Inflammatory Bowel Disease (IBD) comprises a group of disorders, in particular Crohn’s disease (CD) and ulcerative colitis (UC), characterized by chronic inflammation affecting the gastrointestinal tract. The treatment of these conditions is primarily based on anti-inflammatory drugs, although the use of biological drugs with lower side effects quickly increased in the last decade. However, the presence of certain polymorphisms in the population may determine a different outcome in response to therapy, reflecting the heterogeneity of the efficacy in patients. Considering that several studies showed important correlations between genetic polymorphisms and response to biological treatments in IBD patients, this systematic review aims to summarize the pharmacogenetics of biologicals approved for IBD, thus highlighting a possible association between some polymorphisms and drug response. With this purpose, we reviewed PubMed papers published over the past 21 years (2000–2021), using as the search term “drug name and IBD or CD or UC and polymorphisms” to underline the role of pharmacogenetic tests in approaching the disease with a targeted therapy. Inflammatory Bowel Diseases (IBDs) are a group of inflammatory disorders of the gastrointestinal tract, with Crohn’s disease (CD) and ulcerative colitis (UC) being the most widely represented types. IBD can begin at any age and regardless of sex, but the most important peak of onset is concentrated between 15 and 45 years, although a second peak can be found at a later, elderly age. These are severely invalidating conditions, and the incidence is progressively increasing, enough to be considered a global healthcare problem ,3.IBDs are diseases of unknown cause arising spontaneously, although an aberrant dysregulation of the immune response toward commensal and nonpathogenic antigens normally found in the gut is the prevailing pathogenic hypothesis . This miUp to date, there is no definitive therapy for IBDs and, generally, the severity of the disease drives towards the most appropriate therapeutic approach. The main treatment options aim to reduce intestinal inflammation, to relieve symptoms, to keep the disease in remission and prevent its acute exacerbations. Indeed, pharmacological therapy for these disorders involves aminosalicylates, corticosteroids, and immunosuppressants . HoweverTargeting TNF-α reflects its importance in mediating the T-lymphocyte-driven immune response, and the use of monoclonal antibodies to neutralize TNF-α interfere with the inflammatory response and decrease the frequency of flare-ups in two-thirds of patients with moderate and/or severe CD  and UC . Anti-TNSeveral studies showed that some single nucleotide polymorphisms (SNPs) in the gene encoding TNF-α itself can influence the anti-TNF response in patients with IBD. TNF-α is known to be associated with an increased risk of developing IBD. Therefore, several studies demonstrated an association between polymorphisms and different pharmacological responses to treatments, although some results appear to be controversial ,18,19. SGenes encoding proteins that are implicated in the immune response have been a further research target to evaluate responses to anti-TNF agents. For example, Nucleotide-Binding Oligomerization Domain containing protein-2 (NOD2), also known as Caspase Recruitment Domain-containing protein 15 (CARD15) gene, is able to control the innate immune system by functioning as a Pathogen Recognizing Receptor (PRR), subsequently influencing TNF-α expression ,23,24. TAdditional emphasis was laid on genes involved in apoptosis and autophagy for their involvement in response to TNF-inhibitors, which affects the inflammatory immune response in IBD . Among t4β7 integrin, was approved for the treatment of adult patients with moderate-to-severe active CD and UC [Other newly approved targets to treat IBD are the p40 subunit of IL-12 and IL-23. Ustekinumab is a monoclonal antibody against IL-12 and IL-23 approved for the treatment of several autoimmune disorders, including CD, which induced response already after 8 weeks and maintained clinical benefits up to 52 weeks after treatment ,32. UltiD and UC ,34.To understand the role of pharmacogenetics in the treatment of IBDs we reviewed the current literature in order to provide a basis for a proper use of biological therapies in IBD patients.Studies were identified, screened and extracted for relevant data following the PRISMA  guidelines. Literature search, title, abstract and full text screening were conducted independently by RL and GP. PubMed was used to retrieve articles published in the last 21 years (2000–2021) and search terms were “infliximab and polymorphism and Crohn’s disease”, “infliximab and polymorphism and ulcerative colitis”, “adalimumab and polymorphism and Crohn’s disease”, “adalimumab and polymorphism and ulcerative colitis”, “ustekinumab and polymorphism and Crohn’s disease”, “ustekinumab and polymorphism and ulcerative colitis”, “vedolizumab and polymorphism and Crohn’s disease”, “vedolizumab and polymorphism and ulcerative colitis”, “golimumab and polymorphism and ulcerative colitis”.Papers that fulfilled the following inclusion criteria were included: articles on adults or pediatric patients of any gender and ethnicity receiving one of the biological agents used in IBD treatment independently from difference efficacy in drug response. Irrelevant studies were excluded if: (i) biological agents were used to treat other diseases, (ii) articles were written in a language other than English, (iii) papers with unspecified genotypes.Relevant data was extracted and compared through a data extraction sheet. Extraction procedure was conducted by DA and GS. Extracted data included (i) disease, (ii) number of patients, (iii) polymorphic locus, (iv) biological agent, (v) clinical effects, (vi) duration of the study. Missing data entries were marked with N/A (not applicable).The quality of the studies was assessed using the Newcastle–Ottawa Score for the observational studies  and the A number of 153 results were retrieved from Pubmed, and 116 papers were included in title and abstract screening after duplicates were removed. No records were marked as ineligible by automation tools nor other records were removed for other reasons in this phase. Overall, 116 articles were screened, and a total of 30 studies were included for data extraction . The incp = 0.049) [p < 0.05). This would highlight the role of the A nucleotide as responsible for the clinical ineffectiveness of anti-TNF-α therapy [Promoter-level polymorphisms in TNF-α gene, often associated with increased cytokine secretion, would appear to be a plausible explanation for the inefficiency of anti-TNF treatment ,38. In p= 0.049) . Moreove therapy , but in  therapy .p = 0.011) and in Caucasians (p = 0.016). Moreover, in the same study, a positive correlation between the TNF-α -308 G allele and response to TNF-α inhibitors was found (p = 0.0001) [Another TNF-α variant, in position -238, rs361525, has been profoundly studied for its incidence in response to anti-TNF agents, in a meta-analysis comprehensive of 532 studies. In this case, the presence of the G allele, considered a common allele, has been associated with a positive response to these agents in the overall population ( 0.0001) .p = 0.003) [Furthermore, the overall assessment between a common C allele versus a minor T allele in the TNF-α -857 polymorphism, rs1799724, was also estimated. Patients with the -857 C common allele showed a better response to TNF-α inhibitors than those having the minor allele (= 0.003) . In a to= 0.003) . However= 0.003) . Same co= 0.003) .Polymorphisms in the TNF-α receptor genes can influence anti-TNF-α treatment response among patients affected with IBD. However, some data regarding the impact of certain polymorphisms in these genes are divergent. For example, in a study where two cohorts from independent and prospective clinical trials were investigated different SNPs upon TNFRSF1A and TNFRSF1B were studied, and no significant association with the clinical response was found . Moreovep < 0.05), with a downregulated expression of their corresponding genes, consistent with the downregulation of the receptors in CD patients [In a study on 104 subjects, 54 with CD and 50 healthy controls, the frequencies of some SNPs in TNFRS1A  and 1B  were analyzed. The results demonstrated a higher frequency of rs767455, rs1061624, and rs3397 polymorphisms in CD patients compared to controls (p < 0.01) [p = 0.02), while the CC genotype in the rs3397 was significantly higher in responders [The efficacy of anti-TNF-α inhibitors was also studied in 81 CD patients, where TNFRSF1A and TNFRSF1B polymorphisms rs767455, rs4149570, rs1061622, rs1061624, and rs3397 were analyzed. The presence of the G allele of the rs767455 polymorphism was associated with a reduced effect of infliximab, compared with the AA genotype (sponders .p = 0.014), especially in homozygosity (p = 0.006). Differently, the rs1061622 polymorphism positively influenced the response to infliximab (p = 0.014), also during maintenance therapy (p = 0.007) [Furthermore, an observational cohort study conducted on 124 Caucasian CD patients under infliximab maintenance therapy showed that presence of the minor TNFRSF1B rs976881 allele was a negative predictor of efficacy (= 0.007) .In summary, treatments are affected mainly by the presence of the A allele in -308 TNF-α disregarding of the anti-TNF used. Moreover, the presence of the G allele on TNFRSF1A was also associated with a poor response of treated patients, suggesting that a pre-evaluation could be useful for prescribing the most appropriate drug, according to the patient’s genetic profile .Another gene identified as a susceptibility gene for the development of IBDs is the NOD2/CARD15 gene ,52. ThreAlso TLR-4 and CD14 genes have been investigated for their possible involvement in response to anti-TNF therapies. To investigate the possible role of TLR-4 and CD14 polymorphism in the response to adalimumab, 24 patients, from a cohort of 165 CD subjects, were studied. TLR4 896 A/G and CD14 -260 C/T SNPs were screened, without, however, finding any relationship between their occurrence and the impact of treatment response . Same cop = 0.049) [A target molecule in anti-TNF therapy is IL-1β, thus the presence of the rs1143634 polymorphism was assayed in 47 patients with either CD or UC. The results showed that this mutation was related to higher serum IL-1β levels, possibly correlated with a decreased response to infliximab therapy . Likewis= 0.049) . On the = 0.049) .p = 0.05) with beneficial response in the combined study of 482 CD and 256 UC Danish patients [Reduced IL-6 expression due to the polymorphism rs10499563 was borderline associated  [p = 0.04), compared to C/C or C/T genotype [p = 0.0008). Moreover, the presence of rs10210302, was associated with a better response to adalimumab even after 20 (p = 0.004) and 30 weeks (p = 0.04) [Apoptosis and autophagy genes, such as FASL, CASP9 and ATG16L1, have been studied to evaluate primary responses to anti-TNF-α therapy . In a co= 0.002) . Moreovegenotype . A polym = 0.04) . Further = 0.04) . In summUstekinumab is an agent used in patients with moderate-to-severe CD that has been shown to have a significant clinical response. To the best of our knowledge, we found only one study investigating the role of SNPs upon the Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2) gene which