We teach and practice ethical behavior with all clinical and research activities. Notably, we are well educated to treat the subjects participating in research studies with high ethical standards. However, the ethics of interacting with colleagues, or with junior faculty members, are neither well defined nor taught. Dealing with junior faculty has parallels to dealing with vulnerable research subjects such as children, mentally or physically challenged groups, prison inmates or army recruits. Like any other vulnerable population, lower-ranking faculty members are often at the mercy of department chairs or other higher-ranked faculty members. Herein we present some potentially unethical or unfair examples related to academic research. Our goal is to educate the academic community of conceptual paths and to prevent similar untoward occurrences from happening in the future. Unethical behaviors related to sexual misconduct have already been described elsewhere and are not included in this manuscript. This Declaration forms the basis for all later ethical proclamation relating to how “we care for the persons who are our patients, for those who seek advice, and protect those who volunteer in research studies”2. Through repeated training in human research subject protection, researchers recognize the malevolence of exposing unsuspecting prison inmates to syphilis bacteria to study immune responses or use organs from executed criminals for transplantation without their consent. However, the boundaries of ethics in collaboration with colleagues or with the junior faculty members are not well-defined nor discussed. We, hence, discuss the importance of teaching and following a code of ethics in interactions between colleagues in the academic setting. In 2014, the World Medical Association celebrated the 503. The reason is that ethics courses teach mainly abstruse concepts that are vague and intangible. Thus, as he asserts, they stay informative at best but are not strong enough to change a person’s views. Indeed, “ethics” is often used interchangeably with “morality”4, but ethics has more to do with a “philosophical concept” and morality has more to do with human actions5. Aristotle used the word “hexis” to describe moral virtue in relation to ethics6. Becausehexis is an active condition, “virtue”, in this context, implies an action. Again, we arrive at the conclusion that ethics must bring action, namely being transformative7. Therefore, it is necessary to teach ethics in a more action-based and example-based format so that learners can apply ethical judgment in their everyday activities.One of the pioneers of ethical education, Dr. Bertolami, wisely pointed out that our ethics curricula in biomedical education are informative, but fail to be formative and transformative7. We created a similar bell curve using our data and labeled them according to Aristotelian/Bertolamian categorization  were published with null results (no association). Thereafter, the whole world was eager to believe the null results because of the institution’s fame, seemingly excellent methodology and the large sample sizes .In late 1996, several small dental studies published a significant association between periodontitis and cardiovascular diseases (CVD)praejudicium (prejudged or pre-decided without being fairly heard). Researcher A truly believed that making a clear objective conclusion would benefit the research community and his faculty advisors would be pleased with such an endeavor. Researcher A did the project so well and received a standing ovation from fellow classmates and high praise from the instructor. The results were “pure science” without any influence of personal feelings or sense of affiliation. The conclusion was that there was a small but significant association between periodontitis and CVD. More importantly, the mathematical analyses have proven that seemingly poorly conducted studies overestimated the risk by only 13%, while ostensibly well-conducted studies underestimated the risk by 29.7%, thus appearing to be more biased than smaller studies. Later, Researcher A informed his faculty advisor of the results of meta-analysis. This faculty adviser (FA 1) was one of the lead authors of the null-result reporting papers from the θ University. FA 1 showed Researcher A the doctoral thesis which reported similar 30% attenuation in the risk estimates some 4 years earlier. Thus, what Researcher A observed in the meta-analysis was in agreement with the faculty adviser’s thesis. We note that the said faculty adviser did not realize the deficiencies of own thesis which reported 30% underestimation of the true risk. Rather, this faculty adviser thought that the culpability resided in other studies. Consequently, this faculty member wrote several opinion papers criticizing the other small studies.While learning meta-analysis methodology as a Master of Public Health student at the same famous θ University, Researcher A chose this topic as a class project to evaluate the evidence withoutSince the publication of this meta-analysis, which became a sentinel study cited over 560 times, Researcher A’s manuscripts and grant proposals have been harshly criticized and rejected numerous times presumably by these faculty advisors. Because of their institutional fame, these faculty advisors benefited from the privilege of being the most sought-after reviewers. After being rejected so many times, Researcher A’s grant was enigmatically funded when these faculty advisors were facing a legal action from an anti-compound λ group.Researcher E was a classmate of Researcher A at the same θ university and both were mentored by the same faculty advisors. Researcher E collected data for over 5 years and analyzed them. Researcher E found that her results suggested that exposure to compound λ increased the risk of osteosarcoma in adolescent boys. When Researcher E was about to publish the results, said advisors strongly recommended not publishing them citing that her data were of poor quality. This obstruction of the doctoral thesis by said faculty advisors leaked to an advocacy group opposed to the use compound λ and public uproar followed. Moreover, θ University, fearful of any future litigation from the anti-compound-λ group, forced the senior of the two faculty advisors (FA 2) to retire. Collaterally, the poor data collection method as revealed by meta-analysis prevented the younger of the two faculty advisers from winning tenure. Although neither Researcher A nor Researcher E had anything to do with the anti-compound-λ group’s protest on these faculty advisers, the animosity from the faculty advisers towards Researchers A and E deepened without due cause. Although the results showing the association of compound λ and the risk of osteosarcoma were finally published, Researcher E could not find a research position after 6 years of intensive PhD training. Perhaps, the behind-the-scenes evaluation might have had something to do with this researcher’s thwarted research career.It is dangerous to be right in matters on which the established authorities are wrong.” Here, it is worth discussing the objectivity of covert consultation of mentors without the knowledge of mentee’s especially when mentees have differing scientific views from that of their mentors. It is common practice that prospective employers, including post-doctoral research positions, contact the former employer or former faculty advisors about the prospective employee. The sad part is that this important process takes place without the knowledge of the researchers seeking positions. Thus, the nascent researchers have no idea why their job applications were declined. Consequently, what Voltaire had said some 300 years ago was proven true in 2000s: “Our perspectives:To strengthen the fidelity of science, differing opinions should be encouraged. This includes divulging the flaws of faculty members’ methodologies.We find the action of a prospective employer contacting faculty advisor without informing the position seeking researchers unethical although it is quite prevalent. If such an action is elected, they should contact the mentor in the presence of the mentee. Researcher A did not know why his numerous job applications were rejected until one prospective employer called the FA1 in the presence of Researcher A. We find this covert evaluation beyond the written letter of recommendation is despicable and should be abolished.We recognize that it is only human to harbor ill-feelings towards the mentees who exposed one’s own research flaws. We, therefore, suggest that mentors should totally extricate themselves from giving any evaluations of those mentees who provoke ill-feelings. In the case of these two faculty advisors, the younger FA1 delegated reviews of the mentees to the senior FA2 who is more prone to be harsher due to their powerful positions. Technically, FA1 relinquished review of mentees but handed it to a crueler evaluator.Episode 2.Also, take note that until the publication of the said meta-analysis, no one understood the cause for the conflicting results in the periodontitis-CVD association although all the researchers involved were top-notch scientists. This becomes another topic of unethical behavior by another researcher inAs stated above, no one who read the studies reporting the association between periodontitis and CVD knew the cause for the conflicting reports. When Researcher A had proven that non-differential misclassification was the cause of the null results using meta-regression, many people had an “aha!” moment. An “aha!” moment occurs when a simple principle that many people already know explains a complex phenomenon. For this reason, it is easy to claim another person’s intellectual property (IP) that brought an “aha!” moment as their own.In essence, Researcher A proved that the two large studies with meticulous confounding adjustment were more biased than the small, seemingly poorly conducted studies. Because it is expensive to conduct clinical examinations when a sample size is in the tens of thousands, the larger studies used a questionnaire to diagnose periodontitis. When a less-than-precise method, such as a questionnaire, is used, it causes non-differential misclassification. Non-differential misclassification is an elementary epidemiologic concept that can be explained in simple terms as follows: The truly diseased are included in the non-diseased group and some non-diseased persons are included in the diseased group because the measuring instrument was not precise. The result is reduced contrast which, in epidemiology, is referred to as “attenuation of the risk estimate”. It is a well-known epidemiologic principle that non-differential misclassification moves the risk estimates towards the null. However, connecting the dots of non-differential misclassification to the results of the “periodontitis-CVD relationship” was Researcher A’s IP. Let us peruse what has transpired after Researcher A’s meta-analysis.Researcher A wrote in the meta-analysis in 2003: “…potential for underestimation by 2 large epidemiologic studies because of the non-differential misclassification stemming from the use of questionnaires instead of clinical examination of periodontal disease.”Author D wrote quoting Researcher A  in 2005: “potential misclassification of periodontitis in studies not employing periodontal probing for assessment of periodontitis……. Indeed, the attenuation of relative risk estimates due to such misclassification can be quite dramatic”.