Considering that there are many ways to approach animal welfare, we aimed to study the value attributed to the animals themselves in scientific papers about farm animals published in animal welfare (AW) and animal production (AP) journals over time. All the papers were analyzed by five assessors, resulting in moderate agreement because the language used in the animal welfare scientific literature is ambiguous in relation to why and for whom it is performed. The overall mean score was 5.60, showing a low consideration of the interest of animals, close to neutrality. While AW journal scored higher and improved over the decades, the AP average score was below 5.0 and did not improve with time. The statement of the main justification for animal welfare papers, with an explicit declaration of their motivational priorities is important for the improvement of animal welfare science.There are, in the literature, distinct ways to approach animal welfare. The objective of this work was to study the value attributed to farm animals in the scientific papers published in animal welfare and animal production journals at three different points in time, separated by a decade each. The first ten papers mentioning “animal welfare” or “animal well-being” in their objectives or hypotheses from each journal and each focus year were selected. The 180 papers were blindly scored by five assessors between 1 and 10, according to the degree of intrinsic value attributed to animals. The overall mean score and standard deviation were 5.60 ± 2.49, with 6.46 ± 2.29 and 4.74 ± 2.40 for AW and AP journals, respectively, and 5.37 ± 2.44, 5.68 ± 2.52 and 5.75 ± 2.41 for the focus years of 2000, 2010 and 2020, respectively. There was an interaction between focus year and publication area: papers from AW journals scored better over time, in contrast with papers from AP journals, for which scores remained similar over decades. The inter-assessor agreement is moderate, which may reflect the subject complexity, as the language used in the papers studied was ambiguous in relation to why and for whom it is performed. The low overall mean score evidenced that the animal welfare scientific publications are, on average, not prioritizing the interests of the animals. Thus, our results evidenced the presence of animal welfarism in animal welfare science, a problem that seems not to be intrinsic to animal welfare science itself, but rather to the way research is frequently conceived, conducted, interpreted, summarized and applied. Therefore, it seems urgent to further study the motivation for animal welfare research. The statement of the main justification for animal welfare papers, with an explicit declaration of the motivational priorities that constitute each scientific animal welfare study, may be an interesting recommendation for the improvement of animal welfare science. Animal Machines: The New Factory Farming Industry by Ruth Harrison (1964). The author reported the inhumane conditions in which animals were kept in intensive farming and highlighted the physical and emotional suffering experienced by these animals [Animal welfare is a multifaceted concept that has evolved over time to address concerns about the treatment of animals, including, for example, the genuine concerns with animal suffering, registered in the book Harrison 964. The  animals . Its dev animals .While animal welfare science has made important advances in improving the treatment of animals, there are critiques that it does not necessarily view animals as beings with intrinsic value . For insThus, the drive to conduct animal welfare research may be based on a wide range of motivations. Such motivations are necessarily linked to specific ethical views, with varying degrees of recognition of the intrinsic value of animals. In addition, it is not usual for authors in animal welfare science to offer an open statement regarding the motivation for each study, with the main motivation often remaining undeclared. On the other hand, science is not necessarily value-free, as the ideology and political beliefs of the scientists conducting the research shape the way research questions are framed, data is collected and analyzed, findings are interpreted, and main conclusions are built and disseminated . FurtherConsidering the variety of motivations and the distinct approaches to animal welfare according to the context and particular interests involved, the objective of this work was to study the value attributed to farm animals in the scientific papers published in animal welfare and animal production journals in three different points in time, separated by a decade each.Journal of Applied Animal Welfare Science (IF: 1.122), Animal Welfare (IF: 1.550) and Applied Animal Behaviour Science (IF: 2.187) for the AW group; and Livestock Science (IF: 1.700), Poultry Science (IF: 2.659) and Journal of Dairy Science (IF: 3.333) for the AP group, according to IF information available on 14 December 2020.We compared papers published in scientific journals organized in two groups, according to main scope, denominated animal welfare (AW) and animal production (AP) journals and publication in three different decades. To compare the journal groups, we selected three AW journals and three AP journals. To assess how animals were approached over time, we selected three publication years of reference: 2000, 2010 and 2020. Journal selection impact factor (IF) was also considered, and the requirement was an IF equal to or greater than 1.1 to ensure a balance between journal quality and the number of journals available per group. The selected journals were The dataset was composed of the first 10 papers mentioning “animal welfare” or “animal well-being” in their objectives or hypotheses, starting the search with the first issue published by each journal in each focus year and ending when 10 papers were selected. The journals were accessed starting with the focus year up to five years later, except for 2020. If, even so, it was not possible to select 10 papers, the year immediately preceding the focus year was accessed as well. The dataset was complete when 10 papers were selected for each of the six journals and three reference years, with a total of 180 papers. From AW journals, only the papers about farm animals were selected, to maintain consistency with the papers from AP journals in terms of the context of animal studies. All the data was organized in a worksheet, with columns for the paper title, journal title, year of publication, abstract, studied animal species, objective statement and conclusion. Some papers presented very long and non-explicit conclusions, so we had to select the essential information to facilitate the assessment. For the blind rating of each paper, a new coded worksheet was created, containing the paper title, abstract, objective and conclusion, and omitting the journal title. The papers, which were initially entered following a chronological publication order by journal, were randomly re-ordered. The publication year or other date markers that eventually appeared on abstracts, conclusions and objectives were replaced by four asterisks (****). This coded worksheet was then used independently by five assessors to rate each paper.Before assessing the study dataset, a short list of criteria was defined and three pilot assessments were run to refine it. The first pilot worksheet included ten papers and the following two included six papers each; none of such papers were in the study’s main database, as they were selected from different sources. For the pilot assessment results, to keep track of the coherence across scores given by the five assessors evaluating independently the same set of papers, Cohen’s kappa coefficient  was usedLin’s concordance correlation coefficient  was usedp-values adjusted for multiple comparisons, to ensure a global significance level of 5%. To evaluate the effects of the journal group and decade of publication on the score of the papers regarding their approach to animal welfare, we proceeded with regression analysis. A linear mixed regression model  was specAll analyses were conducted in the R environment for statistical computation, version 4.0.2 . The R lIn addition to the planned comparative statistical analysis, to measure how prevalent farm animal welfare papers were in each journal over time, we registered the required publication period and the number of papers checked until it was possible to reach the goal of 10 papers according to the selection criteria, for each focus year and journal.The number of consulted papers and publication period required to gather the sample of 180 papers are shown in p = 0.03) and species (p < 0.001) on the scores attributed. The fitted mean score for each publication area and the decade of publication were summarized in The statistical analysis of the main dataset showed an interaction effect between a journal’s decade of publication and publication area  and of those in the wild whose welfare is compromised by human activities. Papers on related ethical and legal issues are also considered for publication” [The different scores between AP and AW journal groups likely relate to the different main scopes of each journal group, which may function as a stronger underlying driving principle than the inclusion of animal welfare in the objective of each paper. For instance, the scope of the AP journal systems” . On the ication” . Thus, aAn increasing score average over the decades was expected in the face of the notable increase in academic production related to animal welfare science in the past decades, including a growing awareness of animal welfare issues and interest in understanding the science behind the welfare of animals in agriculture settings . Such inThe difficulty in assessing the motivations for each scientific study became evident as successive meetings among assessors with the goal of refining criteria for scoring the papers in terms of the level of consideration of the intrinsic value of the studied animals were needed. Thus, the reason for studying animal welfare was not evident in most papers studied. In addition, after three pilot analyses, the level of agreement among assessors remained only moderate, according to the kappa coefficient. The concordance among assessors remained only moderate for the assessment of the papers in our main dataset as well; however, even under such score variance, significant differences related to our research questions were observed. The lack of clear statements regarding the motivation for animal welfare studies or of an easily understandable motivational context suggests a need for improvement in scientific writing in the animal welfare field, as more clarity is preferable . The proAnimal Welfare, the number of years required decreased with the decade; this suggests that papers approaching animal welfare became more prevalent in AP journals and that papers approaching farm animals became more prevalent in AW journals. Such results seem to show a similar trend of the increasing value for farm animal welfare issues given by society in general, which agrees with the view that animal welfare is a mandated science driven by societal demands. The term mandated science describes research commissioned to guide actions, decisions and policies related to concepts such as welfare, which consist of both scientific and value-based considerations [The number of years and of papers reviewed before reaching the ten required papers per journal in each decade showed some interesting patterns. First, for most journals except erations . Such a erations .In the field of human-animal studies, speciesism is a growing area of study, and language is recognized as an important form of its manifestation ,49,50. TThe expanding investigation area on the psychology of interspecies relationships gives a conceptual theoretical framework capable to describe and explain many tendencies and biases in relation to the form that human groups with different interests understand and approach animals . This arThe difficulties in developing criteria for paper assessment, as well as the moderate concordance among assessors and the relatively high standard deviation for the overall mean score, seem to originate from the fact that the moral consideration of animals by the authors was frequently uncertain, ambiguous, or undeclared, which suggests that animal welfare science is not necessarily focused on the interest of animals, as the concept intuitively imply. This finding is reinforced by the high frequency with which motivations other than the intrinsic value of animals were explicitly stated. Animal welfare science may lose public support and, consequently, the very reason for its existence if it is not centered on animals. In fact, the validity of animal welfare science has been questioned in philosophy, through the concept of animal welfarism . This maOur methodological challenges and results warn about the complexity of the subject addressed in this study. The language used in the animal welfare scientific literature is ambiguous in relation to why and for whom it is performed. Overall, the absence of consideration of the interests of animals is surprisingly frequent for animal welfare papers, in disagreement with the scientific definition of the term animal welfare. The papers from AW journals tended to show a more animal-centered position which improves over decades; on the other hand, the animal welfare literature from AP journals approached animals in a more tangential way, often prioritizing other reasons for improving animal welfare than the animals themselves, with no improvement in the last decades. Thus, our results evidenced the presence of animal welfarism in animal welfare science, a problem that seems not to be intrinsic to animal welfare science itself, but rather to the way research is frequently conceived, conducted, interpreted, summarized and applied. Therefore, it seems urgent to further study the motivation for animal welfare research. The statement of the main justification for animal welfare papers, with an explicit declaration of the motivational priorities that constitute each scientific animal welfare study, may be an interesting recommendation for the improvement of animal welfare science.