could interfere with the response to anti-IL-12 and IL-23 therapy. The presence of SNPs on its locus is associated with chronic inflammatory conditions , therefoIn summary, the role of this polymorphism should be further investigated as a potential biomarker for response to ustekinumab.4β7 integrin which modulates lymphocyte trafficking and therefore should decrease the inflammatory response in IBD patients. To date, there are no studies examining the associations between SNPs and treatment outcome of IBD patients with this biological drug.Vedolizumab is a humanized immunoglobulin G1 monoclonal antibody to αIBD comprises a heterogeneous group of subtypes with different molecular characteristics. Several genetic biomarkers were associated with anti-TNF-α treatment response in IBD and, generally, interpretation of genetic information in a meaningful way may be difficult . The valTherefore, association between SNPs and response to biological treatment in IBDs was investigated by numerous pharmacogenetics studies, finding a linkage between some SNPs and response to biological therapy. In this systematic review, genetic polymorphisms associated with treatment outcome in IBD patients undergoing biological therapy, have been examined to suggest potential pharmacogenetics approach for predictive benefits. To date, there is no recommendation regarding the search for polymorphisms of genes involved in the pathogenesis of IBD as part of therapeutic optimization.Our methodological evaluation suggests the use in clinical practice of some polymorphisms of interest in IBD patients. Particularly, polymorphisms on TNF-α and TNFR genes, such as the rs1800629, were associated with a poor response, as well as the rs1799724, rs767455, rs1061624 and rs976881. Contrarily, other polymorphisms in these genes have been associated with a better response, such as rs361525 and rs3397.Most of the polymorphisms on innate immunity genes did not show any correlation with the clinical response; however, TLR4 rs5030728, rs1554973, IL-1β rs4848306 and IL-17 rs766748 (GG genotype) polymorphisms were associated with a better response, while the IL-1β rs1143634 polymorphism was related to a poor response.Moreover, among autophagy and apoptosis related genes, we found that FasL rs763110, Caspase-9 rs4645983 and ATG16L1 rs10210302 polymorphisms were associated with a better response; on the other hand, ATG16L1 rs2241880 polymorphism was correlated to a poor response. Finally, the role of polymorphisms interfering with the activity of biologicals targeting IL-12 and IL-23 was also assessed despite only one study associated the PTPN2 rs7234029 polymorphism with Ustekinumab non-responders.However, in this systematic review some limitations and biases are present: for instance, environmental factors such as nutrition, lifestyle, and other medication that may interact with genetic susceptibility were not considered. Moreover, the monitorization time, differences between populations, genetic heterogeneity and gene-gene interactions were not taken into account. Likewise, potential statistical errors in the analyzed studies may affect the results. Considering that the included studies were heterogenous for some characteristics , a possible bias can be ascribed. Moreover, some inevitable publication bias might exist in the results because only published studies were retrieved; as a matter of fact, preprint servers, other registries/results database  were not used and the search was limited to studies published in academic journals. We tried to exclude potentially overlapping data, however we may have missed some overlapping data or unintentionally excluded non-overlapping data. Moreover, the severity of disease activity in patients included in studies investigated may have differed between the studies, therefore this may have introduced other biases that have not been accounted for. Furthermore, we cannot exclude that associations were not found because of low statistical power in some studies included in our investigation.However, this systematic review also carries strengths: we evaluated numerous polymorphisms which might modify the efficacy of all biological drugs approved for the management of IBD, providing a broad pharmacogenetic overview of current biological treatment for IBD. Moreover, all the candidate genes that were included in our investigation, having a known biological effect, allowed a logical interpretation of the observed effects. As a matter of fact, our study highlights associations between treatment responses and specific alleles based on a strong biological or clinical effect.In conclusion, from the clinical perspective, improving TNF-α, TNFR and IL-1 pharmacogenetics would be the most suitable way to move towards a targeted therapy for IBDs, even if bias such as ethnicity and different kinds of biological agents used should be considered. Pre-treatment patients genotyping should be incorporated into clinical IBD management guidelines, as it is the most appropriate strategy to select the most suitable biological drug for an individual patient. Finally, clinical implementation of pre-treatment genetic tests could be achieved by investigating the role of target genes which could interfere with the action of other biologicals apart from TNF-α inhibitors, in order to identify more predictive genetic variants.


Bulletin papers. However, we remain keen to present all opinions so have commissioned papers from gender identity experts, which are making their way through the editorial process. When the papers by Griffin et al and Evans were published on First View, they quickly attracted several complaints with demands for their retraction, which as explained in the editorial was not appropriate. Those authors were invited to write opposing articles but unfortunately, for their own reasons, none took up the offer. Hence our decision to publish all the available letters alongside the original papers so readers can evaluate the arguments for themselves. I hope this and the forthcoming papers assures Dr White that no one's voice is relegated to the correspondence section in the BJPsych Bulletin, but letters, such as Dr White's, are also an invaluable element of discourse.I thank Dr Margaret White for the letter in response to my recent editorial ‘Publishing controversy’. It raised important challenges. Why have the trans health papers been published online and in print even though written by non-specialists in gender identity, and should opposing views be relegated to the Correspondence section? First, all papers published online also appear in the paper journal eventually. To do otherwise would have marked these papers out as somehow different. Dr White does not wish to silence debate, but not publishing in print form as usual would be a form of censure even if not censor. Although this is a controversial and contested area, the papers did not express extreme views. In fact, Marci Bowers, president-elect of the World Professional Association for Transgender Health, recently raised concerns similar to those expressed in the


This Perspective discusses the main functions of TNF and its two receptors, TNFR1 and TNFR2, in steady state, as well as in emergency hematopoiesis. It also provides a comparative overview of existing mouse lines with humanization of TNF/TNFR system. These genetically engineered mice allow us to study TNF signaling cascades in the hematopoietic compartment in the context of various experimental disease models and for evaluating the effects of various human TNF inhibitors on hematopoiesis and other physiological processes.TNF is a multifunctional cytokine with its key functions attributed to inflammation, secondary lymphoid tissue organogenesis and immune regulation. However, it is also a physiological regulator of hematopoiesis and is involved in development and homeostatic maintenance of various organs and tissues. Somewhat unexpectedly, the most important practical application of TNF biology in medicine is anti-TNF therapy in several autoimmune diseases. With increased number of patients undergoing treatment with TNF inhibitors and concerns regarding possible adverse effects of systemic cytokine blockade, the interest in using humanized mouse models to study the efficacy and safety of TNF-targeting biologics  Hematopoiesis is the process of blood cell development that in vertebrates is initiated early during embryogenesis and may be divided into 3 phases or so-called distinct waves of hematopoiesis.The first (or primitive) wave takes place in the yolk sac starting in mice at embryonic day 7.5 (E7.5) and generates unipotent blood cell types (The second (or pro-definitive) wave occurs in the yolk sac, embryo proper and allantois of the mouse embryo and gives rise to multipotent progenitors (The third wave of hematopoiesis represents definitive hematopoiesis and is dependent on the activity of hematopoietic stem cells (HSCs), which are the basic units of the adult hematopoietic system. HSCs generated in the embryonic aorta-gonad-mesonephros region first colonize the fetal liver (E10.5) and then shortly before birth (E16) migrate to the bone marrow (BM), where the majority of HSCs reside to sustain steady state hematopoiesis  express Toll-like receptors  and membrane-bound (LTα2β1) lymphotoxins, respectively  in the BM, consisting mostly of lineage-biased multipotent progenitors  CD45.1+ BM cells into lethally irradiated congenic CD45.1+CD45.2+ WT recipients showed enhanced activity of TNFR1-/-TNFR2-/- HSCs as determined by long-term reconstitution by TNFR-deficient HSCs following transplantation. Moreover, in vivo administration of TNF to WT mice led to a decrease in BM cellularity and to reduction in HSC cycling activity in a competitive transplantation assay  HSC expansion rate upon addition of TNF was significantly decreased  and CFU-GMs (granulocyte/macrophage colony forming units), in methylcellulose assay . LSK isoe clones . Thus, Tin vitro in TNF-deficient long-term bone marrow cultures, which were characterized by increased proliferative potential of granulocytic progenitors and increased numbers of CFU-GMs within sorted LSK population, as compared to WT BM cultures  inhibitors proper animal models should be generated and validated. Importantly, in spite of a conservative nature of TNF family of cytokines and their corresponding genes, most hTNF inhibitors do not block mouse TNF . Therefo+ progenitor cells and granulocytes with concurrent increase in the frequency of cells of both lymphoid and monocytic origin in the bone marrow  mice, in which case the mouse r models , 102. Alr models , 102. Adr models . Furtherr models . hTNF inr models . Overallonocytes , we hypoTNFR2 locus, which allowed conditional Cre-mediated deletion of TNFR2 extracellular part in the desired cell type. These doubly humanized hTNF x hTNFR2KI mice showed EAE disease severity and Treg numbers comparable to wild-type mice, confirming the restoration of protective TNF/TNFR2 signaling. Cre-mediated genetic deletion of TNFR2 gene in Treg cells resulted in EAE exacerbation and malfunction of Treg cells. Intrinsic TNFR2 signaling was important for the maintenance of suppressive functions of Tregs by sustaining expression of Treg signature molecules, such as FoxP3, CD25, CTLA-4 and GITR  in a model of NMDA-induced neurodegeneration . AdditioIn summary, there is a growing panel of useful humanized mouse models for evaluation of biologics that affect TNF/TNFR1/TNFR2 systems, including