“potential misclassification of periodontitis in studies not employing periodontal probing for assessment of periodontitis…Indeed, the attenuation of relative risk estimates due to such misclassification can be quite dramatic”.In 2008, Author D wrote quoting his own 2005 publication:Careful readers will recognize that Researcher A in 2003, and author D in 2005, wrote the same scientific construct with small changes in wording. Is Author D not obligated to quote Researcher A for this sentence because Researcher A has already published the very concept 2 years prior? Clearly, Author D had read Researcher A’s article when he quoted Researcher A for “heterogeneity” which is a far less important piece of information than misclassification. It appears that Author D used a two-step “bait and switch” tactic to divert the contest from Researcher A by quoting for something peripheral to the issue and made the more important concept of “non-differential misclassification” in the periodontitis-CVD relationship as his own IP.Researcher A read the above phrase which Author D wrote in 2008 and thought “Whoa, this is exactly what I wrote in 2003. Did he quote me”? But, it was found that Author D quoted his own 2005 paper. So Researcher A traced back to his 2005 publication and discovered that Researcher B quoted Researcher A for very nonessential “heterogeneity” but not for the important “non-differential misclassification”. A majority of meta-analyses report heterogeneity. Thus, it appeared that the construct of “non-differential misclassification attenuated the risk estimate in the periodontitis-CVD association” became his own IP in a two-step ‘bait and switch’ tactic. First, neutralize any protest from Researcher A by quoting the article for an unimportant fact and leaving the real important construct out. And in the second step, he quoted his own paper reporting the important construct without citing the Researcher A, who published this concept some years earlier. This two- step unethical misappropriation of someone else’s IP could be due to unintentional neglects. This is why we need ethical training to recognize that a subtle transgression may be a form of plagiarism.Our perspectives:We must educate ourselves to give credit to prior studies. Even if the concept involves a basic and mundane principle, the first person who used this mundane construct to solve the scientific queries should be given credit.We need to recognize that ethics is a human construct that needs to be taught.Ethical learning curve is applicable to everyone. We first thought citing Aristotle’s Nicomachean Ethics would be adequate citation for our categorization of human ethics . HoweverWe advocate that plagiarism should include not only “word for word” copying but also using other people’s concepts and ideas without appropriate citation.11. Later, a group of investigators reported that the exposure assessment of the original study11 was done by self-report and thus the results are not trustworthy12. Almost everyone with elementary knowledge in epidemiology knows that self-reported data are notoriously unreliable. Do we see the parallels of flawed methodology in the usage of questionnaire and self-report? They are both extremely imprecise, causing biased results. Anyone who has basic epidemiologic knowledge and read the original article11 would have recognized the flawed exposure assessment. Nevertheless, it is our opinion that the first researcher(s) who identified and published this flaw in the original study11 should be given credit as the owner of that IP12.Let us consider another example of a study using a mundane fact to explain a weighty conclusion. In 2012, a study conducted by a group of physicians and epidemiologists reported in a leading medical journal that dental x-rays might increase brain cancer meningioma in childrenResearcher C has been regarded as an expert on the relationship between oral infections and CVD and had published over 30 papers on the topic. Researcher C’s mentor, Dr. Z, known for her fairness and thoughtfulness helped Researcher C’s career transition from clinician to researcher. In appreciation of this mentor’s guidance, Researcher C included the mentor in a number of oral health and CVD-related publications. Subsequently, Dr. Z decided to write a grant proposal for assessing the relationship between oral health and CVD, assuming the role of the principal investigator (PI). In essence, the mentor became Researcher C’s competitor. The mentor appears to think that including researcher C as a co-investigator is fair compensation. However, Researcher C felt that Researcher C should be one of the co-PIs. The differing views on fair compensation caused a rift in their once close relationship.Sadly, ideas, and the associated IP can easily be purloined without any credit given to the source during the discussion because copyright or patent is not possible for these ideas. The concepts Researcher C shared with the mentor during a conversation became the mentor’s idea without any credit given to Researcher C. The concept that forms IP is the fundamental starting point for any scientific endeavor and should come from the PI, not the co-investigators. Often, in open discussions, Dr. Z requested members to provide opinions on how to approach the methodology. This is particularly troublesome when senior faculty members urge junior faculty members to donate their IP although the senior faculty members are the principal investigators. If junior faculty members decline, the senior faculty members are in the position of negatively affecting the junior faculty members’ careers. In this context, the junior faculty members are no different than the vulnerable research study subjects. Therefore, every effort should be made to protect junior faculty members in research collaborations.Our perspectives:An unspoken rule in research is that mentors should stay clear of the mentee’s research domain. This is because mentors are in a position of power and if they compete against mentees, they are in more advantageous locus. A good mentor will place mentees as PIs and put themselves as co-investigators.If the senior faculty is the PI, their role is to develop a study rationale and the research strategy. This requires much reading and critically evaluating prior studies not only supporting his/her study objectives but opposing said objective. The role of PI  should never be delegated.Currently, few researchers consider ethics when writing grant proposals. However, this should change, because a poorly conceived study will waste limited research resources and reduces the probabilities of other worthy studies getting funded. Thus, all researchers have a collective responsibility to conceptualize, prepare and execute a study plan that will benefit society.Most researchers are under tremendous pressure from the institution with which they belong and “getting funded” becomes the ultimate impetus for writing grants without adequate learning and preparation. What was once, “publish or perish”, nowadays becomes “funded or fade away”. In these conditions, the foremost role of the principal investigators (PIs) is accumulating adequate knowledge and developing a rationale so that the co-investigators will follow the well-planned study protocol. Too often, PIs are senior faculty members and sometimes have minimal knowledge of the study’s objectives. Using the position of authority, they delegate important tasks that clearly belong to the role of PI to junior faculty members who do not have enough experience or knowledge. The consequence of this dereliction of duty is an ill-prepared grant that might waste resources.The vertical relationship between senior and junior faculty members in collaboration harbors potential for an abuse of authority. The delegation of tasks can be perceived as an order. Often, senior researchers use words to appeal to the guilty conscience of junior faculty members, such as “this work is good for all of us and good for the institution”. Basically, this can be interpreted as “work hard without getting the credit you deserve”. The unspoken message is “if you do not do what I ask you to do, you are acting against all of us and against the organization we serve.” Forced donations, as suggested by senior faculty members, whether monetary or IP, should be discouraged. We decry sweat shops in South East Asia for their abusive labor practice but very few are speaking against intellectual sweat shops in academia. Is it because the perpetrators and victims both belong to the highly educated echelons of society? Abusive labor practice is abusive, no matter where it occurs.13. This is a clear conflict of interest but the grant review system has been in operation as far as we can remember. Thus, we do not know whether the application has no scientific merit or is opposite of the reviewer’s research theory.There are additional problems with the grant review process. The reviewers are usually those who were funded in the similar domain of research and they often have a hidden agenda to fund the application in the same direction as their own. For example, if the reviewer received funding to investigate oral cancer in relation to human papilloma virus (HPV), it is highly unlikely that this reviewer will fund an application aiming to prove that HPV was not a cause of oral cancer. Thus, whatever biases the reviewer has, will be perpetuated and any proposal that is not concurrent with reviewer’s research direction will likely not be funded. These biases are often unconscious, and the reviewer may not realize that she/he may be blocking a new thesis. Although the National Institute of Health (NIH) is looking for “innovative” research, our experience tells us that reviewers rarely support any innovative idea that may take away their own funding potentials. Others have expressed similar concerns on these non-financial conflicts of interestThe qualifications of reviewers can also sometimes be problematic. If a study involves human-level outcomes, it will be reviewed by epidemiologists who usually do not conduct molecular-level research. And yet, their review determines the fate of many well-thought-out translational research projects. Often, the value of the scientific merit is secondary to the submitter’s personality. Someone who never spoke against another scientist’s flaws will get funded with mediocre science, while scientists who tried to expand our scientific understanding by pointing out deficiencies in other’s research may not be so lucky.14. This is a major scientific misstep because feces are the end results of intestinal activities, not the cause for them16. Consequently, many millions of research dollars have been spent but we are not certain of the benefits these results may bring to society. Reviewers will not support a brand new idea citing “the lack of preliminary data” even in pilot grants. Without a pilot funding, the idea will not be tested at all. The ideas with proven track record will be shunned as “not innovative enough.” Once again, Voltaire was correct in saying: “When the idea is new, people say it is not true. When finally it is proven to be true, people say it is not new.” Wasteful funding may be attributed to the funding agencies’ demand for “innovation” as a key element in grant proposals. “Innovative” research innately encourages charting untested waters. At present, buzzwords ending with “-ome”, i.e., genome, microbiome, proteome, transcriptome, metabolome, etc. are in vogue. These studies are looking at the whole collective constructs, be it microorganisms (over 10 trillion in a human body), genes  or proteins . First, it is nearly impossible to pinpoint any one or several of these as causative of a human pathology. Moreover, the investigators use an easy to access compartment such as the fecal microbiome to estimate the causative microbes in the gut, which may not be similarOur perspectives:The key ethical consideration in grant writing should be whether the grant proposal will improve human health. Thus, selecting causal relationship that will engender better health for all humans is the crucial.The most important tenets of ethical grant review is reviewing the potential for human health improvement. Too often, many studies that will never reach human level results guzzle up a major portion of research funds.There is no easy way to protect the rights of junior faculty members when they collaborate with senior investigators. Educating scientific community on this issue and the senior members of faculty may voluntarily protect the junior faculty members.If you are going to leave the medical profession because you have something to say, you betray your whole life if you don’t [say] and if you don’t say it honestly and bluntly.” For all those who spoke out to make academic research just and fair, whether their honesty and bluntness will contribute to the betterment of academic research ethics will not be known immediately. Only history will tell.We must all take pride in not surrendering to the overwhelming power of the established dogma. As the late columnist Charles Krauthammer said “No data are associated with this study. I concur to index the revised version of the article in its present form.