To examine a series of papers from top ranked orthopaedic journals with respect to the number of citations over a 10-year observation period to identify factors that lead to high citation rates.The Web of Science database was consulted to identify all published papers from the first-year term of 2010 (January-May) from four top orthopaedic journals: AJSM, Arthroscopy, JBJS Am and KSSTA. The database was used to analyze and compare the papers with respect to their characteristics and citations up to 2019. Basic information for each paper was collected including the author, country, study type and average citations per year (ACY). The most (Top20%) and least (Bottom20%) frequently cited papers were identified and differences were extracted.n = 245). On average, a paper received 37.3 citations over the 10-year observation period. The most cited paper was cited 322 times. The most cited study type was randomized controlled trial (RCT) (Ø80.8). The Top20% papers were cited 37 times more often than the Bottom20%. Among the Top20%, the largest group was cohort study (n = 20) followed by case series (n = 19). Among others, the number of authors, the number of keywords and the number of references significantly correlated with the number of citations (p < 0.001).Five hundred sixteen papers were included with a total of 19,261 citations. Most of the published papers were from the United States (Factors influencing citation frequency were identified.The online version contains supplementary material available at 10.1186/s40634-023-00631-x. It is a generally accepted goal of science to publish papers of the highest possible quality . For autCurrently, bibliometrics is being widely applied as a burgeoning method in numerous medical fields , 41, 44.The aim of this study was to investigate which factors influence the citation frequency of orthopaedic papers and to determine which factors impact the citation rate. A further goal was to identify characteristics that distinguish the "top" orthopaedic papers. We hypothesized that studies with a high level of evidence such as randomized controlled trials or meta-analyses are cited most frequently.Although there are already numerous studies that list and examine the most frequently papers on a particular medical specialty, to the best of our knowledge, no study exists that elaborates on the differences between well-cited papers and poorly-cited papers over a long period of time. The results could show authors which factors can be optimized to increase the likelihood of citations after publication.We examined papers from four major orthopaedic journals with high IF: the American Journal of Sports Medicine , Arthrosocopy (IF: 5.973), The Journal of Bone and Joint Surgery American edition  and Knee Surgery, Sports Traumatology Arthroscopy  (as of 2021). All of these are listed among the best 20 in Web of Science's Journal Citation Report in the category orthopaedics [n = 516), were considered and categorized. These papers were then individually analyzed for their characteristics and their citations were determined for every year using the Web of Science database over a total period of 10 years up to and including 2019 . Data collection was carried out by one author from December 2021 to January 2022; a sample check was performed by the second author; the last author provided appropriate quality assurance by cross-checking.All consecutive papers published by the aforementioned journals during the first-year term from January 2010 to May 2010 were entered into a database. The JBJS was published twice per month and all others were published once per month. All papers, including case reports, commentaries, letters to the editor and editorials  independently ranked the titles of the papers according to their subjective informative value on a scale of 1–10, for which 1 was definted as not appealing and 10 was defined as appealing by all measures.Papers were then subclassified by authors according to study field, study type, field of research and study aim Table . The catcomparegroups and glm [n = 516) were divided into a bottom (n = 108), middle (n = 305) and top range (n = 103) using quintiles according to the respective total citation numbers in the years 2010–2019 (Table R > 0.75), good (R > 0.6), or poor to moderate (R < 0.59) [Statistical analyses were performed using the statistical software package R version 4.1.2 and the packages  and glm . As the AJSM; in the field of study type: clinical descriptive: case series; in the field of research: knee; and “Description of new injury patterns, diseases, risk factories, etc.” for the study aim , AJSM , KSSTA  and Arthroscopy . The AJSM had the highest average citation count per paper . Clinical studies were published most frequently (n = 286) Most publications originated from the USA (n = 245) All detailed results can be found in Tables Overall, 516 papers with a total of 19.261 citations were examined. The average citation per paper was 37.3 42.3 SD) and the most frequently cited paper was cited 322 times. The overall collective average citations per year (ACY) was 3.7 (5.1 SD) with 2015 being the year with the highest ACY . Otherwise, the average citation count per paper per year was nearly constant over the ten-year period after about two years after publication. The examined papers came from the JBJS Am  Regarding citation development over ten years, reviews were found to be the leading field of study Fig. .Fig. 1AvAmong the study types, RCTs had the highest citation count  Fig. .Fig. 2Avp = 0.018). In the category study aim, “Review, Description of current state of the art, Guidelines” had the highest citation count   (Table 2.4 SD (Tp = 0.047) and non-RCT , were cited more frequently than the reference case series, whereas biomechanical study  and editorials/letter/other  were cited less frequently. With regard to the subjective ranking of the titles by two sports orthopedists, interrater reliability was good with regard to the assessment of the manuscript titles. Therefore, the assessment was considered reliable. With regard to the other parameters surveyed, the number of authors (p < 0.001), the subjective evaluation of the title (p < 0.001), the title length (p = 0.029), the number of tables (p = 0.014) and the number of references (p < 0.001) significantly correlated with citation frequency; for each reference more in the paper, citations were 1.7% higher over ten years.Considering the dependence of the variables with the citation frequency, it was found that the study type has an especially strong influence on the citation frequency. Clinical analytical studies, such as RCT  was 103 49.6 SD) and the average overall citation count of the Bottom20% (7 or less citations) was 2.8 (2.3 SD), making the Top20% papers 37 times more frequently cited than the Bottom20% papers. The Top20% papers showed a largely constant citation trend after two years . Similarly, the study field was found to be a significant indicator for assessment in the Top20%, Middle60% and Bottom20% (p < 0.01). Reviews were more often among the Top20% (19.4% to 7.4% Bottom20%), whereas editorials/letters/other were found to have 38.9% in the Bottom20% and only 0.97% in the Top20%. In clinical trials, there was a clear tendency towards the Top20% instead of the Bottom20% . The knee joint was by far the most frequently addressed field of research in both the Top20% and Bottom20% groups  and could not be identified as a characteristic of either group. While most of the studies in the Top20% group focused on the study aim "Evaluation of new technique or treatments" (40.8%), the dominant study aim in the Bottom20% group was "Description of new injury pattern, disease, risk factors, etc." (37%). These showed a disproportionate distribution in favor of the respective categories.The publishing journal had a significant effect on the distribution in the Top20%, Middle60% and Bottom20% papers . Despite this fact, a comparison of the distribution in Top20%, Middle60% and Bottom20% showed that no country published a clearly higher proportion of Top20% papers in relation to the total number of papers. Details can be found in Table p < 0.001). In addition, the Top20% were significantly characterized on average by a larger authorship  or had a longer title in words on average . It was less relevant whether or not illustrations were printed in color. In the subjective ranking of titles by two sports orthopaedic surgeons, the Top20% were three rating points on average ahead of the Bottom20% (p < 0.001). Furthermore, it could be proven that Top20% papers show more tables , more Figs.  and more keywords . Detailed results can be found in Table Cohort Studies, RCTs and systematic reviews were disproportionately found in the Middle60% and Top20%, whereas case reports and editorials/letters/other were significantly more likely to be found in the Bottom20%. Biomechanical studies and case series showed no clear tendency regarding their distribution in either group. The most Top20% papers originated in the United States" . It can also be said that even after a time interval of ten years, a high IF has a positive effect on the citation numbers.In addition to the classic method of measuring the scientific influence of a paper by means of documented citations or the classification of a journal by means of an impact factor on databases such as Web of Science, alternative scores such as altmetrics are gaining influence by determining the digital attention of scientific content on the basis of clicks, comments, downloads, or mentions on social media platforms , 21, 35.Recent bibliometric analyses performed by others usually proceeded as following: the best cited papers at a defined time or period on a specific topic , 29, 40 This study is not without limitations: 1) self-citations by the authors were not excluded; 2) articles published in January 2010 have a time advantage and therefore a little more time to be quoted as compared to articles published in April 2010 and therefore may have a few more citations in 2010, however, over the ten-year period, this should only have a minimal impact; 3) the categories study field, study type, field of research and study aim are self-designed and extensive and lack universal reference standards; 4) the four journals examined were selected by consensus of the authors, so the statements in this paper do not necessarily reflect the entire field of sports orthopaedics; 5) the authors would like to emphasize once again that a high IF of a journal or a high number of citations of a publication is not the sole criterion for quality. However, in this study, a bibliometric understanding was used that articles with more citations are more likely to be considered top papers than those with few citations.more extensive authorships are advantageousauthors should aim for longer and therefore more concrete titlesspecifying numerous keywords is favorable and suggests that the study can be found more easilytables and figures are important because they are likely to make the study more understandable and descriptivea broad repertoire of references should be considered, which allows the assumption of a sound basic understanding of the topicRCTs and reviews are confirmed to be more frequently cited than other studiesbasic science has a value in orthopaedics that should not be underestimatedstudies that include an evaluation of a new technique or treatment are often cited by othersIn summary, from our study results, the following recommendations can be made for highly cited publications:The main results of this study are the statistical correlation between the number of citations with the number of authors, number of keywords and number of references, among others. In addition, we found that the Top20% papers showed a largely constant citation trend after two years for up to at least ten years. RCTs and reviews are cited most frequently.Additional file 1: Appendix and Table 2 Part 2.