their effects on hematopoiesis. Furthermore, restoration of affected signaling by humanization of both TNF and its receptor, TNFR2, makes it possible to comparatively evaluate not only anti-TNF drugs, but also hTNFR2 agonists and antagonists. Taken together, humanized mouse models will allow investigators to study efficacy of various TNF/TNFRs-targeting biologics and assess possible side effects on other systems for further clinical translation.For many years TNF was mainly considered a proinflammatory cytokine with its role in host defense, but also with detrimental effects on autoimmunity. However, basic studies on TNF biology, as well as reported side effects in patients receiving anti-TNF therapy, highlighted its homeostatic functions in many physiological processes, including hematopoiesis. Regulation of TNF/TNFRs expression in various tissues modulates the cross-talk between immune and non-immune cells, which subsequently determines the outcome of TNF action.Future investigation of pathological versus regulatory functions of TNF and deciphering its systemic and local effects in tissues may help to improve current therapeutic approaches. Therefore, mouse models with humanized TNF/TNFRs system represent a powerful tool to study side effects of anti-TNF therapy on hematopoiesis.VG, KA, MD, and SAN designed research. VG and KA. performed research and analyzed data. VG, KA, AD, TY, MSD, and SN discussed the concept and wrote the manuscript. All authors contributed to the article and approved the submitted version.Reverse genetics studies were supported by the grant 075-15-2019-1660 from the Ministry of Science and Higher Education of the Russian Federation, myelopoiesis studies were supported by RFBR grant 19-34-51030.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium avium subspecies paratuberculosis (MAP) infection. Surprisingly, anti-TNF-α downregulated ACE2 expression by 0.46-fold and increased TMPRSS2/TACE ratio by 44% in THP-1 macrophages. Treatment of macrophages with rIL-6 also downregulated ACE2 and increased TMPRSS2/TACE ratio by 54%. Interestingly, anti-TNF-α treatment upregulated Notch-1, IL-6, and MCL-1 by 1.3, 1.2, and 1.9-fold, respectively, and increased viability and burden of MAP infection in macrophages. Blocking Notch signaling doubled ACE2 expression, decreased TMPRSS2/TACE ratio by 38%, and reduced MAP viability by 56%. In a small group of patients, ACE2 level was significantly lower in the plasma from rheumatoid arthritis (RA) patients on anti-TNF-α treatment compared to healthy control. The data in this critical study demonstrated that through Notch-1/IL-6 signaling, anti-TNF-α agents decreased ACE2 expression and shedding through TMPRSS2/TACE modulation and increased the susceptibility to infection. Overall, this study warns against anti-TNF-α therapy in some patients with underlining inflammatory conditions during the COVID-19 pandemic. The findings should impact current guidelines regarding treatment decisions of patients on anti-TNF-α during the COVID-19 pandemic.Although millions of patients with underlining conditions are treated primarily with anti-TNF-α agents, little is known about the safety of this standard therapy during the coronavirus disease-2019 (COVID-19) pandemic. In this study, we investigated the effect of anti-TNF-α monoclonal antibodies on the cellular entry mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and increasing the risk of COVID-19 development. We focused on the expression of angiotensin-converting enzyme II (ACE2), type II transmembrane serine proteases (TMPRSS2)/TNF-α converting enzyme (TACE) ratio. We also investigated the involvement of Notch-1 signaling and its downstream influence on IL-6, myeloid cell leukemia sequence-1(MCL-1) in the anti-TNF-α mode of action and increased the susceptibility to  Coronavirus disease 2019 (COVID-19) has emerged as a new respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been associated with substantial morbidity and mortality, especially in patients with underlining conditions, including autoimmune diseases. Autoimmune disease is a group of conditions that are a broad range of related disorders characterized by the dysregulated immune response, which results in excessive activation of immune cells and subsequent uncontrolled inflammation and tissue damage (Mycobacterium tuberculosis (MTB) (Mycobacterium avium subspecies paratuberculosis (MAP) survival in infected macrophages  such as infliximab (IFX), adalimumab (ADA), and certolizumab pegol (CZP) have become the standard treatment of autoimmune diseases . Howeveris (MTB) –5. More is (MTB) . Our labrophages . MAP is rophages , 8. ThesEmerging evidence indicates that anti-TNF-α drugs interact with transmembrane TNF-α and elicit reverse signaling cascade in activated macrophages. A process that could modulate TNF-α converting enzyme (TACE) activity . TACE A, which sTACE has been identified as a principal protease involved in the shedding of angiotensin-converting enzyme II (ACE2), which mediates the entry of SARS-CoV-2 . In contAlthough millions of patients with underlining conditions, including RA and CD, are treated primarily with anti-TNF-α agents, little is known about the safety of this standard therapy and susceptibility to SARS-CoV-2 infection during COVID-19 pandemic. In this study, we investigated the effect of anti-TNF-α monoclonal antibody therapy on the cellular entry mechanism of SARS-CoV-2 and the risk of COVID-19 development. We focused on the expression and dysregulation of ACE2, TMPRSS2, and TACE. We also investigated the mechanism involved in Notch-1 signaling and downstream influence on IL-6, MCL-1, and intracellular infection by MAP in macrophages treated with anti-TNF-α drugs. We also investigated the ACE2 level in plasma from RA patients on anti-TNF-α therapy and controls.The human monocytic cell line THP-1 (ATCC TIB-202) was used to model macrophages. THP-1 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS)  and kept in a humidified 5% CO₂ incubator at 37°C. THP-1 monocytes were differentiated to THP-1 derived macrophages by 50 ng/ml of phorbol 12-myristate 13-acetate (PMA)  for 48 hours.Notch signaling in THP-1 derived macrophages was targeted pharmacologically using γ-secretase inhibitor DAPT [N‐ ‐S‐phenylglycine t‐butyl ester] . To examine the effect of anti-TNF-α therapy on Notch signaling and its downstream effect, THP-1 derived macrophages were treated with two forms of anti-TNF-α agents, non-PEGylated (ADA), and PEGylated (CZP).Clinical MAP strain  was cultured in Bactec MGIT Para-TB medium tubes , containing Bactec MGIT Para-TB supplements  and incubated in BD BactecTM MGITTM 320 Analyzer at 37°C.(−ΔCT) *1000, where ΔCT= [ − ]. The primers (Thermo Fisher) used for the RT-PCR in this study are shown in TRIzol™ reagent was used for total RNA isolation from cells  according to the manufacturer’s instructions. Briefly, 800 ng of total RNA was used to synthesize cDNA, and quantitative real-time PCR was performed using the StepOnePlus™ Real-Time PCR System  with Fast SYBR Green Mastermix  as detection dye. A housekeeping β-actin primer  was used to measure the endogenous baseline CT values. Relative mRNA expression levels were calculated by using the equation 27 CFU/ml) for 24 hours. Cells were then washed with phosphate buffer saline (PBS)  and then collected and lysed with 500 µL of M-PER Mammalian reagent . After sample centrifugation, 100 μL of each sample supernatant was loaded in triplicates in 96-well microplate and mixed with 100 μL of staining reagent mixture (SYTO® 9 green- and Propidium iodide red-fluorescent nucleic acid stains). Following 15 minutes of incubation in the dark, the integrated intensities of the green (530 nm) and red (630 nm) emission and excitation at 485 nm were measured using SpectraMAX® i3x Multi-mode microplate reader. Then the green/red fluorescence ratios were calculated, which detected the Live/Dead MAP ratio.Live/Dead™ Baclight™ bacterial viability assay  was used to measure relative MAP viability in infected THP-1 derived macrophages. Briefly, macrophages were infected with MAP  samples were obtained from 21 subjects . All participants completed and signed a written informed consent prior to enrollment in the IRB-approved study #IRB00001138. Plasma was isolated and maintained at -20C˚ for later use.Measurement of Circulating ACE2 Levels by Enzyme-Linked Immunosorbent Assay (ELISA).® i3x Multi-mode microplate reader.Circulating ACE2 levels were measured in the plasma using Soluble Angiotensin-Converting Enzyme 2 (ACE2) (Human)  following the manufacturer’s instructions. ACE2 levels were determined by reading optical density at 450 nm using SpectraMAXTo determine the statistical significance in this study, all data were pre-tested for normal distribution using the Kolmogorov–Smirnov normality test followed by paired tow-tailed t-test to determine the difference between the treated groups and controls, and unpaired tow-tailed t-test to determine the difference in the plasma ACE2 levels between RA patients and healthy controls. All experiments were performed in triplets, and values are presented as mean ± standard deviation (SD). All statistical analyses were performed using Prism GraphPad software (version 8), with P<0.05 considered significant.To explore the effects of therapeutics approved anti-TNF-α agents on Notch-1 and its downstream effect on IL-6 and MCL-1 expression in macrophages. THP-1 derived macrophages were treated with two forms of anti-TNF- α agents, non-PEGylated , and PEGylated . We found that anti-TNF-α agents induce the expression of Notch-1, IL-6, and MCL-1 in THP-1 derived macrophages in a dose-dependent manner, at 40 μg/ml concentration level, non-PEGylated (ADA): 1.2, 2.2, and 1.7-fold and PEGylated (CZP): 1.3, 1.0 and 1.4-fold, respectively) compared with untreated groups (P < 0.05) (To determine whether anti-TNF-α agents potentiate MAP impact in infected macrophages. THP-1 derived macrophages were infected with MAP (10^7 CFU/ml) for 24 hours. Then the infected macrophages were treated with two forms of anti-TNF-α agents, non-PEGylated (ADA)(0-40 μg/ml) and PEGylated (CZP) (0-40 μg/ml). As shown in We previously reported that anti-TNF-α agents induce MAP survival in infected macrophages. In this study, we have confirmed the involvement of Notch-1 signaling in anti-TNF-α agents’ mode of action 2. To fuTo explore the involvement of anti-TNF-α therapy in the cellular entry of SARS-CoV-2 and the development of COVID-19. THP-1 derived macrophages were treated with non-PEGylated anti-TNF- α  for 24 hours. Then we measured the expression of ACE2, TACE, and TMPRSS2. As shown in To examine the effect of Notch-1 and IL-6 on ACE2, TACE, and TMPRSS2 Expression in Macrophages. THP-1 derived macrophages were treated with DAPT (30 μM) and rIL-6 (500 U/ml) for 24 hours. We found that targeting Notch signaling using DAPT induced the expression of ACE2 and TACE by 0.8 and 1.0-fold compared with untreated groups (P < 0.05)  was examined. As shown in To examine the effect of anti-TNF-α on ACE2 shedding, the circulating ACE2 levels were examined in the plasma of a small number of RA patients on anti-TNF-α therapy. As shown in Notch signaling is juxtacrine signaling regulating critical cellular processes like proliferation, differentiation, and apoptosis . Notch sIn this study, we were able to confirm the involvement of Notch-1 signaling in the anti-TNF-α mode of action. ADA and CZP significantly induced the