即 Y 阳性 p.2595 ; 图 4A , 图 4B 的 结果和作者所用的逻辑推理前提; 再验证作者用于石蜡切片的 Y FISH 方法的漏 检率和 Y/SPC 双 标记方法, 则文中 8 只野生型公鼠肺切片的 Y/SPC 双 标记的结果数据: 1000 分之 1000 的双阳性 ( 请见第一作者 2008 年 3 月 6 日 给我 的电子邮件: 她从来没有将 Y/SPC 双 标记搞工作过, 她的实验室看来连 FISH 也不工作了 !) 和其他 Y/SPC 双 标记的实验结果的命运会如何呢 ? 作者的其他 Y/ 荧光免疫多重标记方法和其相应结果的 命运会如何呢？目前我得先推倒第一 张骨牌, 所以在 此对后续反应不作太多祥细分析。 老板团队在期刊Science上反击 Standford 团队对其文章 Cell 2001的挑战时所用的最大实验依据就是其Y FISH方法的超高检测敏感度(请见我的博文-事件回放), 在接 受了公鼠骨髓移植的母鼠的脾中她的团队检测到大于90%的脾细胞为Y阳性-所谓的 engraftment (意即她的Y FISH方法的检测敏感度本身仍为100%)。 这是一个双重谎言, 其一她的Y FISH方法的检测敏感度离90%还差的远, 漏检率为大百分之几十, 所以她无法在以上所说的脾中测出大于90%的脾细胞为Y阳性, 其二在这种小鼠模型中受体鼠的脾不可能发生大于90%的 engraftment (即大于90%的宿主脾细胞被来自于由供体干细胞变成的脾细胞所替代)。以下是我的实验证据: 因我的专业是免疫学, 出于好奇心我想看看在这种致死照射后接受骨髓移植, 会有多少脾细胞 (T, B淋巴细 胞)被替代, 所以在我做的模型收获时我也同时将脾收获并用我自己的Y FISH方法检测脾。在接受母鼠骨髓移植的公鼠脾中Y阳性 (宿主) 与Y阴性脾细胞成分隔区域 分布, 并且Y阳性细胞并不少, 应该有大百分之几十, 这与我在同一个体的骨髓细胞Y FISH所看的 engraftment不一致, 骨髓片子上只有很小比例的细胞为Y阳性-即绝大部分骨髓细胞已被来自于供体干细胞生成的骨髓细胞所替代。而且所有的模型鼠均是这种现象, 即受体鼠脾细胞被替带的比例并不是很大(我的Y FISH方法也有一定的漏 检率, 但我在受体鼠脾中仍能看到大比例的Y阳性细胞-宿主细胞)。仔细思考后我得出了自认为合理的解释: 致死照射杀死了绝大部分的骨髓细胞, 但并没有杀死大部分相对处于静态的脾细胞, 所以宿主骨髓细胞必须大量快速被供体来源的骨髓细胞所替代, 而其脾中被替代区域应该为生发中心(germinal center) 那里有活跃的淋 巴细胞的死亡和增殖, 但脾中其他相对静态的区域则没理由被替代。 以上的例子也说明其Y FISH方法的高漏检率秘密对老板团队的重要性。她在期刊细胞 (Cell 2001)上发表那篇大作后, 因无人能重复其结果, 所以该领域的主流实验室均质疑其结果。Stanford团队则是在期刊科学 (Science 2002) 上发表文章直接挑战。 老板团队反击 (Science 2003) 的档箭牌就是其Y FISH方法的超高检测敏感度, 即用你们的方法测不到, 但我的方法能测到。Stanford团队在老板团队的反击后, 又在期刊科学 (Science 2003)上对老板的反击进行了回击, 但还是没捉到要点, 因为外人并不知道其Y FISH方法的高漏检率。谁也没有想到其实老板团队一直在她的Y FISH 方法学上唱的是空城计！我作为继任她们研究项目的人当然会知此秘密, 2007年6月我用自己的Y/CK多重标记方法检测正常公鼠肺细胞片子上(Y丢失研究）CK阳性和Y阳性，CK阳性但Y阴性的肺上皮细胞(参见我博文-事件回放)。我有意让老板看我的片子本身, 让她明白在Y FISH方法的漏检率上与我玩把戏没意思并且也没用, 还是留着去骗外人吧 (我的方法检出率本就高于她的方法) 别逼我会将Y FISH 的漏检而结论为Y丢失！她看了我的片子后又与 Erica 一起演双簧给我看, 好象她们从未见过Y FISH在公鼠细胞上的漏检似的 (请见两人2007年6月18, 19日的电子邮 件 )。 成像系统和组织样本细胞上的自发荧光: 在福尔马林或PFA固定过的样本上自发荧光非常普遍而且可以很强。不同的组织可以不同, 肺自发荧光很强, 同一组织中不同的细胞或同一种细胞的不同个体均可以表现不同。自发荧光可以是广谱的, 从短光谱的兰光到长光谱的红光, 红外光, 红, 绿荧光极为常见而且可以很强(主要在细胞浆部分，胞膜上也可有)。老板实验室的成像系统为: 荧光显微镜上配有多个滤光片。滤片1: 允许兰光通过, 用于DAPI所染 的胞核, 滤片2: 允许绿光通过, 用于FITC标记, Alexafluor488等, 滤片3: 允许红光通过, 用于罗丹明-Y的信号, 滤片4: 允许红外光如Cy5通过-肉眼不可见, 滤片5: 允许红, 绿荧光通过, 看片子时一般先用此滤片看红, 绿荧光的标记。照像则为: 从滤片1开 始, 设定暴光时间后OK, 依次重复操作到滤片4, 最后OK后, 则一张彩色电子图像形成, 和各个滤片下所摄的一张黑白图像形成并自动贮存于所联的机算机。 