In 2019, polatuzumab vedotin in combination with rituximab and bendamustine was approved by the United States Food and Drug Administration for the treatment of adult patients with diffuse large B-cell lymphoma who have received at least two prior therapies. Recent Health Authority guidance recommendations for submitting an Integrated Summary of Immunogenicity were followed including a comprehensive immunogenicity risk assessment, bioanalytical strategy, and immunogenicity data to support the registration of polatuzumab vedotin. Key components of the polatuzumab vedotin Integrated Summary of Immunogenicity and data are presented. Validated semi-homogeneous bridging enzyme-linked immunosorbent assays were used to detect anti-drug antibodies (ADA) to polatuzumab vedotin and characterize the immune response in patients with non-Hodgkin’s lymphoma. The overall incidence of ADA observed for polatuzumab vedotin was low across seven clinical trials. The low incidence of ADA is likely due to the mechanism of action of polatuzumab vedotin that involves targeting and killing of B cells, thereby limiting the development to plasma cells and ADA secretion. Furthermore, patients are co-medicated with rituximab, which also targets B cells and results in B-cell depletion. Therefore, the immunogenicity risk is considered low and not expected to impact the polatuzumab vedotin benefit/risk profile.Polatuzumab vedotin, marketed under the trade name POLIVY Diffuse large B-cell lymphoma (DLBCL) is an aggressive form of Non-Hodgkin’s lymphoma (NHL), with a median survival of less than one year if left untreated. Approximately 60% of patients may be cured with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), the current front-line standard of care . Howeverp-aminobenzyloxycarbonyl (mc-vc-PAB) (Polatuzumab vedotin is a CD79b-targeted antibody-drug conjugate (ADC) that preferentially delivers a potent anti-mitotic agent, monomethyl auristatin E (MMAE) , to B ce-vc-PAB) . The pol-vc-PAB) . Polatuz-vc-PAB) , 8–13. T-vc-PAB) , 14–16. -vc-PAB) –19.The polatuzumab vedotin clinical development included a comprehensive immunogenicity assessment and an Integrated Summary of Immunogenicity was provided to support the registration. The Integrated Summary of Immunogenicity included an immunogenicity risk assessment, bioanalytical strategy, and clinical immunogenicity assessment. The clinical assessment included anti-drug antibody (ADA) data obtained from seven clinical studies where polatuzumab vedotin was administered as a single agent or in combination with other agents to patients with relapsed or refractory B-cell NHL. In all clinical studies, polatuzumab vedotin was administered by intravenous infusion over 30 to 90 minutes. These studies have characterized single- and multiple-dose pharmacokinetics (PK) of three key analytes and the immunogenicity of polatuzumab vedotin –25, admiThe purpose of this article is to provide a comprehensive immunogenicity risk assessment of polatuzumab vedotin for human use and to summarize the immunogenicity data included in the Integrated Summary of Immunogenicity that supported the registration of polatuzumab vedotin in combination with rituximab and bendamustine.For pivotal study GO29365, serum samples for ADA assessment were collected at pre-infusion cycles 1, 2, and 4, at treatment completion or early termination, and at post-treatment visits . Sample collection frequency was reduced from the earlier DCS4968g study  based on the median cycle to onset of ADA (cycle 3).The validated immunogenicity assays to detect polatuzumab vedotin ADAs utilized a semi-homogenous bridging enzyme-linked immunosorbent assay format employing biotin- and digoxigenin-ADC reagents, as previously described . BrieflyThe specificity of the immune response for samples that screened positive for ADA was confirmed by competitive binding with polatuzumab vedotin. The validated ADA confirmatory assay method was similar to the screening assay procedure except the appropriate controls and samples were pre-incubated with 30 µg/mL of polatuzumab vedotin prior to co-incubation with the conjugated reagents.The exploratory domain specificity assay method was similar to the screening assay procedure except the appropriate controls and samples were pre-incubated with 30 µg/mL of unconjugated polatuzumab vedotin antibody prior to co-incubation with the conjugated reagents.1050 = 1.70 log titer units.The validated titration assay method was similar to the screening assay procedure except the appropriate controls and samples were diluted to minimum required dilution (MRD) of 1/50 and serially diluted two-fold prior to co-incubation with the conjugated reagents. Antibody titer values were determined using a log titer data reduction program. The minimal reportable titer of the assay was logThe validated polatuzumab vedotin NAb assay utilized a cell-based format . A sample pre-treatment procedure was required to minimize the polatuzumab vedotin interference in the assay. Specifically, polatuzumab vedotin-specific ADAs will be extracted from the samples using a modified BEAD (Bead Extraction with Acid Dissociation) method. A Burkitt lymphoma cell line (BJAB) was selected as the host cell based on its favorable reactivity to polatuzumab vedotin, and the desirable signal/noise ratio in the assay. A rabbit anti-polatuzumab vedotin polyclonal antibody enriched for anti-complementarity determining region (CDR) and anti-MMAE antibodies was chosen as the surrogate positive control. BJAB cells were incubated with polatuzumab vedotin in the presence of BEAD-processed ADA-positive serum samples for 2 days. After incubation, levels of apoptosis in sample-treated cells were measured using a caspase 3/7 activity assay. For samples carrying polatuzumab vedotin-specific NAbs, the NAbs blocked either the binding of polatuzumab vedotin to CD79b or the internalization of the polatuzumab vedotin/CD79b complexes, leading to reduced caspase 3/7 activity. A patient sample is determined to be NAb positive when it showed a caspase 3/7 activity lower than the plate-specific assay cut-point.Samples with responses at or below the assay cut-point  were considered NAb-positive, whereas samples with responses above the cut-point were considered NAb-negative.The presence of polatuzumab vedotin in the sample may interfere with the detection of NAbs in the assay. This assay was determined to detect 1000 ng/mL or 5000 ng/mL of the surrogate positive source material in the presence of either 14.6 μg/mL or 80.0 μg/mL of polatuzumab vedotin, respectively. The relative assay sensitivity was determined to be 650 ng/mL of the surrogate positive source material.®+TDM adjuvant . B cells from lymph nodes were harvested from mice demonstrating antibody activity against anti-CD79b mAb and then fused with mouse myeloma cells (PU-1) as previously described  (clone 2F2.20.1) in MPLescribed . HybridoThe NAb assay positive control source was generated by hyperimmunizing New Zealand White rabbits with polatuzumab vedotin to generate polyclonal antibodies against the complementarity determining region of polatuzumab vedotin .Pooled rabbit sera were purified by affinity chromatography , sterile-filtered (0.2 µm), and stored at -60°C in PBS. Only antibodies that specifically bind to the CDR of polatuzumab vedotin and MMAE were enriched.The studies were conducted in accordance with the principles of the Declaration of Helsinki, the International Conference on Harmonization E6 Guidelines, and the principles of Good Clinical Practice. Approval from the institutional review boards and ethics committees was obtained before study start. The studies were sponsored by Genentech, Inc. Patient consent was obtained before enrollment.Plasma levels of antibody-conjugated MMAE and unconjugated MMAE were measured using validated liquid chromatography detected by tandem mass spectrometry with and without immunoaffinity capture, respectively, as described previously –25.Serum levels of total antibody were measured using a validated sandwich enzyme linked immunosorbent assay. Diluted samples and controls (MRD 1/100) were added to microtiter plates  coated with anti-complementarity determining region antibody against polatuzumab vedotin. After a 2-hour incubation, wells were washed and an anti-framework monoclonal antibody conjugated to HRP was added for detection. TMB was added for color development. The minimum quantifiable concentration was 50 ng/mL.Three PK analytes were used to characterize polatuzumab vedotin PK , 22. IndNext, population PK analysis was performed to assess the impact of ADA on the PK of antibody-conjugated MMAE and unconjugated MMAE, using data from studies DCS4968g, GO27834, GO29044 and GO29365 (N=460). Only 12 (2.6%) of 460 patients of the analysis dataset were ADA positive. Due to this low percentage of ADA-positive patients, this factor was not formally assessed as a model covariate. Instead, the simulated exposures using individual Empirical Bayes Estimates parameters with partial covariate correction method  were obtRecent Health Authority guidelines for biologics recommend the inclusion of an Integrated Summary of Immunogenicity in marketing applications , 29. PriThe immunogenicity assessment utilized a risk-based strategy to evaluate risk factors that may influence polatuzumab vedotin immunogenicity Table 1.For ADCs, a relatively new class of therapeutic, the theoretical immunogenicity risk was initially considered higher compared to standard monoclonal antibodies (mAbs) given their hapten-like structure , 30. ThePolatuzumab vedotin immunogenicity was not assessed in animal models as the humanized mAb component does not cross-react with rat or cynomolgus monkey CD79b . TherefoIn summary, prior to the availability of clinical data, the overall immunogenicity risk for polatuzumab vedotin was initially categorized as medium.The original murine anti-CD79b antibody was humanized using standard procedures, similar to other therapeutic IgG1 mAbs –35, and The clinical trials used two different formulations of polatuzumab vedotin drug product: liquid (4 studies) and lyophilized (3 studies). As is typical for IgG1 antibodies, polatuzumab vedotin contains an N-linked glycosylation site in the Fc region of each of the two heavy chains. All glycans observed are typical of human IgG1 isotype . Some chHigh-molecular-weight forms, which were predominantly molecular dimers of polatuzumab vedotin, were present at low levels (up to 0.8% in lyophilized drug product) and were well controlled by the conjugation process and storage conditions. The extent of this aggregation, which is further controlled by limiting the storage time of the infusion solution, was not expected to contribute to product immunogenicity.Other product-related species, such as charge variants, sequence variants, subvisible particles, low-molecular-weight forms, process-related impurities, and host cell proteins, that may contribute to immunogenicity risks were controlled through a combination of testing and process control.Polatuzumab vedotin liquid drug product was filled into vials and stored as a liquid then administered intravenously and undiluted using a syringe pump. Polatuzumab vedotin lyophilized drug product was reconstituted with sterile water then injected into normal saline for administration to R/R DLBCL patients as an infusion.A minor amount of aggregation (up to 0.1%) was observed following mixing of the reconstituted drug product with normal saline and storage, which can be controlled by limiting storage time in the infusion bag. The slight amount of product aggregation was not expected to increase the risk of immunogenicity relative to administration using a syringe pump and intravenous route of administration