TNF) to improve the precision of anti-TNF biological therapy strategies in patients with ulcerative colitis (UC). Analysis of published literature showed that mucosal TNF could affect the precision of the early identification of candidates who will benefit from anti-TNF therapy prior to treatment, the assessment of response and mucosal healing, and the prediction of discontinuation of anti-TNF biological therapy and relapse after drug withdrawal. Challenges and limitations of using mucosal TNF as a biomarker in applying individualized anti-TNF biological therapy in patients with UC still remain and need to be further investigated.Anti-tumor necrosis factor (TNF) biological therapy has generally been accepted as a standard therapeutic option in inflammatory bowel disease (IBD) patient who are refractory to steroids or immunomodulators. However, the primary and secondary nonresponse rates to anti-TNF bioagents in patients with IBD are high. To improve the response rate, anti-TNF bioagents must be offered to the appropriate IBD patients, and the withdrawal of anti-TNF bioagents needs to be done at the right time. In this context, reliable and reproducible biomarkers can provide important supportive information for clinicians to make correct decisions based on the patient’s individual situation. In this review, we summarized the current understanding of using mucosal TNF transcript ( Ulcerative colitis (UC), that is one of main types of inflammatory bowel disease (IBD), is a group of chronic inflammatory disorders that mainly affect the colorectal tract. Although the exact etiology for UC has not been fully identified thus far, the currently most cited hypothesis proposes that a combination of environmental factors, genetic predisposition, and dysregulated immune response may significantly increase the risk for UC . Recent The traditional approaches for UC management rely on diverse medicines including aminosalicylates, corticosteroids and immunomodulators according to disease activity and severity. The clinical goals of treatments are to suppress inflammation, at best obtain mucosal healing, and finally improve the UC patient’s quality of life. However, being refractory to existing medicines and disease relapse have frequently been reported , 9. In tTNF) as a biomarker of disease activity, the time of switching from ongoing conventional treatment to anti-TNF therapy and selecting candidates, the anti-TNF therapeutic response, and timepoints for drug withdrawal and relapse after anti-TNF discontinuation in patients with UC  levels correlated with the UC disease activity index (UCDAI) score in newly diagnosed UC patients without treatment  compared with the above selected biomarker and clinical parameters. In the second step , the results demonstrated that the validated cutoff values of mucosal TNF with histological index  showed a high reliability and specificity to predict a need for anti-TNF biological therapy within the first year of disease after diagnosis in a cohort with 89 UC patients -6, IL-8, and IL-10 transcripts, and confirmed that the naïve UC . Further with UC . In conj with UC .TNF levels predict the clinical response to anti-TNF biologic agents and can be used for the evaluation of anti-TNF therapeutic efficacy. Tsukada et al. . Indeed, our studies have shown that changed mucosal TNF levels might predict remission and mucosal healing rates in response to IFX in patients with UC   . We founpatients . Our resy levels . Moreoven of IFX . Other cTNF level becomes normalized  or cyclophosphamide therapies  in patients with IBD in the context of precision medicine. Mucosal TNF level alone as a biomarker in precision anti-TNF biological therapy may have its shortcomings. The efficacy of anti-TNF therapy also depended on the formation of anti-drug antibodies, and a high level of anti-drug antibodies might significantly decrease the efficacy of anti-TNF bioagents. Yarur et al. , Norway (Program No. SFP-922-10). The funder did not play any role in paper design, data collection, data analysis, interpretation, writing of the paper. We apologize for not being able to cite all the excellent studies and reviews due to space limitations.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) , c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) , or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders. Tumor necrosis factor (TNF)-α levels are increased in patients with dilated cardiomyopathy and ischemic heart disease but not expressed in the normal heart. The severity of heart failure (HF) and survival rate correspond to the levels of TNF-α in the serum of patients ,2. TNF-αChemokines are a large family of key mediators during immunoregulatory and pro-inflammatory responses. Chemokines are classified as cysteine–cysteine (C–C) and cysteine–noncysteine–cysteine (C–X–C). Chemokine (C-C motif) ligand 20 (CCL20) is also known as macrophage inflammatory protein-3α