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. Here, the authors describe several ethical considerations in academics. This is an interesting topic and the ethical issues related to academic are seldom acknowledged. This is largely an opinion piece, with the conclusions those based on the authors' opinions.  Unconscious bias in academics is an important topic that should be addressed. My only comment for consideration to address is for the authors to somehow make clearer the relationships they describe in their "episodes". I found it difficult to keep track of the various relationships. Perhaps there is a way to do the pictorially.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. In this article, Sok-Ja Janket et al. present several hypothetical cases that resemble real-life situations and pertain to ethical challenges that researchers deal with on frequent basis primarily at the mentor-mentee level. The authors are experienced researchers with significant training record. Thus, both their experience and academic caliber render them suitable to offer unbiased and objective point of view on such an important matter.The use of single letters as hypothetical names is somewhat confusing, especially because multiple exams are listed in the text. Instead of using Researcher Dr. A, it would be easier to process designations such as Dr. Mentor, Dr. Postdoc, Ms. Student etc.Although it is understandable that not all paradigms can be included in the article, it would be important to add one more scenario on "Ethics in assigning leadership positions to junior faculty" e.g. The Society of Research is creating an Early Career Committee to address aspects that pertain to junior faculty. The leadership of the Society of Research has to nominate junior members to operate the ECC. Dr. Past Trainee, who had pursued his post-doctoral training with the President of the Society of Research is selected instead of Dr. Award, who received last year's Outstanding Junior Faculty Award... [CONTINUE]The article would have been even more meaningful if the authors devoted a section on certain and clearly described suggestions about institutional action that needs to be taken. The section should go beyond description of principles and include even some examples of action that has been taken by leadership to serve ethical standards and the authors may be aware of. 1 were translated to health-related applications long time after their discovery, which might not even imply translatability.  I am not positive of the suggestion that research proposals should be reviewed in the light of potential for improvement of human health. Various research findings, such as siRNA that was discovered in plants MINOR: p.6; Typo in Episode 2: Replace "had proved" with "had proven" Here are my suggestions that may improve the content of the article: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.


Because of fast leucocyte degeneration in cerebrospinal fluid (CSF) laboratory examinations of CSF samples should be performed approximately within 30 min after withdrawal. This study examines the storage of canine and feline CSF samples in “TransFix®/EDTA CSF Sample Storage Tubes”  for preventing leucocytes from degeneration, so that routine and flow cytometry examinations are feasible up to 3 days after sampling.After storage in TransFix® tubes, leukocytes could not be adequately stained with Türk’s solution and differentiating between erythrocytes and leukocytes was cumbersome. In addition, the cell morphology could not be sufficiently assessed on cytospin preparations because of shrunken leukocytes and indistinct cell nuclei. In contrast, by flow cytometry, a significantly higher cell count was measured over the entire study period in the samples stored in TransFix® tubes compared to the untreated samples. The antibodies (AB) against CD3, CD4 and CD21, against CD11b and against CD45 showed a good binding strength and thus enabled a good differentiation of cell populations. However, after storage in the TransFix® tubes, monocytes were no longer detectable using an AB against CD14.Based on these results, “TransFix®/EDTA CSF Sample Storage Tubes” can be used for extended storage prior to flow cytometric analysis of lymphocytes and granulocytes in CSF samples but not for detecting monocytes. However, standard examinations, such as microscopic cell counting and morphological cell assessment should be performed on fresh CSF samples.The online version contains supplementary material available at 10.1186/s12917-020-02698-5. For the diagnosis of diseases of the central nervous system (CNS), laboratory examination of cerebrospinal fluid (CSF) is a frequently used method , 16. TheThe function of the CSF is to protect, support and nourish the CNS . PhysiolRoutine CSF analysis includes microscopic white (WBC) and red blood cell (RBC) count and microscopic evaluation of cytospin preparations to differentiate the individual cell populations and thus to determine their percentage distribution , 14. FurBecause of the low protein content of the CSF, nucleated cells degenerate quite fast in cerebrospinal fluid, in particular granulocytes . HoweverCurrently, “TransFix®/EDTA CSF Sample Storage Tubes”  are commercially available. According to the manufacturer’s introductions these tubes prevent cellular degradation in CSF over 72 h , thus enabling investigation at a later date. These tubes include the TransFix® stabilization solution, which contains a buffer, an aliphatic aldehyde and heavy metal salts in combination with ethylenediaminetetraacetic acid (EDTA) . So far,The aim of the current study was to test the use of “TransFix®/EDTA CSF Sample Storage Tubes” in veterinary medicine on canine and feline CSF samples for their use in standard examinations such as leukocyte count and cell morphology assessment, and for their use in flow cytometry. We hypothesize that using the “TransFix®/EDTA CSF Sample Storage Tube” provides an opportunity for longer shelf life and cell stability in CSF samples so that an examination within 72 h after CSF sampling is possible.On day 0, the microscopic total nucleated cell count was simple and unequivocally determined, since the staining with Türk’s solution allowed a clear differentiation between WBC and RBC. On day 1 and 3, the difference between WBCs and RBCs in the untreated stored samples after staining with Türk’s solution was also evident in most of the samples , so that leukocyte and erythrocyte counts could be performed without any problems. In contrast, the distinction between RBC and WBC in the samples stored in TransFix®-solution on day 1 and day 3 was no longer possible. Using Türk’s solution, no cell structures could be stained and none of the visible cells showed a nuclear morphology. All visible cells seemed empty and only a rim was visible . The total number of cells in the untreated samples decreased notably over the investigation period. In addition, the proportion of dead cells in the untreated samples increased significantly over the study period (p < .05). Despite the increase in percentage of dead cells, their absolute number, as well as the total number of cells, was continuously decreasing over the investigation period. In addition, macroscopically visible cell clumps were seen on days 1 to 3 in almost every of the untreated samples. In contrast to the untreated CSF samples, no significant decrease of the total number of cells was observed in the samples stored in TransFix®-solution (p > .05).Table p < .0001). For the percentage of CD3- and CD4 positive lymphocytes the day and the interaction between the day and the type of storage showed a significant main effect in the ANOVA (p < .01) In contrast to the percentage of CD4-positive lymphocytes there was no main effect of the type of storage (p = .2596) in the ANOVA for percentage of CD3-positive lymphocytes.All samples stored in TransFix®-solution could be evaluated for the proportion of CD3- and CD4-positive lymphocytes. The ANOVA showed a significant main effect of the day and the type of storage as well as the interaction between the day and the type of storage on the absolute count of CD3- and CD4-positive lymphocytes (p < .0001), but there were no main effects for the type of storage and the interaction between the day and the type of storage (p > .05) on the percentage of CD21-positive lymphocytes. For the absolute count of CD21-positive lymphocytes the ANOVA showed a main effect of the day, the type of storage and the interaction between the day and the type of storage (p < .01).For the antibody against CD21 all samples stored in TransFix®-solution could be evaluated without any problems. The ANOVA showed a significant main effect for the day (p < .05) on the absolute count and the percentage of the CD11b-positive granulocytes. The effect of the type of storage was only significant for the absolute count of CD11b-positive granulocytes (p < .0001).The evaluation of the granulocyte population with the antibody against CD11b was first carried out by means of a histoplot. However, left-shift of the population of CD11b-positive granulocytes in the histoplot was observed in the untreated samples as well as in the TransFix®-stored samples during the study period. Therefore, samples were evaluated again using the histogram. The ANOVA showed a significant main effect of the day and the interaction of the day and the type of storage (p > .05). On the contrary the ANOVA showed a main effect of all three variables for the absolute count of CD45-positive lymphocytes (p < .0001). For the absolute count and the percentage of CD45-positive granulocytes the day and the type of storage as well as the interaction between the day and the type of storage showed a significant main effect in the ANOVA (p < .0001).The antibody against CD45 was considered separately in the populations of lymphocytes and granulocytes. For the evaluation of the granulocytes in the samples stored in TransFix®-solution the histogram had to be used for four samples, but these were not included in the statistic evaluation. For the percentage of CD45-positive lymphocytes the ANOVA showed no main effect of the day, the type of storage and the interaction between the day and the type of storage  was no longer possible after storage in the “TransFix®/EDTA CSF Sample Storage Tubes”. However, standard examinations, such as microscopic counting of leukocytes and erythrocytes as well as morphological cell assessment on cytospin preparations should be performed on fresh CSF samples within 30 min after sampling.The current study consists of two parts. In the first one microscopic count of leukocytes and erythrocytes and the morphologic assessment of cells on cytospin preparations was evaluated. In the second part of the study blood cell-spiked cerebrospinal fluid samples were analyzed by flow cytometry. In both parts the untreated storage was compared to the storage in TransFix® tubes.All samples were taken at the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover (Germany) between the 20 August 2018 and 4 April 2019. During the study period 43 fresh CSF samples, five feline and thirty-eight canine CSF samples, were analyzed for microscopic cell counting and examination of cell morphology. 