was not expected to impact the risk of immunogenicity.Immunological tolerance in R/R DLBCL patients was not expected to impact polatuzumab vedotin immunogenicity. Next-generation sequencing revealed relatively low numbers of somatic mutations in lymphomas resulting in scant levels of neo-antigens that may limit host immunosurveillance . R/R DLBThe current manufacturing processes and physiochemical and structural properties were not expected to meaningfully impact the risk of immunogenicity. In addition, route of administration and patient and disease-related factors were not expected to negatively impact polatuzumab vedotin immunogenicity. The immunogenicity risk assessment was initially categorized as a medium risk during preclinical development, however, once clinical data were available, the immunogenicity risk was updated to low Table 1.The immunogenicity assessment strategy was consistent with current health authority guidance for biotherapeutics and industry best practices for ADCs , 28, 29.The rationale for the screening assay was to ensure that ADAs to specific domains of the ADC could be detected with sufficient sensitivity and drug tolerance. The confirmatory assay was designed to assess the specificity of the positive response. The domain specificity was developed to assess, by competitive binding with the antibody component of polatuzumab vedotin, whether the ADA responses were primarily to the antibody portion, the linker-drug regions, or neo-epitopes of the ADC. The rationale for the cell-based NAb assay was to detect the inhibition of tumor cell killing (apoptosis assay) due to the presence of NAb .The ADA screening assay minimum required dilution was determined to be 1/50. Acceptable sensitivity and selectivity data observed during development and confirmed during validation indicated that a minimum required dilution of 1/50 was appropriate. A panel of 94 serum samples from polatuzumab vedotin-naïve NHL individuals was run in the ADA screening assay to establish the threshold for ADA-positivity, or screening cut-point. To minimize the potential for false negative results, the screening cut-point was set to yield an untreated positive rate of approximately 5%. The screening assay multiplication (cut-point) factor was determined to be 1.16 times the negative control signal. In neat serum, the relative sensitivities of the polatuzumab vedotin ADA screening assay were estimated to be 60.1 ng/mL using positive control diluted in normal human serum and 1141 ng/mL using an anti-MMAE monoclonal antibody. The screening assay was optimized to tolerate drug interference at the estimated circulating drug level. In the presence of 20 µg/mL of polatuzumab vedotin, two levels of the positive control (90 and 500 ng/mL) tested positive.The screening assay cut-point factor determined in validation was used to generate a clinical baseline screen positive rate using baseline ADA samples from study GO29365. The in-study baseline screen positive rate was 10.9% (15 out of 137 patients), which falls within the variability for false positive rates for a screening cut-point factor targeting a 5% false positive rate . TherefoThe screening assay was assessed for rituximab interference and cross-reactivity since this was a co-medication. Rituximab at multiple levels  was added to multiple levels of the positive control . The samples containing 220-2000 ng/mL positive control and varying levels of rituximab screened positive for ADA, confirming that rituximab did not interfere in the assay. Similarly, the samples containing 0 ng/mL positive control and varying levels of rituximab screened negative for ADA, again confirming rituximab did not appear to interfere nor cross-react in the screening assay.There have been no published reports of circulating soluble CD79b receptors. Therefore, neither CD79b receptor interference nor cross-interference was investigated.The confirmatory cut-point was determined by analyzing a panel of 94 serum samples from polatuzumab vedotin-naïve NHL individuals in the presence of 30 µg/mL polatuzumab vedotin. The same serum panel used to determine the screening cut-point was used to generate the confirmatory cut-point. The cut-point represents the difference in assay signal between a sample with and without polatuzumab vedotin. To minimize the potential for false negative results, the confirmatory cut-point was set to give an untreated positive rate of approximately 1%. The confirmatory cut-point was a 38% reduction in the ADA signal. Samples that were confirmed positive were then further characterized as described below.10 of the sample dilution at which the obtained sample signal would equal the assay cut-point.Samples that were confirmed positive were analyzed in an assay similar to the screening assay to determine titer. Samples were diluted to the minimum required dilution (1/50) and then serially diluted two-fold. Titer values were calculated using the titer offset value  and reported as the logADCs are multi-domain biotherapeutics. As such, an immune response may be directed against any component of the ADC: antibody, linker, drug, or neo-epitope resulting from the conjugation. Samples that confirmed positive were further characterized in a qualified exploratory characterization assay to assess domain specificity , 26, 30.ADA-positive samples from confirmed ADA-positive patients were further assessed for NAb activity. A cell based NAb assay was developed and validated.The immunogenicity of polatuzumab vedotin was assessed in 7 clinical trials. ADAs against rituximab were not monitored in polatuzumab vedotin studies as the immunogenicity of rituximab in patients with NHL has historically been low , 42.For study GO29365, the baseline prevalence of ADAs was 3.7% 5/134, Table 2.ADA titers ranged from <1.7 to 2.71 Table 3.Polatuzumab vedotin total antibody concentrations corresponding to each ADA sample were determined to understand the potential for drug interference in the ADA results. Out of a total of 531 ADA samples that had measurable polatuzumab vedotin total antibody levels, all samples had levels less than 20 μg/mL, the drug tolerance of the ADA assay. Polatuzumab vedotin total antibody concentrations ranged from <0.05 μg/mL to 10.7 μg/mL with a median concentration of 1.77 μg/mL (data not shown).The emergence of ADAs to polatuzumab vedotin did not appear to impact efficacy of ongoing long-term responses despite development of ADAs (results not shown). In this study, a total of 8 patients  developed ADAs. Of the 4 DLBCL patients, 3 were treated with polatuzumab vedotin with bendamustine plus rituximab (pola+BR) and 1 with polatuzumab vedotin with bendamustine plus obinutuzumab (pola+BG). In the first case, a pola+BR patient (Patient A) developed ADAs during Cycle 2 and was assessed with progressive disease during the Cycle 3 Day 15 interim assessment. The other two pola+BR patients responded to treatment (Patients B and C). Patient B had ADAs detected during Cycle 2, completed all 6 cycles, and has an ongoing duration of response (DOR) of 38.2 months. Patient C had ADAs detected during Follow-up Month 6 visit. This patient completed 5 cycles (discontinued study treatment due to grade 1 muscle loss) and has an ongoing DOR of 21 months. The final DLBCL patient  had ADAs detected during Cycle 2, completed 6 cycles, and has an ongoing DOR of 21.1 months.Four FL patients developed ADAs against polatuzumab vedotin: 3 patients were treated with pola+BR  and 1 patient with pola+BG (Patient H). One pola+BR patient (Patient F) had ADAs detected during Cycle 2, had a partial response at Cycle 3 Day 15 interim assessment, and progressive disease at primary response assessment. The other 3 patients had ADAs detected at later time points and after completing 6 cycles: 2 at the treatment completion visit (30 days +/- 5 days after the last dose of study treatment) (Patients E and H) and one (Patient G) at the treatment completion visit. All three of these patients  have ongoing responses  at time of analysis.Safety results for study GO29365 were previously reported . Based oAggregate immunogenicity data for GO29365 and 6 supportive studies for all polatuzumab vedotin treatment groups are shown in The ADA response from patients administered either liquid drug product or lyophilized drug product was compared. The overall incidence of ADAs for patients administered with either liquid drug product or lyophilized drug product was 2.9% (13/441) and 1.1% (1/95), respectively. Refer to Three PK analytes were used to characterize polatuzumab vedotin PK, as described above. For GO29365, the individual PK parameters for antibody-conjugated MMAE, unconjugated MMAE, and total antibody based on NCA for the 8 ADA-positive patients are listed in max), but the difference was not statistically significant as indicated by 90% confidence interval of Geometric Mean Ratio containing 1. The magnitude of difference was much smaller compared to CV% of unconjugated MMAE of 44-58%. ADA-positive status does not appear to have a statistically significant or clinically meaningful impact on the PK of antibody-conjugated MMAE and unconjugated MMAE . The unconjugated MMAE exposures were numerically higher in ADA-positive patients . The antibody titers among the 8 ADA-positive patients were also relatively low (<3.0). For ADA-positive samples, the immune response was characterized by assessing various parameters Table 3.Polatuzumab vedotin demonstrates a low ADA incidence of 2.6% (14/536) across seven clinical trials. For polatuzumab vedotin, the low ADA incidence is reasonable as polatuzumab vedotin targets dividing B cells and induces apoptosis. In addition to receiving polatuzumab vedotin in combination with rituximab, also a B-cell targeted immunotherapy, the majority of patients previously received first- or second-line rituximab-based therapy, further decreasing the risk of an immune response.The other ADC presented in the Carrasco-Triguero analysis, ADC A, that was indicated for NHL had a low ADA incidence of 0.7% (1/142). A listing of 7 non-ADC immunotherapeutics approved for the treatment of NHL or chronic lymphocytic leukemia also shows low incidence of ADA Table 7.Two manufacturing processes were used to supply polatuzumab vedotin clinical trials. A liquid formulation was used to supply early clinical trials and a lyophilized formulation was used for later clinical trials and for commercialization. Manufacturing process changes may potentially influence immunogenicity depending on potential differences in the product quality attributes , 51. HowThe overall risk of generating an immune response in patients treated with polatuzumab vedotin is considered low based on a variety of considerations, including a comprehensive immunogenicity risk assessment and immunogenicity data from 536 treated patients. Multiple clinical trials demonstrated a low incidence of ADA to polatuzumab vedotin. The low incidence of ADAs is reasonable since the mechanism of action of polatuzumab vedotin is to target and kill B cells, which impacts their ability to develop into plasma cells secreting ADAs. Furthermore, patients are co-medicated with rituximab, which also targets B cells. Therefore, immunogenicity is not expected to impact the benefit/risk profile of polatuzumab vedotin.The original contributions presented in the study are included in the article/RD, VN, and SK conceptualized and implemented the assay strategy. RD, DL, GK, GM, and SK conceptualized the data analyses. RD, RB, DL, TL, GK, GM undertook data analysis and interpretation. RD, DL, and TL collected and assembled the ADA and PK data. RD, RB, DL, TL, and SK wrote the manuscript. All authors critically reviewed and edited draft versions of the paper and approved the final version.