or liver activation regulated chemokine, which is a member of the chemokine family. CCL20 elicits its effects by activating the chemokine receptor CC chemokine receptor 6 (CCR6), which is the only chemokine known to interact with CCR6 . It has The majority of cells in the heart consist of fibroblasts and myocytes. Cardiac fibroblasts play a prominent role in maintaining normal cardiac structure and function and participate in cardiac development and heart remodeling during pathological conditions . These cHowever, the molecular mechanisms of TNF-α-induced CCL20 expression in HCFs remain unclear. The objective of this study is to address the mechanisms underlying TNF-α-induced CCL20 expression in HCFs. This study clarifies the role of CCL20 in cardiac inflammation and the mechanisms of TNF-α-induced CCL20 expression. Our results suggest that TNF-α-induced CCL20 expression is, at least partially, mediated through binding with TNFR1 to activate epidermal growth factor receptor (EGFR)/p38 MAPK and c-Jun amino N-terminal kinases (JNK)1/2/forkhead box O (FoxO)1 pathways or stimulate NF-κB signaling in HCFs. These findings can provide potential therapeutic strategies for treating inflammatory heart diseases.To evaluate the effect of TNF-α on CCL20 production, HCFs were treated with various concentrations of TNF-α  for the indicated time intervals . As shown in TNF-α has been shown to regulate cellular functions through TNFR1 or TNFR2 in different types of cells ,4. To deTNF-α-induced expression of various genes has been shown to be involved in the transactivation of EGFR ,16. To aTNF-α can stimulate p38 MAPK phosphorylation leading to the expression of various genes in different types of cells ,17,24. TActivation of JNK1/2 has been shown to trigger the expression of inflammatory proteins induced by TNF-α in various types of cells ,17. To dTNF-α-induced expression of inflammatory proteins has been reported to be mediated through the activation of intracellular signaling components and transcription factors such as FoxO1. To investigate whether FoxO1 regulated TNF-α-induced CCL20 protein production and gene expression, as shown in NF-κB is a master transcription factor involved in the expression of inflammatory proteins induced by TNF-α in different types of cells ,25. To dIn ischemic heart disease, CCL20 has been demonstrated to have higher circulating levels . TherefoTNF-α is a pro-inflammatory cytokine that activates innate immune pathways and triggers inflammatory responses. It regulates several critical cellular functions, including cell proliferation, survival, apoptosis, and differentiation. TNF antagonism is cardioprotective in the in vivo model of HF ,27. IndeRTKs play an important role in the control of most fundamental cellular processes, including the cell cycle, cell migration, cell metabolism, and survival, as well as cell proliferation and differentiation. During cell injury, the inhibition of EGFR also prevents the activation of inflammation, fibrosis, apoptosis, and oxidative stress pathways . TNF-α iEGFR is a part of the signaling networks activated by various stimuli which are independent of EGF. Therefore, EGFR is involved in the cellular responses and signaling events initiated by these various stimuli. A diverse group of G protein-coupled receptors, adhesion receptors, and cytokine receptors can induce EGF-independent tyrosine phosphorylation of the EGFR . It has Chlamydophila pneumoniae infection induces the release of CCL20 through activation of p38 MAPK but not ERK1/2 in human bronchial epithelial cells [Mitogen-activated protein kinases (MAPKs) regulate several kinds of cellular programs by relaying extracellular stimuli to intracellular responses. All eukaryotic cells have many MAPK pathways, which coordinately regulate gene expression, protein synthesis, metabolism, mitosis, motility, cell cycle machinery, survival, apoptosis, and differentiation. In mammals, MAPKs are characterized into seven groups. Conventional MAPKs include the extracellular signal-regulated kinases 1/2 (ERK1/2), JNK1/2/3, p38 isoforms , and ERK5 . So far,The transcription factor FoxO1 has been reported to be activated by TNF-α in both in vitro and in vivo studies . It was NF-κB represents a family of inducible transcription factors which upregulate a series of genes, including cytokines and chemokines, which are involved in various processes of the inflammatory and immune responses. NF-κB has been implicated in the pathogenesis of many kinds of inflammatory disorders. They have also been linked to several pathologic processes in the myocardium, including hypertrophy, apoptosis, cardiomyocyte pro-inflammatory cytokine release, and ischemia/reperfusion injury . CCL20 aTM NT membrane was purchased from Pall Life Sciences . The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer . Anti-phospho-EGFR , anti-phospho-p38 MAPK , anti-phospho-JNK1/2 , anti-phospho-FoxO1 , and anti-phospho-p65  antibodies were purchased from Cell Signaling . Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology . Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology . AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science . AS1842856 was obtained from EMD Millipore . Helenalin was purchased from Cayman Chemical . Recombinant human TNF-α protein was purchased from R&D Systems . TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich .Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific . BioTrace2 atmosphere using the method of Yang et al. [w/v) trypsin/0.53 mM EDTA for 1 min at 37 °C. The cell suspension was diluted with DMEM/F-12 containing 10% (v/v) FBS to a concentration of 2 × 105 cells/mL. The cell suspension was seeded in (1 mL/well) 12-well culture plates, (2 mL/well) 6-well culture plates, and (10 mL/dish) 10 cm culture dishes and made quiescent at confluence by incubation in serum-free DMEM/F-12 for 24 h. The following experiments were carried out using HCF passages from 5 to 7.HCFs isolated from the human heart were purchased from ScienCell Research Lab . They were cultured in a DMEM/F-12 medium supplemented with 10% FBS and antibiotics  at 37 °C in a humidified 5% COg et al. . When thw/v) BSA in TTBS for 1 h. Membranes were incubated overnight at 4 °C with a primary antibody used at a dilution of 1:1000 in TTBS, washed several times by TTBS, and then incubated with 1:2000 dilution of an anti-rabbit or anti-mouse secondary antibody for 1 h. Following incubation, the membranes were washed comprehensively with TTBS. An anti-GAPDH antibody was used as an internal control. Immunoreactive bands on membranes were detected using ECL reagents and captured by a UVP BioSpectrum 500 Imaging System . UN-SCAN-IT gel software 7.1  was used to quantify image densitometry.We used the method of Yang et al.  for the (Ct test gene-Ct GAPDH) (Ct = threshold cycle). The primer sequences were as follows:Total RNA was extracted with 0.5 mL/dish Trizol  from HCFs treated with TNF-α for various time intervals using the method of Yang et al. . When inHuman CCL20: (NM_001130046.2)forward sequence: 5′-TCTGTGTGCGCAAATCCAA-3′;reverse sequence: 5′-CCATTCCAGAAAAGCCACAGT-3′;probe sequence: 5′-TGTGCGTCTCCTCAGTAA-3′.Human GAPDH: (NM_001357943.2)forward sequence: 5′-GCCAGCCGAGCCACAT-3′;reverse sequence: 5′-CTTTACCAGAGTTAAAAGCAGCCC-3′;probe sequence: 5′-CCAAATCCGTTGACTCCGACCTTCA-3′.For the construction of the CCL20-luc plasmid, the human CCL20 promoter, a region spanning −817 to +33 bp , was clo5 cells/mL onto 12-well plates or 10 cm dishes until reaching about 70% confluence. Cells were washed once with PBS and then added to a 1 mL/well or 5 mL/dish of Opti-MEM medium  before transfection using the method of Yang et al. [HCFs were plated at 2 × 10g et al. . The TNFhttps://www.rndsystems.com/products/human-ccl20-mip-3-alpha-quantikine-elisa-kit_dm3a00 (accessed on 1 May 2020)) according to the instructions of the manufacturer . The levels of the optical densities were determined immediately using a microplate reader set to 450 nm .Secreted CCL20 protein levels in culture media of HCFs stimulated with TNF-α were quantified using ELISA development kits (HCFs were cultured in 6-well culture plates with coverslips until reaching about 70% confluence and were serum-free for 24 h. Growth-arrested cells were transfected without or with scrambled and p65 siRNA and then stimulated with TNF-α (5 ng/mL) for the indicated time intervals. The cells were fixed, permeabilized, and stained using anti-phospho-NF-κB p65 antibody (1:200 dilutions) and 4′,6-diamidino-2-phenylindole (DAPI) after washing with ice-cold PBS using the method of Yang et al. . The sta2O and subjected to Real-time PCR amplification performed with the SYBR GREEN assay system  with the primers of CCL20 described below. PCR was performed using a StepOnePlus™ Real-Time PCR System . The relative amount of the target gene was calculated using 2(Ct test gene-Ct GAPDH) (Ct = threshold cycle). The primer sequence is as follows:To detect the association of nuclear proteins with human CCL20 promoter, HCFs in a 10 cm dish were grown to confluence and serum-starved for 24 h. After treatment with TNF-α (5 ng/mL), protein–DNA complexes were fixed by 1% formaldehyde in the medium using the method of Yang et al. . The fixHuman CCL20 ChIP qPCR sequences (NC_000002.12):Forward: 5′-CCTGTGTGGGGCTGACCTTTGTATCG-3′;Reverse: 5′-GGAGCAAACTCTTGGTACAGCACATGG-3′.p < 0.01, and there was no significant variance inhomogeneity. p values < 0.01 were considered to be statistically significant. Error bars were omitted when they fell within the dimensions of the symbols. All the data were expressed as mean ± SEM in three individual experiments (n = 3).GraphPad Prizm Program 6.0 software  was used as a tool for statistical analysis. We used the methods of statistical analysis previously described by Yang et al. . We usedIn the present report, we