22 samples were obtained as part of diagnostic tests in dogs and cats with neurological diseases with written owners’ consent and 4 samples were leftover from a granted animal experiment  from healthy dogs. 17 additional samples were obtained post mortem from patients immediately after euthanasia, due to CNS diseases in 7 cases and due to other diseases in the other 10 cases. The study was performed according to the ethical guidelines of the University and was approved by the Universities thesis Committee. All samples were obtained suboccipitally or lumbar in general anaesthesia or post mortem into plain tubes. Within a maximum delay of 25 min after sampling the examination of the untreated sample on day 0 started and the TransFix®-solution was added to the corresponding amount of the CSF sample as shown in Fig. For the storage the fresh CSF samples respectively the blood-leucocyte spiked CSF pool samples were divided into three parts, as shown in Fig. For the examination 10 μl of Türk’s solution  were mixed with 90 μl of the relevant CSF sample for staining the leucocytes. After 3 min of staining 20 μl of this mixture were transferred to a Fuchs-Rosenthal counting chamber . Leucocytes and erythrocytes were counted via microscopic observation using the 400fold magnification and considering all visible leucocytes and erythrocytes in the squares of the counting chamber.The remaining 80 μl of the mixture of CSF and Türk’s solution were mixed with 40 μl albumin   in PBS) to prepare the cytospin using HETTICH-ZYTO technique . Cells were mounted on specimen slides  by centrifuging  for 3 min at a speed of 1700 rounds per minute (rpm). For drying specimen slides were centrifuged for 1 min at a speed of 3400 rpm with a filter card . Afterwards the preparation was stained using Papenheim coloring . After drying preparations were covered permanently with Eukitt® Quick-hardening mounting medium  and a cover slip, so that the microscopic evaluation with a thousandfold magnification with the use of oil immersion was possible.During the microscopic analysis of the cytospin preparations lymphocytes, segmented and rod-nucleated as well as eosinophilic granulocytes, monocytes, macrophages and blast cells were differentiated and the proportion of the cells was determined.Two and a half milliliter of a canine blood sample were mixed with 5 ml PBS and afterwards layered carefully on 2 ml Human Pancoll  in another 15 ml tube . The falcon tube was centrifuged for 30 min at a speed of 1100×g and a temperature of 10 °C in the Hettich Rotina 35R centrifuge . This centrifuge was used in all following centrifugation steps. After centrifugation, the leukocytes were separated from the erythrocytes and were pipetted in another 15 ml tube with FACS staining buffer. The FACS staining buffer consists of autoMACS™ Rinsing Solution  with 0.5% fetal calf-serum . The new 15 ml tube was again centrifuged for 8 min at a speed of 500×g and at room temperature. Then the supernatant was removed and the pellet was resuspended in FACS staining buffer. In case of erythrocyte contamination of the pellet a lysis of the erythrocytes with distilled water was performed. The cells were washed with PBS and centrifuged two further times for 8 min at a speed of 200×g and a temperature of 20 °C. Afterwards the supernatant was removed completely and the pellet was resuspended in one milliliter of the canine CSF sample pool.6cells/ml.For the determination of the leukocyte content, a cell count was performed with 90 μl trypan blue  and ten microliters of the prepared cell-CSF-mixture. Both components were mixed transferred to a Neubauer improved counting chamber  for microscopic leucocyte count. The cell content of the cell-CSF-mixture was adjusted to 4x10The examination via flow cytometry was done on day 0 and on the three following days day 1, 2 and 3) to compare the untreated storage and the storage in TransFix® tubes. 250 μl of the relevant sample were pipetted into a 3.5 ml tube. After blocking with 13.2 μl Trustain  and thorough mixing, 50 μl of the prepared CSF cell mixture each were used for staining with antibodies and isotypes  in FACS staining buffer for differentiating dead and living cells. Immediately after pipetting the flow cytometric examination was started using the MACSQuant Analyzer 10 .The results were analyzed using the MACSQuantify™ Software . For the examination of the lymphocytic population the antibodies against CD3, CD4, CD21 and CD45 were used. The antibodies against CD11b and CD45 served for analysis of the granulocytes and the population of monocytes was separately examined using the antibody against CD14. The cut-off in the histoplot of each isotype was set at 2% (+/− 0.2%). For the evaluation of the CD11b positive populations and some of the CD45 positive populations a histogram was used in addition and the cut-off for positive cells was set at the point of intersection between the histograms of the isotype and the sample.p-value (Adj P) of <.05.Statistical evaluation was carried out with the SAS Enterprise Guide 7.1. . Descriptive statistics were used to examine the results of the microscopic cell count and the evaluation of the cytospin preparations. The results of the flow cytometric assay were analyzed by descriptive statistics and the mixed model Analysis of Variance (ANOVA). For each antibody, an evaluation was made separately for the percentages as well as for the absolute numbers of positive cells for the respective antibody. If the values of the studentized residuals for the parameter to be examined deviated significantly from the normal distribution, the values were transformed with the decadic logarithm and then the evaluation was repeated. For the post-hoc tests the correction according to Turkey-Kramer was used. The previously logarithmized values were subsequently transformed back, so that in this publication only the inverse transformed values are given. The limit for an existing significance was an adjusted Supplementary Material: Results Part Results of the evaluation of the cytospin preparations compared between day 0, day 1 and day 3 of untreated samples and samples stored in TransFix®-solution.Additional file 1.


Patients with a variety of blood, immune, and metabolic disorders may require an allogeneic hematopoietic stem cell transplant as part of their treatment. However, over 70% of these patients do not have a matched sibling donor and require an alternative donor, such as a matched unrelated donor. We present a multi-part story of a Canadian stem cell recipient who underwent transplantation for treatment of refractory chronic myelogenous leukemia, and the matched unrelated donor who saved his life. The story segments feature excerpts from interviews with the donor and the recipient, along with representative images of both storytellers. The excerpts were optimized for publication on social media and were arranged to build a story arc that parallels the journey of the donor and recipient together. This donor-recipient story may serve as a resource to help raise awareness about stem cell donation and to encourage eligible individuals to register as donors. The story is one of several developed by Why We Swab, a library of stories in stem cell donation in Canada  to support the recruitment of committed unrelated donors. Patients with a variety of blood, immune, and metabolic disorders may require an allogeneic hematopoietic stem cell transplant as part of their treatment. However, over 70% of these patients do not have a matched sibling donor and require an alternative donor, such as a matched unrelated donor. We present a multi-part story of a Canadian stem cell recipient who underwent transplantation for treatment of refractory chronic myelogenous leukemia, and the matched unrelated donor who saved his life . The stoThe story begins by capturing the accounts of the recipient in need of an unrelated stem cell donor (part 1) and his donor joining the Canadian stem cell donor registry (part 2). The donor then recounts giving stem cells from his blood (part 3). The story continues with the recipient’s initial contact with his donor (part 4), and their first in-person meeting (part 5), highlighting the development of their emotional connection. The story concludes with the recipient’s reflection on how his donor has impacted his life (part 6), with a photo of the recipient giving a speech at the donor’s wedding.Sharing stories is an effective tool for promoting health education and awareness . StoriesTo our knowledge, this is the first donor–recipient story in the world which is told in this format. Online communication technologies allow patients to express their experiences of illness to a larger, real-time audience and to facilitate sharing and comments. Some cancer patients maintain online blogs, which allow them to publish and disseminate long-form narratives about their experiences with disease and treatment, often from diagnosis through life as a survivor . We arraFor patients and caregivers, this story can help to inform or give a sense of encouragement about finding a match or going through the transplant process. The emotions and challenges that are captured in this story may help to reassure and provide hope to patients and caregivers that they are not alone in their experience. For healthcare providers, this story can give insight into the experience of stem cell donors and recipients. It can help them to appreciate the complex journey of those impacted by stem cell donation, including how that journey is experienced, understood, and represented. This can support the practice of narrative medicine, which involves mutual learning, understanding, and empathy between patients and health care providers as a means to improve patient care . For pas


The fifteenth author's name is spelled incorrectly. The correct name is: Alfredo A. Sadun.The following information is missing from the Funding statement: This study was also supported by Research to Prevent Blindness Inc. (unrestricted grant).In the Discussion, there is an error in the third sentence of the second paragraph. The correct sentence is: Notably, this observed relationship between CSF protein levels and the retina is similar to that observed in the brain, where CSF Aß is inversely correlated with Aß load and neuropathology, while CSF Tau is directly correlated.The figure legends in S1 Fig(PDF)Click here for additional data file.