expression of Notch-1 in macrophages. Furthermore, ADA and CZP treatment induced the expression of IL-6 and MCL-1 in macrophages, a finding that could explain the poor response of anti-TNF-α agents in a substantial number of patients. Many studies supported the reciprocal modulation between Notch signaling and various pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages, ultimately resulting in the sustained inflammatory response in macrophages . AccordiThus, we are intrigued to study the behavior of anti-TNF-α agents in the presence of MAP infection. We found that ADA and CZP significantly potentiate MAP mediated effect on macrophages by amplifying the expression of Notch-1, IL-6, and MCL-1 in MAP infected macrophages. Thereafter, we hypothesized that anti-TNF-α modulates macrophages defense mechanisms against intracellular pathogens such as MAP through Notch-1 signaling. Thus, we targeted Notch signaling using DAPT, and we found targeting Notch signaling prevents anti-TNF-α induces MAP survival in infected macrophages. This novel finding of the involvement of Notch-1 signaling in the anti-TNF-α mode of action could potentially explain the poor response to these agents, particularly in MAP positive individuals.The association of anti-TNF-α agents and intracellular infection prompted us to extend our effort to examine these agents safety during COVID-19 pandemic. COVID-19 has emerged as a new respiratory disease caused by SARS-CoV-2. COVID-19 has been associated with substantial morbidity and mortality, especially in patients with underlining conditions . Millionin vitro the potential involvement of anti-TNF-α therapy in the cellular entry of SARS-CoV-2 and the development of COVID-19. Specifically, we determined the interaction of anti-TNF-α with TACE and subsequent modulation of SARS-CoV-2 cell receptor ACE2. ACE2 plays a critical role in regulating the renin-angiotensin-aldosterone system, which subsequently regulates blood pressure and fluid balance  is produced after being shed from cell membranes due to membrane protease effects . A high via induction of Notch-1 signaling. Specifically, our results show that anti-TNF-α agents increase MAP viability in infected macrophages through the induction of Notch-1 signaling and its downstream effect on IL-6 and MCL-1. Additionally, we have demonstrated a disadvantage for the use of anti-TNF-α therapy due to its effect on ACE2 expression and shedding mechanism through Notch signaling, a process that favors SARS-CoV-2 cellular entry. Consequently, this exaggerates the viral infection, and significantly increases cytokine production (cytokine storm). Overall, this innovative study demonstrates that anti-TNF-α therapy is a high-risk factor in patients with underlining inflammatory conditions during the COVID-19 pandemic. The outcome of this study will impact current guidelines regarding treatment decisions of patients on anti-TNF-α during the COVID-19 pandemic. Additionally, it provides a powerful and promising strategy  to combat infection and inflammation during SARS-CoV-2 infection. A large-scale study should be performed to look at the level of ACE2 in a large number of blood samples from patients on various treatment options in order to establish a true conclusion about the effect of anti-TNF-α on ACE2 level in patientsIn conclusion, our study significantly extends the current knowledge of the anti-TNF-α therapy mode of action in patients during the COVID-19 pandemic. This is a rapidly changing time and the information we know daily should help address and control the COVID-19 pandemic. The data clearly associates anti-TNF-α therapy with an increase in the risk of intracellular infection, including MAP and SARS-CoV-2 infection The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of central Florida IRB (#IRB00001138). The patients/participants provided their written informed consent to participate in this study.EK designed and performed experiments, analyzed the data, and prepared the manuscript. SB has kindly supervised and provided the clinical samples used in this study and has participated in all aspects of the study. SN is the mentor and primary advisor who supervised all aspects of the work including designing the experiments, interpreting the data, writing and editing the manuscript. All authors contributed to the article and approved the submitted version.This work was funded, in part, by the Florida Legislative Grant and the UCF College of Medicine Dean’s Research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have quite a number of papers in this issue and, honestly, I am lost for choice. None the less a few papers stand out for me.In the infectious disease section, I was touched by Dr. Ombeva's sharing and reflection of his story: the way he caught Covid-19 from a child he was treating and the child's parents. 2It is followed by a meta-analysis on seasonal flu that is highly relevant given that flu impacts the effects of Covid-19. 3H.pylori,C.parvum,20Then follows the attempt by the HIV community to understand the ramifications of the HIV pandemic in Africa despite having lived with it for almost 4 decades now! Hence we have papers on: outcomes on PLHIV in KenyaEven during this Covid-19 pandemic, NCDs are forever with thus: cardio-vascular disease nursing care,In this section we have issues of pregnancy ectopic, labour,458The rest of the papers are on self-medication,61


Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.The online version contains supplementary material available at (10.1186/s13059-021-02349-4). Second, nascent RNA profiling measures not only RNA polymerase occupancy, but also orientation by default, whereas traditional RNA-seq requires specific library preparation steps to capture orientation. Third, nascent RNA profiling measures unstable transcripts, which can be used to infer regulatory element activity and identify promoters and enhancers de novo by detecting bidirectional transcription and clustered transcription start sites (TSSs) [Steady-state transcription levels are commonly measured by RNA-seq, but there are many advantages to quantifying s (TSSs) , 2. Fours (TSSs) –5. With s (TSSs) , 6–10, tHere, we introduce PEPPRO, an analysis pipeline for uniform initial sample processing and novel quality control metrics. PEPPRO features include (1) a serial alignment approach to remove ribosomal DNA reads; (2) nascent transcription-specific quality control outputs; and (3) a modular setup that is easily customizable, allowing modification of individual command settings or even swapping software components by editing human-readable configuration files. PEPPRO is compatible with the Portable Encapsulated Projects (PEP) format, which defines a common project metadata description, facilitating interoperability . PEPPRO PEPPRO starts from raw, unaligned reads and produces a variety of output formats, plots, and quality control metrics. Briefly, pre-alignment steps include removing adapters, deduplicating, trimming, and reverse complementation Fig. . PEPPRO To evaluate PEPPRO on different library types, we assembled a test set of run-on libraries with diverse characteristics Fig. a. Our teTo demonstrate how PEPPRO responds to mRNA contamination, we also generated a set of 11 samples built from a single PRO-seq library (GSM1480327) that we spiked with increasing amounts of RNA-seq data (GSM765405)  into the 3g seqkit  and fqdeg seqkit . PEPPRO g seqkit  to projeOne challenge specific to nascent RNA sequencing is ensuring that the library targets nascent RNA specifically, which requires eliminating the more abundant processed rRNA, tRNA, and mRNA transcripts. Early run-on protocols included 3 successive affinity purifications, resulting in 10,000-fold enrichment over mRNA and over 98% purity of nascent RNA . Newer rnascent purity of RNA, PEPPRO provides two results: an mRNA contamination metric and a rDNA alignment rate. First, PEPPRO assesses nascent RNA purity by calculating the exon to intron read density ratio  plot provides an information-dense look into the genomic distribution of reads relative to genomic features. This analysis is a generalization of the more common fraction of reads in peaks (FRiP) plots produced for other data types [′ UTR, 3′ UTR, exons, and introns  or an HDAC inhibitor (n=3).The focus of PEPPRO is in the pre-processing relevant for any type of biological project. The output of PEPPRO sets the stage for downstream analysis specific to a particular biological question. Perhaps the most common downstream application of nascent transcription data is differential expression analysis. PEPPRO allows the user to easily run a differential comparison using dedicated software like the DESeq2 bioconductor package . To demoTo facilitate differential expression analysis, PEPPRO produces a project-level counts table that may be loaded in R using pepr, and, in a few lines of code, converted quickly into DEseq data sets ready for downstream DESeq analyses  standard may be found at o.4542304.Primary analyses data were downloaded from GEO accession numbers GSM1480327 , GSM1480seqtk, and wigToBigWig [The PEPPRO pipeline is a python script (peppro.py) runnable from the command-line. PEPPRO provides restartability, file integrity protection, logging, monitoring, and other features. Individual pipeline settings can be configured using a pipeline configuration file (peppro.yaml), which enables a user to specify absolute or relative paths to installed software and parameterize alignment and filtering software tools. Required software includes several Python packages  or fastx (https://github.com/agordon/fastx_toolkit), depending on user preference. Because reads are processed uniquely for first and second reads in a paired-end experiment, reads must be re-paired prior to alignment. PEPPRO uses the optimized implementation fastq-pair [Following the removal of PCR duplicates, the UMI is trimmed. For run-on experiments where the sequencing primer sequences the 3stq-pair  to re-paFollowing re-pairing, or starting from processed single-end reads, PEPPRO performs a series of preliminary, serial alignments  before aligning to the primary reference using bowtie2 . As a siSubsequent to these serial alignments, remaining reads are aligned to the primary genome. Primary genome alignment uses the bowtie2--very-sensitive option by default and sets the maximum paired-end fragment length to 2000. The goal with primary alignment is to identify the best valid alignment for reads, sacrificing speed for accuracy. Following primary alignment, low-quality reads are removed using samtools view -q 10. As with the initial prealignments, these parameters can be customized by the user in the pipeline configuration file (peppro.yaml). Alignment statistics  for all serial alignments and primary alignments are reported. For the primary alignment, PEPPRO also reports the number of mapped reads, the number removed for quality control, the total efficiency of alignment , and the read depth. Prior to further downstream analysis, paired-end reads are split into separate read alignment files and only the first read is retained for downstream processing. For both paired-end and single-end experiments, this aligned read file is split by strand with both plus and minus strand aligned files further processed.