Physical activity (PA) is safe for most pregnant women, improving both maternal fitness and birth outcomes. Despite evidence of benefits, most pregnant women eliminate or reduce PA during pregnancy. This systematic review aimed to analyze the factors affecting maternal PA during pregnancy with reference to a socio-ecological model. A systematic search of relevant published studies between 2001 and 2022 was conducted through PubMed, Scopus, Web of Science, Academic Search Ultimate, Medline, and SPORTDiscus with full text via the EBSCO platform. A total of 32 studies that met the inclusion criteria were reviewed. The findings revealed that various study designs can lead to different outcomes in terms of what is identified as a PA facilitator or barrier. The factors that positively influenced PA in pregnant women were: higher levels of education, knowledge, and skills, as well as access to mass media. Conversely, lower levels of education, lack of knowledge and skills, low income, pregnancy discomforts, limited time, safety concerns, and societal perceptions of PA in pregnancy acted as barriers. Additionally, family, colleagues/friends, and partners could either support or hinder PA. Factors affecting overall maternal PA were somewhat different from those affecting the moderate-to-vigorous intensity of PA. Pregnant women receive little organizational and policy support. There is an urgent need to provide accessible information and resource systems for pregnant women. Since most pregnant women are motivated to engage in PA and susceptible to family advice, interventions should not be limited only to pregnant women, but should involve a family member, especially partners. There is a need to take global, systemic actions to promote an active lifestyle in pregnancy. Addressing safety concerns related to PA during pregnancy should be a significant part of these promotional activities. CurrExercise is a subset of physical activity that is planned, structured, and repetitive and has as a final or an intermediate objective the improvement or maintenance of physical fitness. Exercise-related behavior is multifaceted and affected by many factors to varying degrees, which makes it complex to engage in PA . The PA 2The PA of pregnant women is affected by a variety of factors . The mul3The systematic review was conducted using “The PRISMA 2020 statement: An updated guideline for reporting systematic reviews” for the analysis material . The stuThe date of the last search was 15 September 2022. Exclusion criteria included research: (1) taking women with any disabilities or illnesses that could lower their ability in terms of bodily movement as the study population; (2) focusing on nutritional interventions or healthy eating; (3) involving a survey of parturient women; and (4) published only as an abstract, a comment, or a review due to a lack of data for extraction (but reference lists were checked for relevant studies).Data extraction: Two independent researchers  separately searched the databases and assessed the titles and abstracts of articles to determine the initial inclusions. The full texts were then assessed against the inclusion and exclusion criteria to finalize the articles eligible for inclusion in the review. Later, the two researchers independently analyzed all the articles using the extraction tables. If discrepancies were found and could not be resolved between the two researchers, a third researcher (AS) was invited to finalize the assessment.4After removing duplicates and papers irrelevant to the selected topic (judging by the abstracts), 23 papers were finally included in the analysis . Based o4.1N = 24) (N = 18) (N = 17) (N = 3) (A summary of the characteristics of the 32 papers is given in N = 24) , 51–55; (N = 18) , 55; 17 (N = 17) , 55; and (N = 3) , 33, 51.4.24.2.1N = 3 papers) in which older age had a promoting effect, while the effect of a younger age had diverse explanations. Being older appears to be associated with higher MVPA compliance (N = 3 papers). Some studies (N = 13 papers) considered high education a facilitator, and others (N = 6 papers) considered low education as a barrier. Higher education facilitated MVPA (N = 2 papers). Pregnancy discomforts were considered a barrier (N = 12 papers). In addition, authors considered high income as a facilitator (N = 3 papers) and regarded low income as a barrier (N = 6 papers). The first birth was considered a barrier (N = 3 papers), while parity was considered a facilitator (N = 5 papers) and a barrier (N = 2 papers). Regarding other factors such as ethnicity, work, marital status, and BMI, the results were not significantly different and remained controversial. There were differences in MVPA during pregnancy among different ethnic groups (N = 4 papers). Physical occupational work was related with higher amount of MVPA (N = 4 papers). The socioeconomic status (SES) was considered a barrier of PA during pregnancy (N = 2 paper).As shown in N = 2 papers), a lack of knowledge and skills as a barrier (N = 7 papers), and a lack of time as a barrier (N = 6 papers). Some authors considered previous PA as a facilitator (N = 10 papers) and previous lack of PA as a barrier (N = 3 papers). Interestingly, other authors found that previous PA was a barrier (N = 2 papers), and in two papers there was an indeterminate association (N = 2 papers) between previous PA and PA during pregnancy. There were fewer studies about smoking (N = 2 papers) and diet (N = 3 papers).There were five behavioral factors associated with PA during pregnancy, with the knowledge and skills on PA as a facilitator (N = 5 papers) and the perceived benefits of PA (N = 8 papers) were considered facilitators. The lack of perceived benefits of PA was considered a barrier (N = 4 papers). In three papers the authors mentioned specific barriers to PA . Lack of motivation/goal was considered a barrier (N = 4 papers). Safety concerns were the main barrier to PA in pregnancy, mentioned in 13 studies.There were 7 psychological factors associated with PA during pregnancy. PA attitude (4.2.2N = 4 papers) and its lack a barrier (N = 7 papers). Colleague/friend was considered a facilitator (N = 3 papers) and a barrier (N = 2 papers). Some authors identified the public as a barrier (N = 6 papers). Having children was considered a facilitator (N = 8 papers) and a barrier (N = 10 papers). Having a husband/partner was considered a facilitator (N = 9 papers) and a barrier (N = 4 papers).4.2.3N = 11 papers) and, interestingly, also as a barrier (N = 3 papers). In addition, a lack of healthcare provider support was considered a barrier (N = 9 papers).4.2.4N = 4 papers), while a lack of information was considered a barrier (N = 9 papers). Appropriate facilities was considered a facilitator (N = 5 papers), and a lack of appropriate facilities was a barrier (N = 4 papers). In one study the authors mentioned a continuous positive correlation between good access to recreation sites and MVPA throughout pregnancy. The access to mass media was considered a facilitator (N = 8 papers), and living environment was considered both a facilitator (N = 2 papers) and a barrier (N = 2 papers). There were only a few studies on neighborhood safety (N = 4 papers) and the public policy level (N = 3 papers).5N = 16 papers), and the sample for the qualitative evaluation is relatively small (N = 7 papers). It was found that different research types might lead to different results, while the combination of qualitative and quantitative types (N = 9 papers) might lead to a more accurate investigation of influencing factors. In the analyzed material, all studies involved the personal level, and very few concerned the policy level. Through literature analysis, it was also found that there were some differences in the factors affecting overall PA and MVPA during pregnancy, which need to be further verified. It must be underlined that one of the main barrier to PA during pregnancy were the safety concerns.This systematic review aims to examine existing studies on factors affecting PA in pregnancy, referring to the SEM developed by McLeroy et al. . We wantPersonal factors included socio-demographic and biological, behavioral, and psychological factors. Age was not considered to be associated with PA in pregnant women , 54, 55 Opinions vary widely on the impact of pre-pregnancy PA on PA during pregnancy, with one survey finding that previously active participants expressed that they did not continue their active lifestyle during pregnancy . It was Analysis of the interpersonal level, which included family, colleagues/ friends, and husband revealed that these factors could both promote and be a barrier to PA in pregnant women. A commonly reported facilitators were “social influences,” which included encouragement of PA by family and friends. Women’s partners or husbands were the most influential factor, and women enjoyed meeting other pregnant women and expressed interest in PA classes tailored to pregnancy . Having Third, at the organizational level, our analysis focused on the role of health care providers, and the results were consistent. The health care providers were the facilitating factor for engagement in PA during pregnancy , 45, 53.At the community level, both information and resources were considered a facilitator , 44, 53,Finally, in relation to the public policy level, there were only a few studies on PA policies for pregnant women, and policies were not a major concern for pregnant women . HoweverPregnant women face more obstacles to their PA compared to non-pregnant populations. As a result, they cannot comply with PA recommendations, unless these obstacles are overcome . The lacOur literature review is of high practical value. In particular, due to the fact that there is a very low level of PA during pregnancy in different populations worldwide . For exaThis review study has its limitations. First, one criterion for inclusion in the analysis is the publication of papers in English. There may be other studies on maternal PA influencing factors available in other languages. Second, since much of the literature is qualitative, relevant factors are not directly reflected in the results and conclusions, which made potential bias in our review analysis. Third, there are other types of socio-ecological models or theories . Althoug6Through this systematic review, it was found that SEM can provide a wide-ranging overview of factors that influence PA in pregnant women. Nevertheless, a more comprehensive system of factors revision is needed, where a more accurate approach combining qualitative and quantitative methods will be used. The factors that positively influenced PA in pregnant women were: higher levels of education, knowledge and skills, as well as access to mass media. Conversely, lower levels of education, lack of knowledge and skills, low income, pregnancy discomforts, limited time, safety concerns, and societal perceptions acted as barriers. Additionally, family, colleagues/friends, and partners could either support or hinder PA. Factors affecting maternal overall PA are somewhat different from those affecting MVPA.Safety concerns are the main barriers to PA in pregnant women. Therefore, the solutions addressing safety concerns should be a significant issue in promoting maternal PA. What is more, pregnant women receive little organizational and policy support and are exposed to a lack of external drivers to be physically active. There is an urgent need to provide accessible information and resource systems for pregnant women. Since most pregnant women are motivated to engage in PA and susceptible to family advice, interventions should not be limited only to pregnant women, but should involve a family member, especially partners. For pregnant women themselves, physical activity or exercise prescriptions tailored individually by appropriate specialists or trained clinicians may be the most effective means to help them meet PA guidelines, all of which need to be supported by government policies.JS and AS contributed to the conception and design of the study. JS, AS, MP, and AW performed data collection and analysis. JS wrote the first draft of the manuscript. AS and MP screened, reviewed, and revised the manuscript. All authors contributed to the article and approved the submitted version.