G12D mutant remains unclear. Therefore, we simulated the acetylation site on the KRASG12D three-dimensional protein structure, including KRASG12D, KRASG12D/K104A and KRASG12D/K104Q, and determined their trajectories and binding free energy with GEF. KRASG12D/K104Q induced structural changes in the α2- and α3-helices, promoted KRAS instability and hampered GEF binding (ΔΔG = 6.14 kJ/mol). We found decreased binding to the Raf1 RBD by KRASG12D/K104Q and reduced cell growth, invasion and migration. Based on whole-genome cDNA microarray analysis, KRASG12D/K104Q decreased expression of NPIPA2, DUSP1 and IL6 in lung and ovarian cancer cells. This study reports computational and experimental analyses of Lys104 of KRASG12D and GEF, and the findings provide a target for exploration for future treatment.Mutant RAS genes play an important role in regulating tumors through lysine residue 104 to impair GEF-induced nucleotide exchange, but the regulatory role of KRAS K104 modification on the KRAS KRAS activity regulates tumor progression, differentiation, metastasis and survival4. Approximately 90% of KRAS missense mutations occur in codons 12 or 135, and these mutations are associated with tumor formation7. KRAS is mutated in 25–30% of nonsmall cell lung cancer, of which c.35G>A (G12D) and codon 12 mutations are common in lung carcinoma (~ 18%)8 and ovarian cancer (~ 5.8%)9, respectively. The KRAS G12C mutation is not the most common, though it is frequently detected in colorectal and pancreatic cancers11. The c.35G>A mutation, from glycine to aspartic acid, interferes with GEF-induced GTP binding and activates KRAS downstream signaling12. Until recently, mutant KRAS was considered to be a therapeutic target in lung13 and ovarian cancer14.KRAS is a small guanine nucleotide-binding protein (GTPase) that switches between an active form when GTP is bound and an inactive form when GDP is bound. The switch between the two forms is controlled by guanine nucleotide-exchange factors (GEFs) that induce activation by binding to Ras family proteins, including KRAS (and Rho-family GTPases), and catalyzing the release of GDP15. Yang et al. performed molecular dynamics (MD) simulations on a single KRAS structure. Their observations suggest that the K104Q mutation mimics K104 acetylation at the molecular level and completely disrupts the structural integrity of the α2-helix; therefore, the authors predicted that the KRAS–GEF interaction would be hindered by the mutation15. To better understand how the K104 modification alters the intrinsic biochemical properties of KRASG12D, we focused on the KRAS/GEF protein complex structure and investigated its structural dynamics properties, including conformational changes, atomic fluctuations, and binding free energy differences between KRAS and GEF with KRASG12D/K104A and KRASG12D/K104Q mutants.In addition, acetylation negatively regulates KRAS signaling through GEF, as KRAS can be acetylated at lysine residue 104 (Lys104) to impair GEF-induced nucleotide exchange and inhibit oncogenic activityG12D, KRASG12D/K104A and KRASG12D/K104Q and analyzed the trajectory and binding free energy with GEF. We also characterized their cellular biological function and protein–protein interactions in cancer cell lines. This study reveals a novel pathway through which K104Q of KRASG12D may be useful for human cancer therapy.In this study, we predicted the three-dimensional (3D) structure of KRAS16. Human KRAS complexed with a GTP analog  served as the template for modeling the wild-type (WT), G12D, G12D/K104A and G12D/K104Q KRAS structures. The crystal structure of the ternary Ras:SOS complex (PDB ID: 1XD2)17 served as the template for modeling the KRAS structures complexed with GEF. A Ramachandran plot for each structural model was prepared to assess quality18. The steepest descent algorithm19 was used for structural optimization, and PyMOL20 was used to display the 3D structures and charge distributions.The 3D structures of KRAS and the KRAS/GEF complex were predicted using the MODELLER program (version 9v8)21. The OPLS-AA force field was used for energy calculations. Water was described using the TIP3P model, and the models were simulated in a cubic box with periodic boundary conditions. The energy of each model was minimized using the steepest descent algorithm. Each simulation was performed in canonical ensembles (NVT) followed by isothermal isobaric ensembles (NPT) with a position-restrained MD simulation for 100 ps. The MD simulations were performed at constant pressure and temperature for 50 ns using an integration timestep of 2 fs. The cutoff for nonbonded interactions was 10 Å. The coordinates from the MD simulations were saved every 100 timesteps.Molecular dynamics (MD) simulations were performed using GROMACS version 5.1.4 softwarerref, was calculated as i at time t after the last square fitting of the structure to the reference structure. (2) The conformational change of the α2 helix (which is located in the GEF-binding region) and the α3 helix of the WT and mutant structures derived from the equilibrated conventional MD trajectories as a function of the Cα dihedral angles of Met67 and Thr74 (both of which are located in the α2 helix) and Lys104 and Thr87 (both of which are located in the α3 helix). (3) Debye–Waller factors (B-factors) for Cα atoms, which reflect the fluctuation of the atoms about their average positions; the average positions for the Cα atoms were calculated using the coordinates of the last 2 ns of the MD trajectories. B-factors were calculated using the average RMSF values according to the relationship i and a reference position as T is the time over which the RMSF is averaged and i. A large B-factor indicates a highly flexible atom.To understand how amino acid replacements impact the structural and functional characteristics of Lys104 in KRAS, the trajectories of WT and mutant KRAS/GEF complex structures were analyzed for the following structural properties as a function of time. (1) The root mean square deviation (RMSD) of each KRAS backbone atom with respect to its position in the starting conformation, 23. This tool automatically generates hybrid structures and topologies for amino acid mutations that represent the two physical states of the system. The double system (combining both branches of the thermodynamic cycle) in a single simulation box was set to keep the system neutral at all times during a transition22. After obtaining this hybrid structure, we used GROMACS to perform free energy simulations. One hundred snapshots were extracted from the 10-ns equilibrated trajectories, and a rapid 100-ps simulation was performed starting from each frame. Lambda (λ) ranged from 0 to 1 and from 1 to 0 for the forward and backward integrations, respectively, thus describing the interconversion of the WT to the mutant system and of the mutant to the WT system, respectively. Finally, the pmx tool was used to subsequently estimate free energy differences and integrate the multiple curves by the fast-growth thermodynamic integration approach, which relies on the Crooks–Gaussian Intersection (CGI) protocol24. In such a double-system/single-box setup, the estimated free energy corresponds to the ΔΔG of binding.The hybrid topologies of the systems for the free energy calculations were constructed using the pmx tool25, GEF H1 26, RASA1 27, Ac-lysine 29, phospho-AKT 30, AKT 31 and β-actin 32, for western blotting.Protein was extracted from cells in lysis buffer that contained a protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined using reagents from a BCA Protein Assay kit . For immunoprecipitation experiments, 2 mg of cell lysate was incubated with specific antibodies and immobilized on protein A/G PLUS-agarose beads (Santa Cruz Biotechnology). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and blotted with specific antibodies, including those against RAS To evaluate the amount of active guanosine triphosphate − bound Ras, we used an Active Ras Pull-Down and Detection kit (Thermo Fisher Scientific). In brief, cells were homogenized in lysis buffer and incubated with GST-Raf1-RBD agarose beads at 4 °C for 1 h. The beads were washed with PBS, and the eluate was immunoblotted using K-, N- and H-Ras-specific antibodies.4 cells were seeded into the wells of 96-well plates, and at the indicated times, the culture medium was replaced with CCK-8 reagent. The absorbance of each sample was measured at 450 nm and used to determine the number of viable cells in each culture. Values are presented as the mean ± standard deviation from three independent experiments.Cell numbers in culture were analyzed using a Cell Counting Kit (CCK)-8 kit . Briefly, 1 × 105 cells were seeded in the wells of 6-well plates and cultured for 24 h. Each cell layer was scratched with a new, sterile 200-μl pipette tip. After an additional 24 h, the extent of wound healing was assessed by visualization under a microscope. Invasion assays were performed in a 24-well Transwell invasion chamber  that was coated with Matrigel (BD Bioscience) on the upper chamber. The top chamber of each well was seeded with 5 × 104 cells. After 24 h, the lower membrane surface was fixed with methanol and stained with 0.1% crystal violet. Invading cells were observed by microscopy.For wound-healing assay, 5 × 10Human Whole Genome OneArray v7  containing 29,204 DNA oligonucleotide sense probes of 50–60 nucleotides in length was recommended based on previous studies and used according to the manufacturer’s instructions. A total of 28,264 probes and 940 controls corresponding to the annotated genes from the RefSeq v70 and Ensembl v80 databases were included.