′ end and may optionally be scaled by total reads. PEPPRO may alternatively use seqOutBias [Following read processing, alignment, strand separation, and quality control reporting, aligned reads are efficiently converted into strand-specific bigWig files by default. For PRO-seq and similar protocols, reads are reported from the 3qOutBias  to correqOutBias . K-mer cqOutBias . CorrectqOutBias . As suchqOutBias .for every gene. The overall measure, the mRNA contamination metric, is the median of all genic exon to intron density ratios.PEPPRO provides an mRNA contamination histogram for quick visual quality control, and a BED format file containing gene by gene exon:intron ratios for detailed analysis. To calculate this metric, PEPPRO utilizes annotation files derived from UCSC RefSeq gene files. Because promoter-proximal pausing inflates these ratios, PEPPRO excludes the first exon from the calculation. Otherwise, the reads per kilobase per million mapped reads (RPKM) is calculated for all exonic and intronic sequences on a gene by gene basis. Then, the ratio of exon RPKM to intron RPKM is determined each identified TSS per gene. We determine the read density at every annotated pause window per gene and identify the predominant, singular pause window as the pause window with the greatest density. This singular pause window is used to calculate the pause index for that gene. The corresponding gene body is defined as the region beginning 500-bp downstream from the predominant TSS to the gene end. We found that lowly expressed genes represent a significant portion of genes with a low pause index. At low sequencing depth, these lowly expressed genes experience greater dropout and fluctuation in pause index calculation, skewing the metric upwards at low depth. To address this, we restrict the pause index calculation to the upper 50th percentile of genes by expression, which eliminates the variability due to depth. Finally, PEPPRO plots the distribution of pause indices for each remaining gene in a histogram and provides a BED-formatted file containing each gene’s pause index for more detailed analyses.Pause indices are calculated as the ratio of read density in the promoter proximal region versus read density in the gene body. To calculate these values, PEPPRO utilizes annotation files derived from Ensembl gene set files. Pause indices can vary widely depending on the defined pause window and how a pause window is determined . PEPPRO defines the density within the pause region as the single, most dense window +20–120 bp taken from all annotated TSS isoforms per gene. This is necessary as some genes contain multiple exon 1 annotations and because this region is where most polymerase pausing occurs, PEPPRO identifies the predominant exon 1, based on density, and calculates the pause index using this window density. This means that for genes with multiple TSSs, we define the pause window as the region +20-120 bases from H9 PRO-seq experiments were conducted as described previously . The HDASynthetic sequencing depth variant libraries were constructed for single-end and paired-end PRO-seq libraries using either the K562 PRO-seq (GSM1480327) or H9 PRO-seq 2 (GSM4214081) as source libraries, respectively. For K562 PRO-seq subsamples, seqtk sample -s99 was called on the raw fastq files to generate libraries between 2 and 10%, in 2% increments, and between 10 and 100%, in 10% increments. For the H9 PRO-seq libraries, seqtk sample -s99 was called on the raw fastq files to produce libraries between 10 and 100%, in 10% increments. Lower percentage K562 PRO-seq libraries were generated to yield libraries of total size comparable to low percentage H9 PRO-seq libraries.RNA-seq spike-in libraries were also produced using the command seqtk sample -s99 on raw fastq files using combinations of the K562 PRO-seq library utilized prior and a corresponding K562 RNA-seq library (GSM765405). RNA-seq libraries were sampled between 10 and 100%, in 10 percent increments, and concatenated with the sampled K562 PRO-seq libraries to generate mixed libraries composed of 0–100% RNA-seq.Low complexity libraries were similarly constructed. Thirty million total read libraries were generated by using seqtk sample -s99 on the H9 PRO-seq 2 library and sampling at 50, 80, 90, 92, 94, 96, 98, and 100%. At each percentage of original H9 PRO-seq 2 library sample, the remainder represents duplicates of the original raw reads composing the opposite percentage, producing libraries with varying levels of duplicated reads.Additional file 1 Example R code to generate a gene counts table and supplemental figures.Additional file 2 Review history.


Discrimination and species identification of meat has always been of paramount importance in the European meat market. This is often achieved using different conventional analytical methods but advanced sensor-based methods, such as the electronic tongue (e-tongue), are also gaining attention for rapid and reliable analysis. The aim of this study was to discriminate Angus, domestic buffalo, Hungarian Grey, Hungarian Spotted cattle, and Holstein beef meat samples from the chuck steak part of the animals, which mostly contained longissimus dorsi muscles, using e-tongue as a correlative technique with conventional methods for analysis of pH, color, texture, water activity, water-holding capacity, cooking yield, water binding activity, and descriptive sensory analysis. Analysis of variance (ANOVA) was used to determine significant differences between the measured quality traits of the five-meat species after analysis with conventional analytical methods. E-tongue data were visualized with principal component analysis (PCA) before classifying the five-meat species with linear discriminant analysis (LDA). Significant differences were observed among some of the investigated quality parameter. In most cases, Hungarian Grey was most different from the other species. Using e-tongue, separation patterns could be observed in the PCA that were confirmed with 100% recognition and 97.5% prediction of all the different meat species in LDA. Meat provenance and meat quality are important areas of focus for the European meat market, which provides approximately 30% of the available calories in the European Union (EU) through meat consumption . Meat quMeat from dairy breeds has been generally considered of inferior eating quality, compared to British and European cattle breeds , such asCurrently, these quality markers are being monitored and determined through many different methods but the most common are spectrophotometric techniques (color) , pH diffThe International Union of Pure and Applied Chemistry (IUPAC) defines the electronic tongue as “a multisensory system consisting of a number of selective sensors and uses advanced mathematical procedures for signal processing based on pattern recognition and/or multivariate data analysis” . In compOne if its major advantages is that it is non-subjective and can be used for both targeted and non-targeted analysis, due to their partial/non-partial selectivity and sensitivity to different ionic compounds in solution . In spitThus, the aim of this study was to characterize nine important physico-chemical properties  of four different beef breeds and domesticated buffalo using conventional methods and sensory analysis. The potential of e-tongue to rapidly discriminate the different meat species and predict the nine physical parameters was also performed, in order to determine the feasibility of using the e-tongue both independently and as a correlative technique in combination with conventional methods.Angus, domestic buffalo, Hungarian Grey, Hungarian Spotted cattle, and Holstein breed meat samples were collected from two slaughterhouses in central Hungary (Pest county) on the day of slaughtering. The animals were kept at two farms in the central part of Hungary (Pest county). The animals were of the same sex (bull) and same maturity: ready for slaughtering .Feeding and housing were also identical (semi-intensive fattening) for all the tested animals, and they were slaughtered in the same way: resting time, stunning method, cooling technology, etc., were identical. Samples were stored uniformly at 5 °C until the experiments.Three parallel animals were used for each of the five studied breeds, resulting in three replicate samples per breed. The left chuck steak part of the animals, which mostly contained longissimus dorsi muscles, was tested . The samples were each, subjected to different physico-chemical analysis, sensory profile analysis, and electronic tongue measurements .The pH was determined as described by Prommachart et al. , using aThe color properties were determined as described by Aroeira et al. . A hand-According to the international scale, the following ranges can be applied in the total color difference (ΔE): not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3.0), well visible (3.0–6.0), and great difference (6.0–12.0).2 (50 × 50 mm) area filter paper. The filter paper and the samples were placed between two glass plates and pressurized with 0.5 kg weight for 5 min then the filter paper was dried. The formed spot (moisture from the sample) was cut out from the filter paper and the paper without spot was weighted. The released water from sample was calculated as the proportion of area and weight of spot and sample and was expressed in [mm2/mg] dimension. Three replicates were analyzed.The WHC was characterized according to Huff-Lonergan et al. . An amouDetermination of water activity was carried out by using the LabMaster-aw neo equipment . A thin layer of meat was placed in the jar belonging to the apparatus, such that it covered the whole jar before the water activity values were recorded after 25 min.Moisture was determined using the AOAC method of proximate analysis . Twenty Texture was measured by Warner–Bratzler shear force (WBSF) with a SMS  TA.XT plus texture analyzer. Before the measurement, 150–200 g of meat from the five different species were heat-treated until the core temperature of the meat reached 72 degrees celcius (°C), then cooled to 4–6 °C after heat treatment. They were then cut into sizes of 1 × 1 × 10 cm before measuring and recording their texture values .The meat samples were first vacuumed, then placed in a 72 °C water bath for 1 h. Afterwards, they were cooled on an ice bed and baked for ten minutes on a baking tray preheated to 200 °C, before commencing sensory evaluation.Sensory quality was evaluated with the profile analysis method . The panForty grams of the same body part (chuck steak part) was taken from each of the three different cattles for each breed under study, resulting in three replicate samples per breed. The meat samples were separately cooked in a pot containing 600 mL of distilled water, while covered, until their core temperatures reached 72 °C.The meat extracts obtained after cooking the three different meat samples from each breed was diluted up to 50% using distilled water to obtain a total of 100 mL solution per each sample . All the diluted samples were then transferred in 100 mL beakers before putting them in the e-tongue autosampler for analysis. The volume of each tested sample during the measurement was 100 mL, the sampling time was 120 s, the sampling frequency was 1 s, and the cleaning time with distilled water was 20 s. All experiments were performed at room temperature.The e-tongue used for the measurements was a potentiometric electronic tongue (e-tongue) with food grade sensors  and was configured according to the manufacturers  recommendations prior to each adulteration measurement. To configure the instrument, a conditioning was performed