concluded that TNF-α-induced CCL20 expression is, at least partially, mediated through binding to TNFR1, leading to transactivation of EGFR. Activated EGFR promoted the phosphorylation of p38 MAPK- or JNK1/2-dependent FoxO1 activation, which further bound with the FoxO1 response element (FRE) on the CCL20 promoter. In addition, TNF-α also activated NF-κB transcription factors. Either FoxO1 or NF-κB activation could enhance the expression of CCL20 induced by TNF-α, which may be engaged in the inflammatory responses in HCFs. A better understanding of mechanisms underlying the regulation of the CCL20 gene by TNF-α will support more opportunities to develop anti-inflammatory therapeutic strategies for treating cardiac inflammation. However, the limitation of the present study is the lack of in vivo data. It is worth exploring animal models further in the future.


The study of cytokine storm in COVID-19 has been having different edges in accordance with the knowledge of the disease. Various cytokines have been the focus, especially to define specific treatments; however, there are no conclusive results that fully support any of the options proposed for emergency treatment. One of the cytokines that requires a more exhaustive review is the tumor necrosis factor (TNF) and its receptors (TNFRs) as increased values of soluble formats for both TNFR1 and TNFR2 have been identified. TNF is a versatile cytokine with different impacts at the cellular level depending on the action form (transmembrane or soluble) and the receptor to which it is associated. In that sense, the triggered mechanisms can be diversified. Furthermore, there is the possibility of the joint action provided by synergism between one or more cytokines with TNF, where the detonation of combined cellular processes has been suggested. This review aims to discuss some roles of TNF and its receptors in the pro-inflammatory stage of COVID-19, understand its ways of action, and let to reposition this cytokine or some of its receptors as therapeutic targets. COVID-19 is an infectious disease caused by the Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2); it was declared a pandemic by the World Health Organization (WHO) in March 2020 . On 18 MEpidemiological evolution shows that of those SARS-CoV-2 infected, about 80% present mild or moderate disease, 15% of patients developed a severe disease requiring oxygen, and near to 5% evolve to critical disease where complications such as respiratory failure, acute respiratory distress syndrome, sepsis, and septic shock, thromboembolism, and even multi-organ failure can manifest ,7. CurreSince the beginning of the pandemic, cytokine levels emerged as indicative of disease progression, suggesting that the pro-inflammatory cytokines play a determining role in the pathogenesis of COVID-19. The pro-inflammatory cytokines modify the immune system and, if it is out of control, in some cases, lead to fatal consequences ,11.The absence of regulatory mechanisms to modulate the inflammatory process is responsible, at least partially, for favoring a cytokine storm that progresses to organ dysfunction and even death ,12,13. TThe concept “cytokine storm” was published for the first time in 1993 in graft-versus-host processes; later, it was appropriated in viral and some bacterial infections . It has Under the context of non-infectious diseases, such as those with an autoimmune origin that display a robust inflammatory process such as systemic lupus erythematosus and systemic juvenile idiopathic arthritis, the cytokine storm is associated with macrophage activation syndrome (MAS) . In thisIn infectious diseases such as COVID-19, the cell entry used by the virus impacts the activation of the immune response; this is related in particular to the receptors used . The angACE2 is shedding on the membrane cell surface by the effect of TMPRSS2 and the disintegrin and metalloprotease 17 (ADAM17), and this last also is responsible for TNF, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2) shedding ,32. RepoOnce the virus is inside the cell, the activation of cell death mechanisms such as apoptosis, necroptosis, pyroptosis, and PANoptosis induce the release of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs), mainly Toll-like receptors (TLR); molecules expressed on natural killer cells, dendritic cells, macrophages, and neutrophils ,14,36,37In the infection site, chemoattractants, such as CCL2 and CXCL8, are produced to help in cell mobilization as monocytes and neutrophils, which can also induce inflammatory mediators; epithelial and endothelial cells also contribute to both inflammation and induction of cell death mechanisms ,38. SingAnother consequence that maintains a constant inflammation status in the infection site is the inflammasome activation by the generated PAMPs and DAMPs. NLRP3 is the central protein platform for caspase-1 activation, leading to the proteolytic