−ΔΔCT method.RNA was isolated from cell lines using TRIZOL reagent (Invitrogen). cDNA was synthesized using a cDNA Synthesis kit . Quantitative PCR was performed with 10 ng of cDNA using Power SYBR Green PCR Master Mix (Applied Biosystems), and the cRNA was analyzed with an RT-PCR System kit ; 18S was used as the reference gene for normalization. All values were calculated using the 25) were seeded in a 6-well plate and incubated at 37 °C for 24 h. Plasmids were transfected with TurboFect transfection reagent  following the manufacturer’s protocol. pBabe-KrasG12D was purchased from Addgene (#58902).Cells  as the template Fig. A. Of theThe trajectories from MD simulations of the WT and mutant KRAS/GEF complex in explicit solvent models were calculated. The RMSD values of the backbone atoms for the WT and mutant KRAS/GEF complex with regard to the initial structures were plotted Fig. A, and thTo understand the structural changes of KRAS in the GEF-binding region caused by these mutations, we detected the conformational difference between the WT and mutant KRAS/GEF complex structures by measuring the dihedral angle of the α2-helix and α3-helix   when compared with WT and with the G12D and G12D/K104A mutants Fig. . This is24. The GEF binding affinity differences (G1 (GEF-bound) – ΔG2 (GEF-free), as shown in the thermodynamic cycle that was distributed between two simulation boxes for λ = 0 (green) and λ = 1  using site-directed mutagenesis. Active Ras binds to the Ras-binding domain (RBD) of Raf1, leading to Ras activation. The results of the analysis showed increased interaction of KRASG12D/K104Q with Ras GTPase-activating protein 1  but decreased interaction with GEFs  and acetylation. According to the results, the levels of RAS of KRASWT, KRASG12D, KRASG12D/K104A and KRASG12D/K104Q were the same. However, ac-lysine of RAS was not detected. KRASG12D and KRASG12D/K104A exhibited increased levels of p-AKT compared to WT and KRASG12D/K104Q  and migration ability in H1299 (P < 0.0001) and MCAS (P = 0.02) cell lines. These results suggest that K104 modification regulates KRASG12D-mediated cell growth and motility in lung and ovarian cancer cell lines.To investigate whether K104 modification affects KRASwth Fig. A,B, wounwth Fig. C,D and iwth Fig. E,F. KRASG12D/K104Q affects cellular biological functions by associating with GTP binding. However, the related downstream genes were not clear. Therefore, we sought to identify downstream genes of KRASG12D/K104Q that are reduced compared with KRASG12D/K104A using a whole-genome cDNA microarray to screen KRASG12D (> twofold), KRASG12D/K104A upregulation (> twofold) and KRASG12D/K104Q downregulation (> twofold) of gene expression in the lung cancer cell line H1299 and the ovarian cancer cell line MCAS 35 showing that KRAS K140Q decreases the SOS-catalyzed nucleotide exchange rate.MD simulations of the KRAS/GEF protein complex structure revealed that the conformational changes of the G12D + K104Q mutant were significant at the α2- and α3-helices when compared with the WT, G12D and G12D + K104A KRAS/GEF complexes . According to RMSD results, the most populated dihedral angle of the G12D + K104A KRAS/GEF complex (green) is ~ 5° larger and more widely distributed than that in G12D KRAS (red)  represses downstream signaling and inhibits cell growth through HDAC6 and SIRT2 in NIH 3T3 cells36. Previous studies as well as our own observed that KRASG12V and KRASG12D have the same function with respect to regulating the KRAS pathway and cellular function when K104 is acetylated. Although KRASG12D can activate cancer cells, progression to malignancy requires additional genetic lesions37. Among cancers with the highest mortality rates, KRAS-activating mutations occur in ~ 90% of pancreatic cancers, 40% of colon cancers and 30% of lung cancers. Therefore, it is important to consider cancer therapies that target KRAS mutations. Research has predominantly focused on EGFR mutation for lung cancer-related drug development, and there are many EGFR antagonists that have been used for clinical treatment. Unfortunately, the mortality rate for lung cancer remains high throughout the world. Indeed, epidemiological statistics indicate that EGFR and KRAS mutations are present in ~ 50% of lung adenocarcinomas, of which ~ 20% are EGFR mutations and 26% KRAS mutations38. KRAS mutations occur in 3.7–36.4% of endometrioid ovarian cancers40, though therapies targeting KRAS have rarely been used to treat ovarian cancer. Hence, it is very important to focus on KRAS mutations for the development of future therapies to treat lung and ovarian cancer.We also show that KRASG12D/K104A regulates GEF to activate KRAS signaling and promote cell growth, invasion and migration in lung and ovarian cancer cell lines. Mutationally activated KRAS (through G12D and G12V) is still a difficult pharmacological target. Next, we will analyze K104 acetylation of KRASG12D using histone deacetylase inhibitors (HDACis), which regulate cellular functions, including cell growth, invasion, migration and apoptosis, by repressing expression of HDAC, leading to increased acetylation of lysine residues in target proteins42. The HDACi trichostatin A (TSA) is a naturally derived hydroxamic acid, which has been studied as a potential nontoxic anticancer drug44. It is hoped that an HDACi such as TSA—either alone or in combination with other chemotherapy approaches—may provide a viable strategy for clinical targeting options in lung and ovarian cancer.Although expression of NPIPA2, DUSP1 and IL6 was decreased after acetylation of KRAS, it is still unclear whether this is related to the ubiquitin system. This possibility will be pursued in our future studies. Finally, our findings suggest that KRASSupplementary file1.


Dear Editor,MYC gene rearrangements or MYC (MYC proto‐oncogene) and BCL2 (B‐cell lymphoma 2) dual translocations.Central nervous system (CNS) involvement in diffuse large B‐cell lymphoma (DLBCL) patients correlates with dismal outcomes, and the detection sensitivity of conventional diagnosis of lymphoma is restricted.Pim‐1 proto‐oncogene , lysine methyltransferase 2D , BCL2 (27.5%), myeloid differentiation primary response 88 , B‐cell translocation gene 2 , and tumor protein p53 . Majority of altered genes were involved in four important pathways including epigenetic regulation , BCR (breakpoint cluster region gene) and NF‐kB (Nuclear factor‐kappa B) signaling pathway , apoptosis/cell cycle , and immunity . SNV was the most frequently observed type of alteration, whereas CNV had the tendency to be enriched in patients with lower CNS‐IPI including BCL2 and MALT1 (mucosa‐associated lymphoid tissue lymphoma translocation protein 1) amplification. Interestingly, cell‐free DNA (cfDNA) concentration in CSF‐in DLBCL patients showed a trend of increase from low to high level CNS‐IPI. By dividing patients into 0‐3 and 4‐6 CNS‐IPI, we observed a significant elevation in CSF‐cfDNA concentration, suggesting CSF‐cfDNA might be an indication of CNS involvement . Genomic landscape of this DLBCL cohort in systemic tumor tissue is shown in Figure t Figure . Plasma‐t Figure .In the 20 CSF‐cfDNA‐positive patients, a comparison of gene alterations (GAs) in matched systemic tumor tissue, CSF, and plasma was performed Figure . DespiteBTG2 S31N, was found in the PCNSL cohort.Compared to GAs only found in tumor tissue  were identified in more than 20% of total PCNSL cases. Meanwhile, 31 DLBCL patients with high risk for CNS had at least one alteration in the BTG2, PIM1, DUSP2, ETV6, and CXCR4 genes from CSF and/or plasma, including 14 patients with level4‐6 CNS‐IPI. Ten high‐risk DLBCL patients (nine were CSF‐cfDNA‐positive) were later confirmed CNS involvement by magnetic resonance imaging (MRI) or flow cytometry (FCM). Among them, seven patients were identified with multiple alterations of the five CSF‐CNS genes in CSF (Figure Then we look into the CSF signature of high‐risk DLBCL in the gene level rather than mutation level. Here, we referred the genes with alterations found exclusively in CSF of high‐risk DLBCL as CSF‐CNS genes. As shown in Figure F Figure . MoreoveLimitations of this study included the lack of CSF and plasma samples from low‐risk group to assess the status of CSF‐CNS genes. Close monitoring of CNS relapse in the high‐risk cohort will be also of great help to further validate our observation. Potential blood contamination or normal cfDNA in the CSF sample might also have some impact on the result interpretation.In summary, our study provided evidence for the association between CSF‐cfDNA concentration and CNS‐IPI score, highlighting the importance of CSF‐cfDNA in the detection of CNS tumors in DLBCL. Five CSF‐CNS genes in CSF was found to be associated with CNS risk in DLBCL patients, which deserve further investigation to determine their relevance among the diagnosis, treatment, and outcome.The National Science Foundation of China; Grant Number: 81970176; Natural Science Foundation of Guangdong Province; Grant Number: 2019A1515011943.Study design: WXX, GY, and HHQ. Data acquisition: WXX, SCG, LMY, ARY, WXX, and CLB. Data analysis: WXX, HHX, GY, RY, and QO. Manuscript writing: WXX, HHQ, RY, XW, and YWS. Study supervision: HHQ.Ruoying Yu, Qiuxiang Ou, and Xue Wu are the shareholders or employees of Geneseeq Technology Inc. Canada. Xiaoxia Wang and Yang W. Shao are the shareholders or employees of Nanjing Geneseeq Technology Inc. The remaining authors have no conflict of interest to declare.SUPPORTING INFORMATIONClick here for additional data file.SUPPORTING INFORMATIONClick here for additional data file.