using 0.01 M hydrochloric acid solution and distilled water then, a calibration using the solution prepared from the extraction of meat samples. The purpose of this, was to achieve good sensor signals from the instrument during measurement and so as to allow rapid detection of low concentration differences in the meat mixture samples otherwise, known as fingerprinting. The main operating principle of measurement for the e-tongue is based on the difference in potential changes of sensor probes  against a reference electrode in zero-current conditions.Each diluted meat extract was measured four times, resulting in 12 readings in total, from the e-tongue sensor for each beef species. The last 10 s of the sensor signals was then exported for multivariate data analysis because the signals at this point are considered to be the most stable. All data analysis was performed in R-project statistical software.p < 0.05 as significant. Sensory data were also analyzed with one-way ANOVA and Tukey’s test for least significant difference tests.Physico-chemical results of all the different breeds were analyzed in excel using bar charts and standard deviations. Analysis of variance (ANOVA) was used to determine significant differences between the breeds, based on their measured parameters. The Tukey method was applied for multiple comparison among breed group means considering E-tongue results were analyzed by using linear discriminant analysis (LDA) for multi-class classification of the different breeds in R-project. Cross validation was performed for the LDA model to evaluate their robustness in predicting the breeds. For this, the data were divided into a training set and a validation set. The training set was made up of two-third of the data; thus, the sensor signals from the first and second replicates of each sample , representing six e-tongue readings. The validation set was made up of sensor signals from the third replicate  representing three e-tongue readings. The data splitting was performed three times, such that each sample was used at least once in the calibration and validation set.Partial least squares (PLS) regression was used to develop models, in order to regress on some of the physico-chemical properties of the meat samples. Cross-validation was also used to test the robustness of the models by splitting the data into two groups: the calibration and validation sets. The training set consisted of two-thirds of the data, which included sensor signals from the first and second replicates of each meat breed. The validation set consisted of sensor signals from the third replicate. The data splitting was repeated as many times as possible to ensure that each replicate was used for calibration and validation at least once. The statistical parameters used to evaluate the performance of the PLS regression models were the root mean square error of calibration (RMSEC) and the coefficient of determination (R2C). For cross-validation, it was root mean square error of cross-validation (RMSECV) and coefficient of determination after cross-validation (R2CV). The optimum number of latent variables was determined based on the minimum RMSECV value.p < 0.05) between the pH of the Hungarian Grey species and Angus, domesticated buffalo, Holstein, and Hungarian Spotted cattle.With an average pH of 6.04, the Hungarian Grey species had the highest pH among all the other species, whilst the domestic buffalo had the lowest with an average pH of 5.49 . Angus, p < 0.05) between the redness  of Angus and Holstein, and also between Holstein, Hungarian Grey, and Hungarian Spotted cattle. Hungarian Grey and Hungarian Spotted cattle were also significantly different in terms of redness. Holstein and Angus were also significantly different in terms of color a*. Yellowness  of Hungarian Spotted breed, Holstein, Hungarian Grey, and Angus were significantly different (p < 0.05). There were also significant differences between the color b* values of the domestic buffalo, Holstein, and Angus. For color L*, which represents lightness and brightness, significant differences (p < 0.05) were observed between the Angus and domestic buffalo, as well as between the Angus and Hungarian Grey breeds. Hungarian Spotted cattle were also significantly different from the Hungarian Grey. The Hungarian Grey cattle breed was used as the reference for the determination of total color difference because it had the highest pH. Based on the calculated average of the total color differences, there was a clearly visible difference (3.0–6.0) between Angus, domestic buffalo, and Angus species. There was also considerable difference between the Hungarian Spotted cattle and the other breeds as proven in the Anova analysis where total color difference was significant (p < 0.05) between Angus, domestic buffalo, and Hungarian Spotted cattle meat. There was also significant different the Hungarian Spotted cattle, Holstein, and Hungarian Grey. There was no significant difference between the domestic buffalo and Hungarian Spotted cattle meat in (Color parameters: a* (a), b* (b), L* (c), and total color difference (d) measurements of the five different breeds are presented in  meat in d.2/mg, compared to the lowest water-holding capacity of 1.10 mm2/mg recorded for the Hungarian Grey breed (p < 0.05) between the water-holding capacity of the Hungarian Grey breed and all the other breeds. The water-holding capacity of the Hungarian Spotted breed and Holstein were also significantly different.The Hungarian Spotted breed had the highest average water-holding capacity of 2.11 mmey breed . Angus, p < 0.05) between the water activity of Angus and domestic buffalo cattle meat and also, between the domestic buffalo and Hungarian spotted cattle breeds, as shown in Angus had the highest average water activity of 0.99, whilst the domestic buffalo had the lowest (0.94) . Holsteim/m  were only observed between the dry matter content of Angus and all the other breeds.All the five different breeds had dry matter content higher than 23% m/m . The HunThe Warner–Bratzler shear force was only significantly different between the Hungarian Grey cattle and the other breeds, as shown in p < 0.01), while in the vividness of the color the panel detected no difference. Among the texture attributes the chewability of the domestic Buffalo had the lowest scores, both the Hungarian Grey cattle and the Hungarian Spotted cattle were easier to chew (p < 0.05). The cooked beef odor was the most intense in case of the domesticated buffalo, followed by the Hungarian Spotted cattle and the Hungarian Grey cattle. In the remaining attributes there were no significant differences.Based on the sensory perception  of the cThe confusion matrix  for thisIn our study, only the Hungarian Grey cattle breed had a significantly higher pH; the pH range of all the different breeds were in agreement with those generally reported in the literature for carcasses from bovine species ,34. AccoWater-holding capacity (WHC) is also a meat quality parameter generally related to the concept of isoelectric point (pl); as the pH of meat decreases due to the buildup of lactic acid, the pH approaches the isoelectric point, which is basically the point of pH where positive charges are equal to negative charges . When poThe influence of pH and water activity (aw) on microbial growth is an area of interest in food quality that has been widely studied. Generally, low water activity and high pH in food is recommended for microbial control. The pH and water activity were inversely related to each other in this study. For example, Angus and Hungarian Spotted breed had the highest water activity, although they had the lowest pH among all different meat breeds in this study. Hungarian Grey breed also had the lowest water activity, although it had the highest pH. Watanabe et al.  reportedDry matter content, which can refer to the material remaining after the removal of water, was highest in the Hungarian Grey breed. The dry matter content of meat has been often related to the Warner–Bratzler shear force of meat, which is a criterion of measurement for meat tenderness. The Warner–Bratzler shear force results were in agreement with chewability results in the sensory analysis, where meat from the Hungarian Grey breed was the only one that was significantly different from the other breeds. Hungarian Grey is one of the most famous Hungarian breeds, a breed native to Hungary; parts of the body that are valuable for meat production are immature, i.e., the rump is poor in muscle, the thighs are thin, and the flesh is dry and less fatty. Dry matter content was only significantly lower in the Angus breed, which is known for traits that can make a real difference in cow herd profitability, including calving ease, marbling, growth, and superior milking capabilities, particularly with high fertility rates; however, they are not usually reared for their meat .Meat quality, as perceived by consumers, is a subjective, multidimensional, and dynamic concept. For this reason, other analytical instruments, such as the electronic tongue, have been recommended for objective validation of meat quality. Classification models developed with the electronic tongue showed that the instrument could classify Angus, Holstein, Hungarian Spotted, and Hungarian Grey breeds with 100% accuracy. Only domestic buffalo meat showed misclassifications as meat from the Hungarian Spotted breed. This suggests that, although meat quality parameters of the domestic buffalo have been reported in literature  and alsoThe electronic tongue on the other hands, is a quick and reliable method with a very short analysis time. As a correlative technique in our current study, the electronic tongue could also predict, pH, water activity, and dry matter content of all the different meat breeds with acceptable accuracies. The pH could be predicted with the best accuracies in all the different meat breeds. This is particularly important because, as reported in the literature and proven in our results, a low ultimate pH results in meat proteins having decreased water-holding capacity and a lighter color. Conversely, a higher ultimate pH will give a darker color and less drip loss, which also affects eating quality characteristics, such as juiciness, tenderness, and taste .Modeling and determination of meat origin and physico-chemical properties is a promising step that can help monitor increasing cases of meat fraud (misrepresentation) and also help consumers make the right decision, with regards to meat preference. In our study, the purported meat quality differences between five prominent cattle breeds were ascertained using both conventional and advanced rapid methods. Our results suggest that, although physico-chemical analysis and sensory evaluation have both proven to be effective methods of meat characterization, the electronic tongue presents higher sensitivities of component detection that be used to rapidly discriminate meat quality in the studied different beef breeds. This especially important as meat origin and quality have the potential to impact both economic revenue in the meat industry, as well as consumer perception. It is also important to note that our study only proves the applicability and effective of this corelative technique. Further studies would be required if they are to be applied to discriminate other breeds. Further study is, however, recommended to strengthen the robustness of LDA and PLSR prediction models.