Candida infections. Oropharyngeal microbes are believed to play an important etiologic role. This study was undertaken to determine whether next-generation sequencing (NGS) can be used to simultaneously analyze bacterial and fungal communities. Buccal samples were collected from intubated ICU patients. Primers targeting the V1-V2 region of bacterial 16S rRNA and the internal transcribed spacer 2 (ITS2) region of fungal 18S rRNA were used. V1-V2, ITS2, or mixed V1-V2/ITS2 primers were used to prepare an NGS library. Bacterial and fungal relative abundances were comparable for V1-V2, ITS2, or mixed V1-V2/ITS2 primers, respectively. A standard microbial community was used to adjust the relative abundances to theoretical abundance, and NGS and RT-PCR-adjusted relative abundances showed a high correlation. Using mixed V1-V2/ITS2 primers, bacterial and fungal abundances were simultaneously determined. The constructed microbiome network revealed novel interkingdom and intrakingdom interactions, and the simultaneous detection of bacterial and fungal communities using mixed V1-V2/ITS2 primers enabled analysis across two kingdoms. This study provides a novel approach to simultaneously determining bacterial and fungal communities using mixed V1-V2/ITS2 primers.Intubated patients in intensive care units (ICUs) too frequently contract ventilator-associated pneumonia or  Pseudomonas aeruginosa, Enterobacteriacea, Acinetobacter, S. aureus, and Streptococcus spp. are markedly elevated in VAP patients  samples. Overall, a significant correlation was observed between V1-V2 and mixed V1-V2/ITS2 primers  and real-time PCR (RT-PCR). Bacterial B and C. albicans C abundanChao1, Faith PD, and Shannon indices were used to evaluate alpha diversities depending on the primers. Similar Chao1 and Shannon index was observed between V1-V2 and bacterial reads from mixed V1-V2/ITS2 primers and between ITS2 and fungal reads from mixed V1-V2/ITS2 primers A,B. HoweC. albicans and Clavispora lusitaniae were 1.46% ± 1.26% (max: 4.05%) and 0.33% ± 1.25% (max: 6.05%), respectively.Next, bacterial and fungal relative abundances determined using the mixed V1-V2/ITS2 primers were simultaneously plotted for individual samples A. In intC. albicans showed positive correlations with several bacteria, including Mycoplasma salivarium, Fusobacterium nucleatum subsp. Vincentii, and Desulfomicrobium orale. In addition, C. lustitaniae formed a dense network with oral bacteria and its abundances were positively correlated with those of Treponema socranskii, Staphylococcus aureus, Saccharibacteria (TM7) spp., and Streptococcus anginosus and negatively correlated with Rothia mucilaginosa, Haemophilus parainfluenzae, Lautropia mirabilis, Streptococcus sanguinis, and Neisseria oralis.Finally, a microbiome network was constructed and showed highly dense inter- and intrakingdom interactions B. IntereThe human oral microbiome is a most diverse ecosystem, comprising numerous communities . In thisThe use of high-throughput sequencing for 16S/ITS targeted amplicon high-throughput sequencing and metagenomics has allowed for a closer examination of the bacterial aspect of the human microbiome to better comprehend its function in health and disease ,33. The Total read counts obtained using mixed V1-V2/ITS2 primers were significantly higher than those obtained using V1-V2 or ITS2 primers (Table). Since all samples were run simultaneously and equal concentrations of libraries were applied to chips, it is not clear why the V1-V2/ITS2 primer produced significantly higher read counts. Furthermore, the input to nonchimeric read count percentage was significantly higher for the ITS2 primer than the V1-V2 or V1-V2/ITS2 primers. Since all the samples were worked up using the same PCR conditions, PCR cycles are probably not responsible for these differences. On the other hand, primer specificities or target DNA abundances within samples might explain these differences .After preprocessing, the relative abundances of bacterial and fungal communities were compared to determine whether mixing primers influenced results . When reC. albicans, the copy number of 18S rRNA is 55 [C. albicans abundances showed significant correlations, which indicated the adjustment optimally predicted bacterial and fungal abundances in clinical samples.Next, a microbial DNA standard was used to test the ability of the V1-V2/ITS2 primer mix to simultaneously predict the abundance of bacterial and fungal species . When weNA is 55 , which sFinally, we plotted bacterial and fungal relative abundances obtained using mixed V1-V2/ITS2 primers. When alpha diversity was determined, a similar Chao1 and Shannon index was observed between V1-V2 and bacterial reads from mixed V1-V2/ITS2 primers and between ITS2 and fungal reads from mixed V1-V2/ITS2 primers A,B. ThesC. albicans and Clavispora lusitaniae were determinable within bacterial populations  spp., and Streptococcus anginosus and negatively correlated with those of Rothia mucilaginosa, Haemophilus parainfluenzae, Lautropia mirabilis, Streptococcus sanguinis, and Neisseria oralis. In a previous study, Rothia, Haemophilus, Lautropia, and Neisseria were more frequently found in healthy individuals, whereas Treponema, Staphylococcus, and Saccharibacteria (TM7) were more frequent under disease conditions, such as periodontitis [A microbiome network was constructed to visualize bacterial and fungal interactions and revealed strong inter- and intrakingdom interactions B. Intere. mutans ,42, whicsubjects , and S. patients . Thus, odontitis . The bioThis study is limited by a relatively small sample size caused by the requirement for family consent for the collection of oral samples from ICU patients. Additionally, only elderly patients were included, which could have introduced bias due to the limited number of participants. Therefore, we recommend a larger, well-designed study be undertaken to validate our findings in the future.C. lustitaniae and C. albicans, among a few other taxa, as potential keystone taxa of the oral microbiome of ICU patients. Overall, our study has facilitated an understanding of the determining factors and cross-kingdom interactions of the human oral microbiome. These findings will lead to the development of more effective approaches for diagnosing complex clinical infections. In the bacterial and fungal microbiome analysis, data regarding technical issues related to primer development are still lacking. In this study, we demonstrated that analyzing bacterial and fungal communities simultaneously in human oral samples can reduce analysis costs and enable the identification of infectious causes across two kingdoms. By performing network analysis, we suggested that bacterial–fungal interactions are present in the oral microbiome. Moreover, we identified


Bacillus thuringiensis strain RC340, isolated from an environmental microbiology experiment soil sample is presented here. B. thuringiensis strain RC340 sequenced by GridION consists of a single genome consisting of 5.86 million bases, 8,152 predicted genes, and 0.23% contamination.The complete genome sequence of  B. thuringiensis has several mechanisms to gain a competitive advantage including bacteriocin production and antimicrobial resistance (AMR) proteins  course at the University of Massachusetts Amherst . All sofr v2.9.2  producinr v2.9.2 . The drar v2.9.2  and polir v2.9.2  to generr v2.9.2 , QUAST vr v2.9.2 , and Cher v2.9.2  determinr v2.9.2  . The ANI estimate was 95.865% which classifies RC340 genome as the same species.The taxonomy for RC340 from Classify Microbes with GTDB-Tk v1.7.0  is DomaiI v0.1.3  was utilBt strain RC340 bacitracin resistance genes may provide a competitive advantage in the battle for nutrients in nutrient-depleted soils due to climate change stress (To assess the presence of bacitracin resistance genes in the RC340 genome and the nine closest related neighbors, we used Compare Genomes from Pangenomes v0.0.7 . The pane stress , 24.