Concordance between CSF biomarkers and amyloid-PET was higher in FTD patients compared to AD cases. None of the AD patients presented both negative Aβ biomarkers. Conclusions: CSF Aβ42/40 ratio significantly increased the diagnostic accuracy of CSF biomarkers. On the basis of our current and previous data, we suggest a flowchart to guide the use of biomarkers according to clinical suspicion: due to the high PPV of both amyloid-PET and CSF analysis including Aβ42/40, in cases of concordance between at least one biomarker and clinical diagnosis, performance of the other analysis could be avoided. A combination of both biomarkers should be performed to better characterize unclear cases. If the two amyloid biomarkers are both negative, an underlying AD pathology can most probably be excluded.Background: The aims of this study were to compare the diagnostic accuracy, sensitivity, specificity, and positive and negative predictive values  of different cerebrospinal fluid (CSF) amyloid biomarkers and amyloid-Positron Emission Tomography (PET) in patients with a clinical diagnosis of Alzheimer’s disease (AD) and Frontotemporal Dementia (FTD); to compare concordance between biomarkers; and to provide an indication of their use and interpretation. Methods: We included 148 patients (95 AD and 53 FTD), who underwent clinical evaluation, neuropsychological assessment, and at least one amyloid biomarker . Thirty-six patients underwent both analyses. One-hundred-thirteen patients underwent Apolipoprotein E (ApoE) genotyping. Results: Amyloid-PET presented higher diagnostic accuracy, sensitivity, and NPV than CSF Aβ Alzheimer’s disease (AD) is the most prevalent type of dementia and it has an increasing incidence worldwide . AD progUntil 2007, AD diagnosis was based on clinical symptoms and cognitive examination, while a defined diagnosis was possible only by postmortem pathological confirmation  whose pr1–42) [42/40) [11C]Pittsburgh compound-B (PIB), [18F]Florbetapir, [18F]Florbetaben, or [18F]Flutemetamol, which allows one to directly visualize fibrillary amyloid-β deposits in brain tissue Florbetaben (FBB)—Bayer-Piramal; [18]Flutemetamol (FMM)—General Electric). Images were rated as either positive or negative according to the criteria defined by the manufacturers.Fifty-eight patients (39 AD and 19 FTD) underwent amyloid-PET. Amyloid-PET imaging was performed according to standard national and international guidelines , with an1–42, t-tau, and p-tau were measured with ELISA kits , and INNOTEST HTAU AG.). Cut-offs for normal values were: Aβ1–42 > 600 pg/mL; t-tau < 300 pg/mL; and p-tau < 60 pg/mL [1–42, Aβ42/40 ratio, t-tau, and p-tau were measured using a chemiluminescent enzyme immunoassay (CLEIA) analyzer LUMIPULSE G600 ). Cut-offs for normal values were: for Aβ1–42 > 670 pg/mL; Aβ42/40 ratio > 0.062; t-tau < 400 pg/mL; and p-tau < 60 pg/mL [The CSF samples were collected by lumbar puncture, then immediately centrifuged and stored at −80 °C until performing the analysis. Two different methods of analysis were used to establish CSF measurements. In 59 patients (39 AD and 20 FTD), Aβ60 pg/mL . For 67 60 pg/mL . ReagentA standard automated method  was used to isolate DNA from peripheral blood samples. ApoE genotypes were investigated by high-resolution melting analysis (HRMA) . Two sethttp://bioinfo.ut.ee/primer3-0.4.0/primer3/) [All the coding exons and the intron/exon boundaries of the familial AD genes  and frontotemporal dementia genes (GRN and MAPT) were amplified by polymerase chain reaction (PCR) using primers designed with Primer3 software (rimer3/) . The anap < 0.05.Patient groups were characterized using means and standard deviations (SD). We tested for the normality distribution of the data using the Kolmogorov–Smirnov test. Depending on the distribution of our data, we used t-tests or non-parametric Mann–Whitney U tests for between-group comparisons and Pearson’s correlation coefficient or non-parametric Spearman’s ρ (rho) to evaluate correlations between groups’ numeric measures. We used a Chi-square test to compare categorical data and calculated the effect size by Cohen’s d for numeric measures and Cramer’s V for categorical data. All statistical analyses were performed with SPSS software v.25  and the computing environment R 4.0.3 . The significance level was set at Raw data that support the findings of this study are not presented due to ethical and patient data security reasons but will be shared upon request from any qualified investigator.2 = 7.077 p = 0.008) , age at CSF analysis, age at amyloid-PET, family history of dementia, sex, years of education, MMSE, and HDRS. Proportion of ApoE ε4+ was higher in the AD group compared to FTD (χ= 0.008) . In two 2 = 48.42 p < 0.001).Seventy-seven AD (81.05%) and 12 FTD (22.64%) patients were Aβ+. Aβ+ pathology proportion was higher in AD compared to FTD (χ1–42 (χ2 = 22.83 p < 0.001); 31 AD (75.60%) and 5 FTD (17.85%) had positive Aβ42/40 (χ2 = 22.24 p < 0.001). Fifty-one AD (64.55%) and 9 FTD (19.14%) were CSF Aβ+ (χ2 = 24.36 p < 0.001). Aβ1–42 and Aβ42/40 were both positive in 28 AD patients (70.00%) and in 4 FTD patients (14.81%).Concerning CSF biomarkers, 48 AD (60.75%) and 8 FTD (17.02%) had positive Aβ2 = 27.37 p < 0.001).Out of 58 patients who underwent amyloid-PET, 35 AD (89.74%) and 4 FTD subjects (21.05%) had positive scans for amyloid deposition (χ2 = 21.23 p < 0.001).Considering the group of patients who underwent both CFS biomarkers analysis and amyloid-PET, 23 AD (100%) and 4 FTD (30.76%) were Aβ+  . CSF Aβ p = 0.034)  .1–42 was available for 10 cases: from a qualitative point of view, CSF Aβ1–42 levels were lower than 700 pg/mL in 9 cases, only slightly higher than the cut-off values. CSF Aβ42/40 ratio was available for three cases: similarly, two of them had CSF Aβ42/40 ratio values less than 0.7, slightly higher than the cut-off values. Moreover, they underwent CSF analysis more than 3 months before amyloid-PET. On the other hand, the only CSF+/PET- case underwent CSF analysis 2 months before amyloid-PET.As regards the AD CSF-/PET+ patient subgroup, CSF Aβ1–42 and Aβ42/40 was 89.55% (95% C.I. 82.33–96.88) in the whole cohort, 92.25% (95% I.C. 84.34–100) in AD patients, and 85.19% (95% C.I. 71.75–98.59) in FTD patients . When we considered the subgroup of patients who underwent both CFS biomarkers analysis and amyloid-PET, the concordance was 88.89% (95% C.I. 78.62–99.16).p = 0.065), which was more frequent in Aβ+ FTD patients.In order to assess if the discordance between amyloid biomarkers and clinical diagnosis was influenced by demographic features, we divided patients into two groups: the first including Aβ+ AD and Aβ- FTD (concordant cases), the second including Aβ- AD and Aβ+ FTD (discordant cases). There were no statistically significant differences between the two groups with respect to demographic features, MMSE, and ApoE ε4 prevalence. We also found no differences when we considered AD and FTD patients separately for this analysis. Considering Aβ+ FTD patients, we found a trend to significance for the ApoE ε4 allele , but not than CSF Aβ42/40 ratio  and CSF Aβ    , but not the Aβ42/40 ratio . We did not detect any differences in specificity between amyloid-PET and CSF Aβ biomarkers  and CSF Aβ   and CSF Aβomarkers  (Table 25–66.21)   processing, Aβ production [1–42 measures soluble forms of Aβ, and a low concentration suggests that significant parenchymal deposition has occurred, whereas amyloid imaging directly identifies fibrillar Aβ in brain tissue [Another aim of our study was to compare the concordance between the amyloid-PET and CSF Aβ biomarkers in the group of patients who underwent both the analyses. Regarding AD patients, we found a very low concordance 39%), which was different from previous works showing higher levels of concordance: Leuzy et al. reported a concordance around 60% , Jung etoduction ,62,63, aoduction . Moreoveoduction ,66. Finaoduction . On the oduction . False-noduction , amyloidoduction . Moreove9%, whichn tissue .1–42 and of the Aβ42/40 ratio (when available), slightly higher than cut-off values. These data are in accordance with previous reports, which suggested that amyloid-PET scans could have clinical utility in addition to CSF examination, where results are borderline and diagnostic uncertainty remains [Despite previous works describing a higher prevalence of CSF+/PET- discordant cases compared to CSF-/PET+ ,57,58, o remains .Nevertheless, our results showed that none of the patients with a clinical diagnosis of AD presented both negative Aβ biomarkers, in accordance with previous works showing that no subjects diagnosed with AD during life had a non-AD pathological diagnosis .In our cohort, concordance between CSF Aβ biomarkers and amyloid-PET was higher in FTD patients than in AD patients, since the two biomarkers were both negative in 70% of cases. According to a multicenter European memory clinic study , the conInterestingly, we found a trend toward significance for the ApoE ε4 allele in FTD patients with Aβ positivity. It has been proposed that the presence of an ApoE ε4 allele could increase AD co-pathology across neurodegenerative diseases .In the FTD group in which both amyloid biomarker analyses were performed, all Aβ+ cases had a clinical diagnosis of sv-PPA. It is well known that PPA is a group of neurodegenerative diseases with heterogeneous neuropathologic causes. A recent work showed that Aβ positivity was detected in 25% of sv-PPA cases and in 40% of patients with mixed PPA with overlapping linguistic features for sv-PPA and lv-PPA (s/lv-PPA) . A recenOn the basis of our current and previous data , we woul42/40 ratio is positive, underlying Aβ pathology can be reasonably suspected and amyloid-PET might be avoided. Similarly, if amyloid-PET detects cortical amyloid deposition, CSF analysis cannot be performed. In conclusion, as also reported in a recent study [If CSF analysis including Aβnt study , the mat○If CSF shows Aβ-, amyloid-PET should be performed, also considering previous results highlighting an advantage of amyloid-PET when used as a second biomarker .