Boundary objects can add value for innovative design and implementation research in health care through their organizational focus and the dynamic structure between ill-structured and tailored use. However, when innovation is approached as a boundary object, more attention will need to be paid to the preimplementation phase. Research and design thinking pay attention to the preimplementation stage but do not have a social or organizational focus per se. The integration of boundary objects in design methodologies can provide a more social and organizational focus in innovative design projects by mapping out the mechanisms that occur at boundaries during design. Four dialogical learning mechanisms that can be triggered at boundaries have been described in the literature: identification, coordination, reflection, and transformation. These mechanisms seem suitable for integration in innovative design research on health.Focusing on innovation in health, this study aims to find out whether the different learning mechanisms can be linked to studies on health innovation that mention boundary objects as a concept and assess whether the related mechanisms provide insight into the stage of the design and implementation or change process.The following 6 databases were searched for relevant abstracts: PubMed, Scopus, Education Resources Information Center, PsycINFO, Information Science and Technology Abstracts, and Embase. These databases cover a wide range of published studies in the field of health.Our initial search yielded 3102 records; after removing the duplicates, 2186 (70.47%) records were screened on the title and abstract, and 25 (0.81%) papers were included; of the 13 papers where we identified 1 mechanism, 5 (38%) described an innovation or innovative project, and of the 12 papers where we identified more mechanisms, 9 (75%) described the development or implementation of an innovation. The reflective mechanism was not identified solely but was present in papers describing a more successful development or implementation project of innovation. In these papers, the predetermined goals were achieved, and the process of integration was relatively smoother.The concept of boundary objects has found its way into health care. Although the idea of a boundary object was introduced to describe how specific artifacts can fulfill a bridging function between different sociocultural sites and thus have a social focus, the focus in the included papers was often on the boundary object itself rather than the social effect. The reflection and transformation mechanisms were underrepresented in the included studies but based on the findings in this review, pursuing to trigger the reflective mechanism in design, development, and implementation projects can lead to a more fluid and smooth integration of innovation into practice. However, not striving for consensus but identifying and addressing needs on the front end requires a fundamentally different approach. Much more attention will need to be paid to the preimplementation phase, which is seldom included in frameworks  OR boundary cross*[tiab]) AND  mentioned boundary objects, (2) involved an empirical study, or (3) otherwise focused on a newly developed or implemented innovation. Papers meeting these criteria were selected for full-text screening.The following exclusion criteria were used for full-text screening: (1) non–peer-reviewed papers such as abstracts, conference posters, or trade journals; (2) papers with full text not available; (3) papers in languages other than English; (4) monographs or short reports; and (5) papers with not sufficient information in the abstract.After removing the duplicates, the papers were screened based on title and abstract using Rayyan . A totalOur initial search yielded 3102 records. Of the 3102 records, after removing the 916 (29.53%) duplicates, 2186 (70.47%) records were screened based on their titles and abstracts. Next, of the 65 records, we screened the full text, leaving 25 (38%) papers for inclusion  ,93 descrIn 8% (2/25) of the papers, the identification mechanism was deduced in more innovative projects. Nielsen and Mengiste  describeBjørn et al  describeIn 16% (4/25) of the papers, we identified the coordination mechanism to facilitate cooperation in daily practice. Approximately 50% (2/4) of these papers presented controlled clinical vocabulary to facilitate and coordinate collaboration between different professionals ,87. ApprIn 12% (3/25) of the papers, we identified a coordination mechanism to promote innovation. The coordination mechanism was identified in the first of 2 included papers from Sajtos et al , where tGregory et al  evaluateWe identified no papers which merely reported the reflection mechanism.The transformation mechanism was identified in 8% (2/25) of the studies. Both studies described a more extended transformational development in retrospect. Fox  describeBoundary objects are not merely passive vehicles that allow communication between communities of practice or knowledge but elements that encapsulate the broader social meaning of a concept, theory, technology or practice, and the underlying relations surrounding its development and adoption.This study described the surgery profession’s transformation process, partly through boundary objects, from only a healer of disease to a healer of disease who was also a safety procurer.The second historical perspective was written by Stewart and Watson , who deseveryday practices, 9 (75%) identified multiple mechanisms that focused explicitly on developing or implementing a new tool, 6 (50%) were concentrated mainly on professionals and professional collaborations, and 3 (25%) actively focused on processes involving clients or patients at the center.Of the included 25 studies, in 12 (48%) studies, we identified ≥1 mechanism. Of these 12 papers, 3 (25%) focused more on structuring Jentoft  describeKeshet and Popper-Giveon  explicitMelo and Bishop  describe.”This section describes the 24% (6/25) of papers where multiple identified mechanisms focused on developing or implementing a new tool to enhance or trigger professional collaboration. In 33% (2/6) of these papers, we discovered the identification and coordination mechanisms. Marabelli et al  describebringing the existing differences to the foreground, applying the identification and coordination mechanism. In addition to the software, which the authors explicitly called a boundary object, many sessions, workshops, test sessions, and prototypes were described, which also had the characteristics of boundary objects.Mengiste and Annestad  reportedIn the paper by Håland, Røsstad, and Osmundsen , coordinIn 12% (3/25) of the papers focusing on professionals and professional collaboration, we identified all 4 mechanisms. Jensen and Kushniruk  presenteClinical simulation was used as a boundary object and thereby achieved mutual clinical agreement on the content. By using clinical simulation, knowledge was transferred and transformed between the different communities of practice to support gaining a shared understanding.This was mainly to overcome organizational barriers. As they presented in their case study, the clinical simulation might have helped form “shared mental models and shared understanding of user requirements, work practice and organizational requirements” within an innovation project. The boundary objects approach helped analyze vital issues and triggered a reflective approach to improving solutions. This case study showed that the adoption and acceptance of new technology might be significantly improved by leading end users and other important stakeholders within the organization through all mechanisms.Sajtos et al  introducin-between versions of the tool acted as boundary objects. During implementation, 2 events occurred. The first event resulted from new circumstances, which were illustrative of solutions: problems are not static. This event was overcome during the project. The second event led to a breach of trust between the stakeholder groups and, thus, to the project’s end. The initially overcome differences between the stakeholder groups were reinforced again by triggering the identification mechanism in a different way than the first time.Kajamaa  reportedOf the 25 studies, 3 (12%) actively focused on processes involving clients or patients at the center. In these studies, clients or patients actively participated, and 3 mechanisms were identified. Islind et al  applied The narratives, in the forms of patient stories, played a central role for understanding the patient group and the healthcare professionals as the needs of both user groups needed to be accommodated for.In a way, the narrative became the container of the essence of being a patient.metaphorical phase, boundary objects facilitated conversation, collaboration, and consultation among stakeholders, aligning with the coordination mechanism. In the structured phase, the boundary objects matured more as prototypes. They triggered a conversation about the platform’s future functions, aligning with the first signs of transformation. Their conclusion stated the following:In what Islind et al  called tDesigning with boundary objects might slow down the design process initially but actually speed up the programming process as fewer aspects will come as a surprise during the software development when everything has been negotiated thoroughly on beforehand.In a study by Islind and Snis , the focMediating tool for a deepened caring conversation-in-practice where interactions and realizations generate new emerging properties and opportunities. The boundary object-in-use proved to function as a conversation starter where the use facilitated fruitful conversations between the elderly and caregivers about new aspects of grocery shopping..” This reshaping of the home care practice affected the caregivers’ role, “evolving into a more meaningful caretaking and nurturing role.”In addition, new diet and “nutrition explorations were interpreted and negotiated via their evolved conversationTerlouw et al  describebarricades and mazes that generate conflict and reinforce boundaries and existing differences, something that Langley et al [This review shows that the concept of boundary objects has found its way into health care. The use of the concept has been growing since 2008, with a significant number of papers describing boundary objects from the past 5 years. In the reviewed studies, we see that boundary objects are mainly used to shape and organize multidisciplinary work, close to the original explanation of Star and Griesemer , or to sey et al  also desey et al , we saw designated boundary objects and boundary objects in use [object in use. The analogy can also be made by applying a boundary object from a more ill-structured to a more well-structured context. In a second included paper of Islind et al [Although the concept of a boundary object was introduced to describe how specific artifacts can fulfill a bridging function between different sociocultural sites  and thuss in use ,104,105.nd et al , they deThe reflection and transformation mechanisms are underrepresented in the included studies. Of the 25 studies, 2 (8%) describe the transformative effect of boundary objects from a historical perspective ,99, descOn the basis of the findings of this study, for future design and implementation projects, the social focus of boundary objects can add value to innovation projects. Pursuing to trigger the reflective mechanism can lead to the benefit of more fluid and smooth integration of innovation into practice. Here, the boundary object perspective avoids the pursuit of consensus, which often proves unfeasible in complex practices with many stakeholders. The reflective mechanism creates a shared awareness that there are multiple perspectives and needs. This awareness can lead to a shared change space in which innovation can flourish.As seen in previous research , little Another observation was that many innovations in the included studies were more administrative systems, such as electronic patient files. These are pre-eminently systems with which different disciplines must work, and boundary objects are thus helpful; however, 12% (3/25) of studies showed that boundary objects are also of added value in research in which clients or patients have an active role. This observation raises the idea that there are still more gains that can be found by involving end users earlier in design processes.The included papers were subject to the interpretation, discussion, and consensus of the reviewers . To counteract subjectivity as much as possible, papers were double-blind reviewed by 2 reviewers in the title and abstract scan (GT and DK). They were only included in the consensus of both reviewers. In the full-read phase, 20% (13/65) of the papers were double-blind reviewed by 2 reviewers (GT and LV) before they were discussed. No disagreements on inclusion occurred during the discussion.The concept of boundary objects has found its way into health care. In this review, we saw that boundary objects in health are primarily used to shape and organize multidisciplinary work or to surface differences in, for example, the interpretation of a concept from different contexts or disciplines. Although the concept of a boundary object was introduced to describe how specific artifacts can fulfill a bridging function between different sociocultural sites and thus have a social focus, the focus in the included papers was often on the boundary object itself rather than the social effect. The reflection and transformation mechanisms were underrepresented in the included studies; however, based on the findings in this review, pursuing to trigger the reflective mechanism in design, development, and implementation projects can lead to the benefit of more fluid and smooth integration of innovation into practice.