The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined. The UBASH3 gene/protein family was defined through the independent effort of several research groups approximately 20 years ago. The first report on a UBASH3 family member published in 2001 was focused on characterization of human UBASH3A, a novel gene, whose possible involvement in the Down syndrome-related autosomal recessive deafness was ruled out in this study ,5. Thus,The genes and proteins of this family are annotated in databases as UBASH3A and UBASH3B due to the presence of ubiquitin-associated (UBA) and Src-homology 3 (SH3) domains in their structure. Several synonyms have been introduced to denote them in the original reports, and these synonyms are frequently used. UBASH3A has been termed STS-2 for Suppressor of T-cell Signaling, TULA (or TULA-1) for T-cell Ubiquitin Ligand, and CLIP4 for Cbl-Interacting Protein 4. UBASH3B has been termed STS-1, TULA-2, and p70. In this publication, the terms TULA-1 and TULA-2 are always used, regardless of the term used in the original papers, solely for the sake of consistency with our other publications and throughout this review.The structure of TULA-family proteins exhibits a unique combination of functional domains, which includes UBA and SH3 interactive domains and the histidine phosphatase domain . The UBAC. elegans and Drosophila [The C-terminal region of TULA proteins was initially recognized as an area of homology (i) between the TULA orthologues from human, osophila  and (ii)osophila . Subsequosophila , which posophila ,19,20,21osophila .In the TULA family, the HP domain exhibits the ability to hydrolyze multiple low-molecular weight phosphatase substrates, phosphotyrosine (pY)-containing peptides, and pY-containing proteins ,20,23,24The C-terminal-most region of TULA proteins mediates their dimerization, which has been established using size-exclusion chromatography , crystalThe latest addition to this family’s set of functional domains is a 2H phosphoesterase domain, termed this way due to the presence of two histidine residues that are essential for its phosphodiesterase activity . The 2H Functional roles of TULA domains have been analyzed in multiple and diverse experimental systems. Briefly, the HP domain and its role have been studied very thoroughly. It has been shown that HP is essential for the functions of the TULA family. Mutations inactivating its PTP activity impair the ability of TULA-2 to suppress signaling and cell activation mediated by the Syk-family of protein tyrosine kinases (PTK), which in T cells, where the original studies have been conducted, is represented by ZAP-70 ,20,21,31The two TULA-family members demonstrate a substantial degree of the overall amino acid sequence similarity , which is only slightly lower than that in such two-member families as the Syk/ZAP-70 PTKs or the SHP-1/SHP-2 PTPs . Expectedly, inside the functional domains, sequence similarity between TULA-1 and TULA-2 is even greater see .Despite the structural similarities, differences between the two family members are also apparent. The initial studies demonstrated that TULA-2 is ubiquitously expressed in various cells and tissues . It was Despite the initial negative results, expression of TULA-1 was later demonstrated in myeloid cells which mediate innate immune responses: monocytes/macrophages, neutrophils, and dendritic cells . This isAnother noted difference between the two family members is related to their specific PTP activity. A high degree of sequence similarity between TULA-family members, especially inside their HP domain see , consistIn agreement with these findings, a library screening using peptides containing a central pY residue surrounded by random flanking sequences easily yielded multiple pY-peptide substrates of TULA-2 (see below for details), although no pY-peptide was identified to be dephosphorylated by TULA-1 . FurtherDespite a significant disparity in PTP activity between the two family members, their key cellular functions overlap. Indeed, a very strong up-regulatory effect of the TULA-family double knockout (dKO) on TCR-mediated signaling, proliferation, and cytokine secretion in T-cell culture, as well as an exacerbation of severity of experimental autoimmune encephalomyelitis in vivo, was demonstrated in the seminal study characterizing dKO mice ; a stronIt should be noted that, although the effects of TULA-1 and TULA-2 deficiency in dKO on various immune responses in vivo and in vitro are clearly synergistic ,19,36, tConsistent with the results outlined above, not only dKO, but both TULA-1 and TULA-2 sKO mice demonstrated the improved resistance to systemic Candida infection . The hosIt should be noted that TULA-1 also exerts regulatory effects that appear to be mediated by its interactions with the NF-κB activation pathway in T cells ,42. ThisOverall, immunity, especially immunity dependent on T-cell responses, is a major biological phenomenon regulated by TULA-family proteins. However, immunity is not the only major biological function influenced by the TULA family. Platelets have been shown to express TULA-2 at a level substantially exceeding that in other blood cells , in agreConsistent with these results, TULA-2 sKO and TULA-family dKO exerted equally strong enhancing effects on platelet signaling and activation, while TULA-1 sKO platelets demonstrated responses indistinguishable from those of WT platelets . The lacIt should be noted that platelets, being a critical element of hemostasis and thrombosis, are also involved in immune responses ,55,56. IThe regulatory role of TULA-2 in the bone-resorbing function of osteoclasts, while less extensively studied, clearly resembles that observed in immune cells and platelets. It has been shown that bone volume is decreased in mice lacking TULA-2 due to an increase in both the number of osteoclasts and their activity. This biological effect correlates clearly with a negative effect of TULA-2 on ITAM-mediated signaling required for osteoclast development and function. Consistent with this effect of TULA-2, expression of a PTP-defective form of TULA-2 increased osteoclast activity .(i)TCR-induced phosphorylation of ZAP-70, indicative of the activation of this PTK, is significantly enhanced in T cells which lack TULA proteins. This effect is much stronger in dKO T cells ,19,24,36(ii)Phosphorylation and activation of Syk, as well as Syk-dependent signaling induced by various ITAM-bearing receptors, is significantly enhanced in the cells lacking both TULA proteins or TULA-2 alone ,21,35 or(iii)Screening of libraries containing peptides with a pY residue surrounded by random flanking sequences yielded multiple pY-peptide substrates of TULA-2 and revealed the structural specificity determinants of this PTP ,35 Figu. Based o(iv)WT TULA-2 and its substrate-trapping mutant form bind to Syk and ZAP-70 in a strong and selective fashion in cell lysates from platelets ,49, T lyIn immune cells and platelets, where the role of the TULA family has been examined extensively, the molecular basis of the observed effects is thought to be related to the ITAM-/hemITAM-mediated signaling dependent on ZAP-70 and Syk PTKs, which form a two-member Syk family , as well as the corresponding sites in ZAP-70, are critically involved in the regulation of activity and functions of these PTKs ,76,77,78An additional possible element of the regulatory mechanism by which TULA-2 modulates Syk activity is the Syk pY317 site, clearly exhibiting positive substrate specificity determinants see , and whoIn addition to well-characterized PTP-mediated effects, TULA-family members exert some biologically significant effects that are mediated by their interactive domains and may likely be independent of PTP activity. For example, both TULA proteins have been shown to reduce production of HIV-1 by inhibiting the late steps of its life cycle in a UBA-dependent fashion. This effect appears to be mediated by binding of TULA proteins to ABCE-1, a host factor of HIV-1 assembly . Also, TFurthermore, TULA-2 has been shown to form a complex with the mitotic kinesin-like protein 2 and Aurora B, a protein kinase that regulates chromosomal segregation. The recruitment of Aurora B, which appears to be mediated by the interactions between its ubiquitin residues and TULA-2 UBA domain, induces anaphase . The welFindings suggesting that TULA-family proteins play a key role in the biological processes that take place outside the immune/hematological compartment and might be unrelated to ITAM-mediated signaling continue accumulating. Thus, TULA-1 has been implicated in the regulation of human kidney development; in this process, TULA-1 may interact with AIF-related proteins . AnotherThese findings suggest that both family members exert some PTP-unrelated effects, but this ability may be especially important for TULA-1. Although TULA-1 appears to be capable of acting through dephosphorylation of ZAP-70 ,36, a drThe effect of TULA-1 in type-1 diabetes may be mediated by down-regulation of NF-κB, linked to the interactions of TULA-1 SH3 with NEMO and TAK1, key elements of NF-κB signaling in T cells, which are known to be poly-ubiquitylated, as well as to the interactions of TULA-1 UBA and poly-ubiquitin chains . The dowConsidering strong effects of TULA proteins on key biological and pathological processes, their targeting appears to represent a promising therapeutic tool. One may speculate that TULA-1 can be targeted to treat autoimmunity and/or fight infections, while TULA-2 can be targeted to treat hemostasis-/thrombosis-related conditions. However, this area is not extensively studied, and some key issues remain unclear. Thus, the specificity of potential targeting effects is not obvious, because both family members are involved in the regulation of immune responses ,37,100, Several TULA-2 inhibitors and even their classes have been identified, but none of them are ready for the immediate therapeutic use, due to insufficient potency and/or specificity ,101,102,Together, the findings discussed above strongly suggest that the TULA-family proteins play a key regulatory role in many disparate biological processes. Although most of the discussed studies focus on T lymphocytes and, more recently, on immune cells of myeloid nature and platelets, this emphasis may, at least in part, be due to the circumstances of the initial characterization of these proteins and the interests of the researchers involved. The role of the TULA family in biological regulation may be substantially broader than our current knowledge suggests. Several other questions still remain to be answered despite the extensive studies of the TULA family conducted over the last two decades. Thus, the reason for employing two family members in signaling regulation, which is especially evident in T lymphocytes, is not obvious, since TULA-2 alone can regulate receptor signaling in other cell types. Can this be explained by differences in the regulatory mechanisms of the individual Syk-family members, thus requiring different sets of TULA-family regulators? It also remains enigmatic how the overlapping functions of the two TULA-family members in T cells relate to the significant difference in their PTP activity. It is especially puzzling, considering that a low-activity member appears to be involved in a large panel of very diverse autoimmune conditions, while only a single autoimmune disorder has been found to be linked to a high-activity member.One possible independent approach that may facilitate further understanding of this family and its biological functions rests in inquiring into the issues related to its origin. This approach may help us to evaluate this family’s ‘reason for being’ by conducting the comparative analysis of TULA