○42/40 ratio is suggested.If amyloid-PET is negative for cortical deposition, CSF analysis including AβIn case of a mismatch between clinical diagnosis and one amyloid biomarker, we suggest performing the other analysis, due to the low NPV of both CSF and amyloid-PET. In particular:In conclusion, if both the analyses show Aβ-, an underlying amyloid pathology could most probably be excluded, and a revision of clinical diagnosis should be considered. On the contrary, if the second analysis detects Aβ+, and matching between clinical diagnosis and biomarkers is achieved, amyloid pathology can be reasonably suspected.In case of a clinical diagnosis of AD  Figure a:If CSFIf CSF analysis or amyloid-PET shows Aβ-, an underlying amyloid pathology could most probably be excluded. Thus, the performance of the other biomarker is not required, due to the high concordance between these two analyses in FTD cases.If CSF or amyloid PET detects Aβ+, some factors should be taken into account, in particular ApoE genotyping, age, and a deep neuropsychological evaluation of language to exclude a diagnosis of PPA. Moreover, the presence of comorbidity and co-pathology should be considered, as also suggested by other recent reports .In case of a clinical diagnosis of FTD  Figure b:If CSFSeveral studies have highlighted that biomarkers strongly improved the diagnostic accuracy of clinical diagnostic criteria, and it has been suggested that their combination seems to add accuracy to clinical evaluation . However42/40 ratio was not available in all cases. Moreover, the subgroup of patients who underwent both the analyses was relatively small, making it difficult to perform multivariate analysis to correct for possible confounding factors. Furthermore, CSF and amyloid-PET were not performed at the same time. Another limitation was that FTD patients who underwent both amyloid biomarkers had amyloid-PET performed before CSF analysis, so we cannot exclude that differences in the proportion of Aβ positivity between AD and FTD were influenced by the time between the two analyses. Moreover, APOE genotyping was not available for all patients.The main limitation of our study was the lack of autopsy confirmation of Aβ pathology. We only tested the associations between amyloid biomarkers and clinical diagnosis and the concordance between CSF Aβ biomarkers and amyloid-PET, and it is possible that some subjects were clinically misdiagnosed with AD. However, we detected GRN pathogenetic mutations in two cases with nfv-PPA, so we can diagnose these patients with PPA with defined pathology . SecondlHowever, our study presents some remarkable strengths. First of all, a relatively large number of patients of our cohort underwent at least one amyloid burden biomarker. Another strength of this study is that it was based on a clinical setting cohort; therefore, it provides realistic information on the use and interpretation of amyloid biomarkers. Third, all patients were evaluated by an extensive neuropsychological battery and their clinical diagnosis was well defined.42/40, in cases of concordance between at least one amyloid biomarker and clinical diagnosis, performance of the other analysis could be avoided; on the other hand, a combination of both amyloid-PET and CSF biomarkers should be performed to better characterize unclear cases of dementia. If the two biomarkers of amyloid burden are both negative, an underlying AD pathology could most probably be excluded.In conclusion, due to the high PPV of both amyloid-PET and CSF analysis including Aβ


Granulocyte‐colony stimulating factor‐producing uterine cervical cancer is a rare aggressive disease, which may be genetically distinct from other uterine cervical cancers. Granulocyte‐colony stimulating factor‐producing uterine cervical cancer is a rare aggressive disease, which may be genetically distinct from other uterine cervical cancers. We report a case of G‐CSF‐producing UCC treated with chemoradiotherapy. Target‐capture sequencing results indicate that G‐CSF‐producing UCC is genetically distinct from other UCCs, warranting further research to establish specific treatment strategies.UCC is a common cancer that causes >311 000 deaths worldwide each year.Although UCC is a common cancer, a subset that produces G‐CSF is rare. G‐CSF‐producing UCCs are highly aggressive.218F]fluoro‐D‐glucose (FDG)‐positron emission tomography (PET) showed abnormal FDG uptake in the primary tumor  and high levels of serum C‐reactive protein . Immature granulocytes suggestive of leukemia were not detected in the peripheral blood. Serum G‐CSF was abnormally high . Based on these data, the clinical diagnosis was G‐CSF‐producing tumor.The patient had a high‐grade fever (39.1°C) with no symptoms of infection. Blood tests showed a high white blood cell (WBC) count (28.3 × 102) was administered weekly for a total of three cycles starting on day 29 after improvement of the fever and inflammatory response. The patient completed the radiotherapy regimen as planned  and intracavitary brachytherapy (ICBT). EBRT was administered as follows: (a) whole pelvic irradiation with 50 Gy in 25 fractions ; (b) prophylactic irradiation targeting PALN regions at a dose of 40 Gy in 20 fractions; and (c) boost irradiation to the enlarged lymph nodes (8 Gy in four fractions for pelvic lymph nodes and 16 Gy in eight fractions for PALNs). ICBT was delivered at a dose of 24 Gy in four fractions. Cisplatin  Figure . Serum G3/mm3) suggested that the G‐CSF‐producing property of the tumor was preserved at the metastatic sites  2 months after completion of the treatment. CT examination showed multiple metastases to a left supraclavicular lymph node, the liver, and the lung Figure . The pats Figure . The patPIK3C2B (T879N), KDR (V297I), and TET2 (P29R) were registered in the Catalogue of Somatic Mutations in CancerTo better understand the biological basis of this aggressive tumor, we analyzed mutation profiles. DNA was extracted from a pretreatment biopsy specimen in which the tumor cell content was higher than 50%. The exons of 409 cancer‐related genes Table  were seq3d is controversial because studies indicate that the sensitivity for this criterion is not high .uterine cervical cancer” AND “granulocyte colony stimulating factor”, followed by full‐manuscript examination, identified only 14 cases of G‐CSF‐producing UCC reported to date . Therefore, treatment strategies specific for this type of cancer need to be established. One candidate strategy is the use of molecular‐targeted drugs selected based on actionable mutation profiles. As a limitation of this study, we were unable to assess the expression of G‐CSF in the tumor tissue using immunohistochemistry because of insufficient sample material.4We reported a case of G‐CSF‐producing UCC that showed rapid disease progression after definitive chemoradiotherapy leading to an overall survival period of 5 months. The aggressive behavior of this tumor against conventional treatment was in line with the findings described in previous reports on G‐CSF‐producing UCCs. This study is the first report describing the somatic mutation profile of G‐CSF‐producing UCC. The data presented will expand our biological understanding of this cancer subset, which warrants further investigation.None declared.SS and KA: treated the patient and drafted the manuscript; YY: analyzed sequencing data; YT: performed pathological review; T. Oike: analyzed sequencing data and finalized the manuscript; T. Ohno: supervised the study.The enrollment of the patient was approved by the Institutional Ethical Review Committee of Gunma University Hospital . The Institutional Ethical Review Committee waived the requirement for written informed consent from the patient because of the retrospective and noninvasive design of the study. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.Tab S1Click here for additional data file.Tab S2Click here for additional data file.