也就是说在各个滤片下可以设定不同的暴光时间以改变不同颜色光在最后所形成的图像中的强度。所以这种实验手段和获取图像的方式为制作假电子图像留下了巨大的空 间。例如在小鼠肺石蜡切片或细胞片(cytospins)一个具有红, 绿自发荧光的细胞 (在 滤片5下, 肉眼可见为黄色), 只需调整滤片2和滤片3下的不同暴光比例 , 则这个细胞在最后形成的彩色图像中即可以是红色荧光标记, 也可以是绿色荧光标记。也就是说将胞浆中的自发荧光做假成特异性抗体标记的信号在这种电子图像中并不难。辩别这种假图像的最好方法就是与真正的阳性对照细胞图像作比较并且用全景图像-含有内参照系统。因为真正由抗体所产生的荧光信号来自于一种纯的荧光分子所发射, 如Alexafluor488, 这种荧光的光谱很窄, 所以颜色看起来很纯而与广谱的自发绿荧光不同。请见我上传的Y/荧光免疫多重标记的全景图像和图解, 尤其是英 文的图解 (Fig.legend)。作假的图像往往只能显点而不能显面, 即有意裁 切掉内参照系统-片子上其他细胞的信息。 ----- Forwarded message from Erica Herzog erica.lyndrup@yale.edu ----- Date: Thu, 06 Mar 2008 14:51:55 -0500 From: Erica Herzog erica.lyndrup@yale.edu Reply-To: Erica Herzog erica.lyndrup@yale.edu Subject: slides To: bugen.hu@yale.edu hi bugen i left the slides and results for you on the bench. were they in any way correct? i never was able to get pro-spc to work with fish because of the autofluor but if you are able to that's great. i always did prospc first and detected that way so the signal was really bright and then did fish after doing confocal. your control looked nice and like real signal. the few experimental cells that i saw that were possibly spc+ didn't look exactly like the control. anyway let me know how this compared with your results also since my lab cannot seem to get fish to work diane said you guys would be willing to collaborate if i gave you some slides for staining - if so can i bring you the slides sometime soon? best, erica Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注： 问我她的判片结果怎样？她不能将 Y/SPC 双 标记搞工作的原因是其 Y FISH 方法本身和抗体本身不工作（ Chemicon, AB3428). autofluor( 自 发荧光)总是存在的, 这 根本不是她的 Y/SPC 双 标记不工作的原因！在实验方法学上此人一贯狡辩。 其 Y/SPC 双 标记的方法都不工作, 但敢在文章 FASEB 2007 中将此双 标记结果写成 1000 分之 1000 的双阳性（ 8 只野生型公鼠肺石蜡切片 表 -2 p.2596). 她所 说其 lab 不能将 FISH 搞工作, 应该是指很高的漏检率。当时她已不敢再对我说她们的 Y FISH 是 100% 的 检出率了, 因为她还等着我帮她一把。请见她 2007 年 6 月 18 , 19 日的 电邮, 她是怎样说其 Y FISH 方法的 检出率。 当 时她应该还不知道我老板已将我逼到死角, 我已别无选择, 准备打假了, 否则她不会给我送这电子邮件。 ----- Forwarded message from Diane Krause diane.krause@yale.edu ----- Date: Tue, 19 Jun 2007 06:32:54 -0400 From: Diane Krause diane.krause@yale.edu Reply-To: Diane Krause diane.krause@yale.edu Subject: Re: Y loss in males To: Erica Herzog erica.lyndrup@yale.edu What % of the male into male transplants were Y-? At this point Bugen is using cytospins. So far, he's only done older transplanted and untransplanted SPC-/- and untransplanted WT males. So far, with cytospins, we never see 20% without a Y. Diane On Jun 18, 2007, at 9:29 PM, Erica Herzog wrote: age matched but not irradiated truly less than 1% were Y neg unlike the male into male transplants how old are his mice - are they the old ones for the Y loss project also depending on fixation, size of probe, length of permeablization etc there can be issues.depending on the PFA strength, age of reagents there can be big issues. how is his X staining? Hi Erica In rereading the FASEB paper, I see that we don't have the % of SPC+ cells in males that are Y- by confocal. Did you do this with any males? If so, were they age-matched or younger? I am concerned that we don't know the sensitivity of the assay to detect for Y loss after cell fusion . Bugen is seeing epithelial cells have no Y on lung cytospins from male mice. Thanks for any info that you have. Diane -- Diane Krause MD, PhD Yale University School of Medicine Associate Professor, Department of Laboratory Medicine PO Box 208035 New Haven, CT 06520-8035 Phone: (203) 688-4829 Fax: (203) 688-2748 Office: BML 462 Administrative Associate, Pat Sember: (203) 688-3265 Pager: (203) 412-0805 Krauselab website: http://info.med.yale.edu/labmed/faculty/labs/krauselab/index.html Erica Herzog MD, PhD Assistant Professor Yale University School of Medicine Internal Medicine - Pulmonary and Critical Care Division 333 Cedar St TAC 441-S New Haven CT 06511 (203)785-3207 ----- End forwarded message ----- 注： 这是老板看了我用自己的 Y/CK 多重 标记方法所做的公小鼠肺细胞片子后, 她与 Erica 之 间就所谓 Y FISH 方法的 电子邮件, 但有意转给我。她两人在 Y FISH 方法漏 检率的问题上给我演双簧, 其实就是想逼我将我所做的公鼠肺细胞上的 Y 漏 检当 Y 丢失的结论。老板已知道我当时刚建好的多重标记方法已突破了她们文章 FASEB 2007 中方法的技 术瓶颈, 想让我用自己的方法做出 Y 丢失的结果去掩盖文章中谎谬 的 Y 丢失结果和结论。两人装着一副好象不知道她们的 Y FISH 方法有漏 检率似的 (因她们总是说其 Y FISH 方法具有 100% 的 检出率）。我当然是装糊涂, 对这种双簧毫无反应, 所以老板也拿我没办法。请见 Erica 于 2008 年 3 月 6 日 给我的电子邮件, 看她是如何说她们的 Y FISH 方法。 老板 说步根在公鼠肺细胞片子上已看到 CK 阳性但 Y 阴性的细胞（其实就是 Y 漏 检）也应该是警示 Erica : 其 实我已知道她们的秘密只是不说而已。 这里 Erica 的所 谓解释在技术上均是一派胡言。
标签: 造假案