maturation of IL-1β and IL-18. The inflammasome induces an inflammatory cell death termed pyroptosis ,36,40,41Although the initial reports indicated a relationship between the severity of the disease and high inflammatory cytokine levels, recent evidence questions whether COVID-19 activates a cytokine storm. Some authors described that pro-inflammatory cytokines levels are not higher than in other pathologies with acute respiratory distress syndrome (ARDS), and others suggest that there are lower levels in COVID-19 than those observed in non-COVID-19 pathologies ,44,45,46Several efforts have focused on identifying cytokine profiles that predict a poor outcome to avoid mortality. In this regard, at the beginning of the pandemic, several reports converged in identifying increased levels of individual cytokines like IL-6 as potential biomarkers or targets for therapy. However, it later was proposed that the impact of the combined effect of two or more cytokines such as IL-6, IL-1β, TNF, IFN-γ, and CXCL10, among others, must be considered ,12. IL-6The conventional approved immunotherapy to mediate neutralizing the inflammatory function through monoclonal antibodies (mAbs) has been used in different pathologies . mAbs arWorldwide, there are around eleven antibodies targeting SARS-CoV-2 or modulators of the deregulated immune response that have received approvals both as emergency use or authorized . The comAnakinra is another mAb used to modulate the pro-inflammatory response; it is an interleukin receptor antagonist that binds to the IL-1 type I receptor; it has been considered in combination with tocilizumab for COVID-19 clinical trials ,67. In tThus far, therapeutic antibodies targeting the virus, some pro-inflammatory cytokines, or one of their receptors are highlighted as a focus of attention in COVID-19.TNF is one of the most critical pro-inflammatory cytokines of the innate immune response and mediates pleiotropic effects, which implies action on diverse cells subpopulations to mediate a wide range of activities such as the production of inflammatory mediators, cell proliferation, and cell death. TNF is produced by macrophages, T-, B- NK-, dendritic cells, and fibroblasts . TNF is In addition to the ADAM17 activity, the soluble formats of TNFR1 and TNFR2 may result from alternative splicing . The solAnti-TNF therapy was approved by the U. S. Food & Drug Administration (FDA) in 1998, and currently, it is efficiently administered as a treatment for diverse inflammatory processes like rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, Crohn´s disease, plaque psoriasis, among others ,76,85,86There are five types of biological TNF inhibitors approved for use; the difference between them is the nature of their origin . The firLegionella pneumonia and Listeria monocytogenes [Mycobacterium tuberculosis and hepatitis B reactivation, anti-TNF inhibitor antibodies, and neurological impact [The anti-TNF biological agents target both tmTNF and sTNF, and all of them trigger diverse effects such as neutralization, apoptosis, and modulation of the immune system; several facts may affect the efficacy of every anti-TNF inhibitor as pharmacokinetic, tissue penetration, affinity, and avidity, among others , reasonsytogenes ,85. Evenl impact ,92,93. IVarious pronouncements have been made to support the administration of anti-TNF agents in COVID-19 patients; they are based on the principle that the increase of this cytokine has severe effects on diverse cell subpopulations and its function blocked with the anti-TNF therapy is efficient in diverse autoimmune diseases ,19,48,94At present, there are ongoing clinical protocols to identify the efficiency of the anti-TNF therapy in COVID-19, some of them suggesting that COVID-19 patients treated with anti-TNF agents have a better prognosis ,99. In tIn 2020, the Crohn´s & Colitis Foundation of America recommended that patients with Inflammatory Bowel Disease (IBD) that develop COVID-19 stop the anti-TNF administration (biologics or biosimilars) until they recover. It was supported by the National Scientific Advisory Committee of the USA, the International Organization for the Study of Inflammatory Bowel Diseases (IOIBD), and the American Gastroenterological Association ,103,104.Mycobacterium avium subspecies paratuberculosis (MAP) infection has suggested that the Notch-1 signaling pathway induced by the anti-TNF therapy impacts IL-6 and MCL-1 expression in macrophages. Additionally, the authors showed that, in particular, adalimumab increases the TMPRSS2/ADAM17 ratio, suggesting the infection facilitated by TMPRSS2 [Recently, Keewan et al. suggested that PEG-, non-PEG-Certolizumab, and Adalimumab favor the infection of SARS-CoV-2 because the mode of action of these agents induces the Notch-1 signaling pathway promoting a pro-inflammatory environment . In addi TMPRSS2 .Although successful cases of immunotherapy with TNF inhibitors have