Heart failure with preserved ejection fraction (HFpEF) has become a major health issue because of its high mortality, high heterogeneity, and poor prognosis. Using genomic data to classify patients into different risk groups is a promising method to facilitate the identification of high-risk groups for further precision treatment. Here, we applied six machine learning models, namely kernel partial least squares with the genetic algorithm (GA-KPLS), the least absolute shrinkage and selection operator (LASSO), random forest, ridge regression, support vector machine, and the conventional logistic regression model, to predict HFpEF risk and to identify subgroups at high risk of death based on gene expression data. The model performance was evaluated using various criteria. Our analysis was focused on 149 HFpEF patients from the Framingham Heart Study cohort who were classified into good-outcome and poor-outcome groups based on their 3-year survival outcome. The results showed that the GA-KPLS model exhibited the best performance in predicting patient risk. We further identified 116 differentially expressed genes (DEGs) between the two groups, thus providing novel therapeutic targets for HFpEF. Additionally, the DEGs were enriched in Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways related to HFpEF. The GA-KPLS-based HFpEF model is a powerful method for risk stratification of 3-year mortality in HFpEF patients. Heart failure (HF) is the leading cause of death and disability worldwide among older adults . Over 50While numerous risk assessment models have been developed in cohorts with HFrEF or a mixture of HFrEF and HFpEF, risk prediction in HFpEF patients has been less studied . This maWith advances in sequencing and computer technology, high throughput expression data can be extracted without limits. Genomic measures of gene expression offer rich information about the underlying disease mechanism and have provided new possibilities of using these molecular data to understand the disease gene function and further predict disease outcomes . Based oThe purpose of this work is to evaluate six different risk stratification models and to predict the survival risk of HFpEF patients based on gene expression profiles using data from a high-quality epidemiologic study, the Framingham Heart Study (FHS). We applied five advanced machine learning methods  to build an optimal risk stratification model. Identification of patients with a high risk of HFpEF will be helpful for targeted interventions and clinical trials to further improve the survival of HFpEF patients.http://dbgap.ncbi.nlm.nih.gov). The FHS has recruited participants from Framingham, MA, United States, to undergo biennial examinations to investigate cardiovascular disease and its risk factors since 1948  transformed and then standardized (d = [2n/log(n)] features; The expression data contained 17,873 gene expression probes. We mapped these probes to genes following the annotation from the Affymetrix Human Exon 1.0 ST GeneChip platform, which yielded 17,358 genes. The gene expression data were dardized . A variaThe clinical outcome was defined as a good or poor outcome based on patients’ survival status. The good-outcome group had event-free survival for at least 3 years [survival time was measured from the time of admission for HFpEF diagnosis to the time of last follow-up (2011) or time of death from cardiovascular disease]. The poor-outcome group included patients who died because of cardiovascular disease during the 3-year period. We further explored the differentially expressed genes (DEGs) between the good-outcome and poor outcome groups using significance analysis of microarrays  and thenNR into a high-dimensional feature space ℱ, and therefore, original data that cannot be linearly separated in NR can be separated in ℱ , sensitivity (Se), specificity (Sp), accuracy (ACC), Youden index, G-means, and Matthews correlation coefficient (MCC). The MCC and AUC were mainly used to evaluate the model performance because they are more comprehensive evaluation criteria. We employed one-way ANOVA, followed by Dunnett’s multiple-comparison test, to compare the performance of the GA-KPLS and the five other models . Statistical significance was indicated by a value of At the end of the 3-year period, 42 patients (28.19%) met the study endpoint of cardiovascular disease-related death, and 107 patients (71.81%) had survived. There were 91 males (61.07%) and 58 females (38.93%). The average age was 75.02 (±8.02) years old. We compared the classification performance of the six models: GA-KPLS, RF, LASSO, RR, SVM, and Logit. The evaluation index of the six models was summarized as the average value obtained by repeating the data partition 1,000 times. mean = 0.921). The Kaplan-Meier curves based on the original and predicted data yielded significantly different survival probabilities (p < 0.0001). To demonstrate the clinical significance of identifying high-risk patients, we selected the prediction result of one random split with 120 training samples and 29 testing samples, which gave an MCC = 0.920  is an important member of the CTRP protein family. C1QTNF9 knock-out mice were protected from left ventricular dilatation and contractile dysfunction; however, C1QTNF9 overexpression promoted ventricular remodeling and systolic dysfunction. TECRL was recently suggested to play a key role in the electrical activity of the heart. TECRL affects the electrical conduction system of the heart by causing mutations in a calcium-processing protein, which eventually leads to arrhythmia , G protein-coupled receptor signaling pathway , and protein binding . The plasma membrane plays important roles in maintaining homeostasis, cell material exchange, and information transmission  study risk scores  and MetaSecond, because of the highly heterogenous nature of HFpEF, a consensus has not been reached on which predictors can be used to reliably predict HFpEF. We demonstrated that gene expression can be used to predict HFpEF survival status with high accuracy using the GA-KPLS prediction model. With the availability of increasing types of omics data , we can further improve the prediction accuracy by integrating different data sources with the GA-KPLS model. Our study illustrates the development of new machine learning methods for HFpEF risk prediction by integrating different omics data types.Current studies have focused on single or multiple clinical indicators to identify patients at high risk for HFpEF. However, most methods can only achieve an AUC of 0.7, which is unrealistic for application in clinical practice . Many reRisk prediction in HFpEF patients using the GA-KPLS model may (1) serve to motivate patients to adhere to recommended treatments and lifestyle modifications ; (2) helHowever, our study had some limitations. First, because of the lack of additional external data on HFpEF, we cannot validate our findings in another data set. Second, we focused on gene expression data in our study. As lifestyle is an important risk factor for HF, further research should be performed to predict HFpEF risk by integrating both clinical and genomic data to improve the prediction performance because potential interactions may exist between these factors. Third, the HFpEF data set is imbalanced, with a ratio of 28:72 between the poor-outcome and good-outcome groups. However, the GA-KPLS and SVM methods performed well, with high sensitivity and specificity. If either low sensitivity or specificity becomes a concern, the SMOTE algorithm can be applied , which iIn conclusion, the GA-KPLS-based HFpEF prediction model using gene expression data represents a valuable tool to improve the prognosis of HFpEF patients with different risk levels. The discovered transcriptional biomarkers of HFpEF provide new insight to the understanding the complex mechanism of HFpEF, leading to the development of novel targeted therapies for HFpEF. It is expected that integrating multi-omics and clinical data can further improve HFpEF outcome prediction, leading to the development of targeted, adaptive, and precision treatment of HFpEF patients with different risk levels.http://dbgap.ncbi.nlm.nih.gov.The data analyzed in this study is subject to the following licenses/restrictions: The data analyzed in this study require NIH approval through the dbGap website. Requests to access these datasets should be directed to LZ and ZG performed the study and drafted the manuscript. BW, YW, ZL, RF, and HtY participated in the data processing and analysis. HY provided the clinical interpretation. HC and YC conceived of the idea and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Generally, the symptoms of normal pressure hydrocephalus (NPH) are defined by the Hakim-Trias, i.e., gait disturbance, cognitive impairment, and urinary incontinence . The gaiThe cognitive impairment of NPH patients corresponds to a subcortical-frontal dementia. Affected areas of cognition are attention, concentration, psychomotor velocity, working memory, executive functions, as well as spatial and constructive ability and flexibility of thinking , 14, 21.Urinary incontinence in patient with NPH is very common and occurs in up to 91% of the cases with a gender bias towards females and includes always detrusor over reactivity . But, thPostural instability is part of the gait disturbance from which NPH patient suffer from. The origin of the disturbance of balance is not clear and seems to contain several different domains. There are some studies which suggest a central vestibular origin , 13; othAs above mentioned not only the lower extremity motor function is affected by the disease but also that of the upper extremities. Introduced test are the Grooved Pegboard Test , 22, the