proteins in diverse species and taxa. A close look at the TULA family tree reveals the existence of a TULA-like protein-encoding gene in almost all the reviewed invertebrate taxa. These taxa are extremely diverse; hence, it appears that the presence of a TULA-like protein is typical for invertebrates .There is one notable exception to this rule. Most TULA-homologous sequences annotated in roundworm genomes lack an extensive N-terminal portion characteristic of all other TULA-type vertebrate and invertebrate genes. Several annotated roundworm genes possess an N-terminal part, but it is quite dissimilar to the TULA genes of all other species examined. Furthermore, the key histidine residue of mammalian TULA-family PTPs (His-380 of mouse TULA-2), which is conserved in their vertebrate and invertebrate homologues, is not fully conserved in roundworms. However, it cannot be ruled out that the observed anomalies in the annotated roundworm sequences are due to the failure of the prediction algorithms in this case.The origin of the TULA family is clearly associated with the origin of vertebrates; all vertebrates have genes unambiguously recognized as encoding either TULA-1 (UBASH3A) or TULA-2 (UBASH3B). Such diverse vertebrate taxa as mammals and other tetrapods, lobe-finned fish , and cartilaginous fish (sharks) possess both genes, thus exhibiting a conserved family structure. These findings are consistent with a duplication of the TULA-family ancestral gene at the root of the vertebrate tree, likely as a result of whole-genome duplication .Lepisosteus oculatus of the ancient Holostei class has a single TULA-2 gene. The sterlet Acipenser ruthenus (class Chondrostei) has two genes which encode TULA-2-related proteins, but their protein products are different by a single amino acid, suggesting a gene duplication that has not been followed by differentiation yet.Whereas jawless vertebrates, such as lamprey, have a single family member, it is substantially dissimilar to the invertebrate TULA-like protein and clearly belongs to the TULA-2 branch of the family, suggesting that, following a duplication of the invertebrate gene, one family member has been lost in this lineage. A scenario similar to lampreys is also apparent for jawed vertebrates; while both cartilaginous fish, which are considered to be a taxon closest to the vertebrate ancestral form, and lobe-finned fish, which represent a sister group of tetrapods, have a two-member TULA family similar to that in mammals, no species among the examined ray-finned fish has a TULA-1-encoding gene, arguing that TULA-1 has been lost at the root of the ray-finned fish tree . ConsistDanio rerio and Xiphophorus maculatus, two common fish experimental models, respectively; these differences are very close to those between human and shark TULA-2 (75–77% identity). Furthermore, the salmonids also have TULA-2a’, a TULA-2a-like gene, arguing that an additional duplication occurred in this branch , whereas TULA-1 is significantly less conserved . The presence of TULA-family members in all vertebrates, the highly conserved nature of this family, and, in particular, the highly conserved sequence of TULA-2 within vertebrates is consistent with the idea of critical involvement of this family in the central biological functions of vertebrates that distinguish them from invertebrates.Bombyx mori [Daphnia magna has been shown using RNAi-depletion of EPP message in the developing egg of this crustacean; its development is blocked by an EPP-targeting RNAi [Whereas vertebrate TULA-family proteins have been studied extensively in human and mouse systems, little is known about invertebrate TULA-like proteins. Some arthropod TULA-like proteins have been characterized as ecdysteroid-phosphate phosphatases (EPPs) ,107, whibyx mori . Furthering RNAi .However, it is unclear whether ecdysteroid-phosphates represent major biologically relevant substrates of the EPPs, since mammalian TULA-2 can also dephosphorylate ecdysteroid-phosphates, while EPP can dephosphorylate pY-peptides and pY-proteins ,107. FurWhile invertebrate TULA-like proteins cannot be identified exactly as either TULA-1 or TULA-2, representing a lineage clearly separate from both vertebrate family members see , sequencThe conserved nature of invertebrate TULA-like protein sequences suggests that these proteins are conserved functionally as well. Considering that the differences in anatomy and physiology between invertebrate taxa are very dramatic, it is reasonable to hypothesize that the common regulatory targets of TULA-like proteins play a role in the fundamental biochemical processes which are universal for all metazoans.Whereas invertebrate species have a single gene encoding for a TULA-like protein, the two-member TULA family is typical for vertebrates see . Thus, tThe effect of the TULA family on cells which mediate innate immune responses has also been shown ,41,100, Another key regulatory function of the TULA family in vertebrates has been demonstrated in platelets, whose major role is to constantly survey the integrity of the blood circulatory system and to mediate hemostatic responses by forming an aggregate, which seals off a damaged area in a blood vessel ,35,49 , and Holostei (gars).  has two TULA-2-type genes, its protein products differ by a single amino acid substitution, which is conserved, thus rendering the difference between protein family members in this group negligible.) Both fish and lampreys possess the closed circulation system equipped with thrombocytes, which is characteristic of all vertebrates ,122,123.Of further interest is the unique TULA-family structure in teleosts; despite the apparent adaptation of lampreys and ancient fish taxa to the loss of TULA-1, a new form of this family is restored in teleosts as an apparent result of UBASH3B duplications. Most teleosts have two members of this unique family—TULA-2a and TULA-2b, while salmonids have three—TULA-2a, TULA-2a’, and TULA-2b. The difference between TULA-2a and TULA-2b within species exceeds that between TULA-2 of some very distant species, but is still less extensive than the difference between TULA-1 and TULA-2 see  above. TMost TULA-family functions in both lymphocytes and platelets that have been studied in detail are critically dependent on ITAM-/hemITAM-mediated signaling through ZAP-70 and Syk, which are strongly regulated by TULA-family proteins, especially TULA-2 (see above). Therefore, a close look at a possible link between Syk-family PTKs and TULA-family PTPs in various taxa is warranted. The vertebrate Syk family is represented by Syk and ZAP-70 genes; both PTKs bind to phosphorylated ITAM-bearing receptors and play a key role in propagating signals from such receptors. However, ZAP-70 is expressed in T and NK cells, while Syk is expressed more ubiquitously; hence, ZAP-70 is an essential PTK of T-cell signaling, while Syk plays this role in B cells, platelets, and many myeloid cells ,131,132.In contrast to vertebrates, invertebrates have only a single Syk-like gene. In addition to this gene, the HTK16 gene , which possesses extensive regions of Syk homology but also an ankyrin-repeat region, is present in invertebrates and is unique to them, being dissimilar to any vertebrate PTK. These observations may lead to the hypothesis that the invertebrate PTKs homologous to vertebrate Syk and ZAP-70 are targeted by the invertebrate TULA-like PTPs. However, the major TULA-2 substrate site of mammalian Syk-family PTKs homologous to mouse Syk pY346 is not conserved in either invertebrate Syk or SHARK. Furthermore, not all invertebrate Syk-homologous PTKs possess tyrosine residues in the interdomain B region, which are targeted by TULA-2 in mammalian Syk-family PTKs. Therefore, if invertebrate TULA-like proteins act indeed as PTPs, their targets are likely to be different from those of mammalian TULA-2.The current considerable knowledge of the TULA family in mammals and a few studies of arthropod TULA-like proteins, together with the genome sequencing data on TULA in various taxa, encourage us to address several key issues related to the biological role of this family by using comparative analysis of TULA protein functions in various distinct metazoan taxa. Using this approach, we should be able to answer the question of whether the regulation of immunity and hemostasis is indeed a common function in all vertebrate taxa, as was hypothesized. Perhaps, comparison of dissimilar vertebrate taxa will allow us to identify the role(s) of TULA-family proteins outside of immunity or hemostasis.We may also ask whether the functions of this family are related to the regulation of Syk-family PTKs in all vertebrate taxa. What are the other dephosphorylation targets at a molecular level? Are these putative non-Syk targets common for all vertebrate taxa or are they taxon-specific? This approach would complement any systematic unbiased substrate screening conducted in mammalian systems.Are the PTP-independent functions of TULA proteins, including an adaptor function, common or specific for various taxa? Do other regulatory systems, such as NF-κB signaling, play a role in the common vertebrate effects of TULA proteins? The comparative analysis of TULA-family functions in various groups of vertebrates is also expected to help us in determining the specific contributions of PTP-dependent and -independent effects of TULA proteins to the overall biological role of this family.Does the regulation of immunity and/or hemostasis represent a major TULA function in the vertebrate groups demonstrating deviations from the classical conserved family structure? What is the mechanism by which a single family member mediates the regulatory functions in such groups? Are such differences in TULA functions, if observed, underlined by substantial differences of T cell and/or thrombocytes between mammals and ‘single-member taxa’, or do these taxa demonstrate additional molecular mechanisms, substituting for the absence of TULA-1? Finding these mechanisms would considerably advance our understanding of the TULA-1 function.What is the extent of TULA-family members’ differentiation in teleosts? How distinct are individual members of the unique teleost TULA family in the regulation of immunity and hemostasis? Does a pair of TULA-2-derived genes  function as the TULA-2/TULA-1 pair in mammals, or does it substitute for TULA-2 only? Comparison of this unique family to the typical one is likely to reveal the potential as well as the limits of TULA-family member differentiation.What are the functions of invertebrate TULA-like proteins? Do they act as PTPs by dephosphorylating regulatory targets? Are these functions related to invertebrate Syk-like PTKs? If so, what is the TULA-like PTPs’ target at the level of specific pY-sites? If not, what are the invertebrate substrates of TULA-like PTPs? Are these targets common for all invertebrates or specific in different taxa? Do the invertebrate systems/functions regulated by TULA-like proteins and the vertebrate systems regulated by the TULA family appear to be connected by some evolutionary links, or are these systems totally distinct and unrelated?Addressing these issues by analyzing diverse metazoan taxa should be very beneficial to our understanding of the functions of TULA-family proteins in mammals, a vertebrate group naturally attracting our utmost attention. Perhaps by taking a look at the broader picture, we will better understand the TULA family ‘reason for being’, and this, in turn, will assist us in fine-tuning our research program. It should be noted that this approach has received very limited attention up to date and, thus, requires a considerable amount of work focused on organisms never analyzed before in connection to the functions of TULA proteins.