been well documented for years, indeed, some adverse events are also known. A meta-analysis documented that the use of adalimumab, golimumab, infliximab, certolizumab, and etanercept, significantly increases fungal, viral, and bacterial  infections . DemyeliTNF gene might direct or predict the patient’s responsiveness to anti-TNF therapies, and TNF signaling pathways may be restricted to epigenetic regulation [TNF-308G>A polymorphism in the promoter region could be associated with a poor response to TNF inhibitors [Epigenetic control of the gulation . In partgulation . The TNFhibitors . At the hibitors . In thishibitors .In the case of COVID-19, there are two main unknowns related to TNF inhibitors: the risk of immunosuppression in the SARS-CoV-2 infection and the possibility that these agents could facilitate virus entry into target cells. Therefore, further molecular and clinical studies are necessary to understand the real potential of anti-TNF inhibitors in COVID-19. Meanwhile, novel treatment targets must be considered for evaluation, alone or combined with antiretrovirals . In thisScientific evidence shows that the TNF is not the alone-worker in COVID-19; data indicate that the couple TNF/IFN-γ synergizes to induce crosstalk of several types of cell death such as pyroptosis, necroptosis, and apoptosis (collectively named PANoptosis). It is regulated by signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), inducible nitric oxide synthase (iNOS), and nitric oxide (NO); and Caspase-8/RIPK3, all together induce an inflammatory cell death that is in detriment to the host. Under the COVID-19 context, it has also been identified that this TNF/IFN-γ synergism could be blocked with the mAbs co-administering .At present, it is well known that TNFR1 signaling is related to apoptotic cell death, but additionally, this receptor also promotes the release of pro-inflammatory molecules, including cytokines, chemokines, adhesion molecules, and matrix metalloproteinases ,117. ThuEfforts have been made to identify if sTNFR1 could represent a potential biomarker in COVID-19. A first report indicated that COVID-19 patients in an intensive care unit (ICU) have a higher level of a pro-inflammatory profile , including high sTNFR1 levels . PosteriThe use of anti-TNF therapy requires an integrative comprehension considering the roles of TNFR1 and TNFR2, as well as the soluble or transmembrane formats of the proteins . Based oAlthough the current TNF inhibitors are highly efficient, it has been well documented that total TNF inhibition may lead to severe side effects such as tuberculosis reactivation ,128. An TNFR1 inhibitors include a wide range of molecules, including antibodies, small molecules, and aptamers, that selectively inhibit TNFR1 but without affecting TNFR2 function. The first group of inhibitors includes derivatives of antagonist mAb that interact with the cysteine-rich domain 1 of TNFR1, competing with high affinity with the natural ligand ,136. To Muteins are mutant proteins of TNFR1-selective antagonists; structurally, they have peptide-linkers to enhance their stability and bioactivity and polyethylene glycol (PEG) modification to limit the reactive sites. These mutants showed good avidity and thermal stability for selective binding to TNFR1; using the arthritis murine model, muteins in plasma have an extended half-life, and regulatory T cells are increased compared to etanercept ,142,152.Though High-Throughput Screening (HTS) has been developed to identify selective small molecules TNFR1 ,146. Lo Another example of TNFR1 inhibitors is GSK1995057; it is a domain antibody (dAb) representing the minimal fraction of antigen-binding units, although it has a low molecular weight (10–13 kDa) is highly stable. One study reported the safety of the administration of GSK1995057 by nebulization; it was evaluated in both non-human primate models of acute lung injury and healthy subjects, suggesting that it could be a therapy for the prevention of acute respiratory distress syndrome .Thus, the previous evidence supports the hypothesis that the TNFR1-selective inhibitors, which do not affect the immunomodulatory functions of TNFR2, have the potential to be used as therapy for diverse inflammatory diseases, including lung affections as COVID-19. Despite the clinical successes of the anti-TNF therapy, reports also indicate that it can induce side effects because TNFR1 mediates cell death; the inflammation process could be a novel drug candidate, although it is necessary to provide pre-clinical data to support further its clinical use.Reports have shown diverse effects of anti-TNF agents in the context of COVID-19, some of which are beneficial, whereas others have suggested conflicting results based on the complexity of the nature of TNF and TNFRs pathways. More extensive research may be developed to understand the role of this pivotal axis in the innate immune response against SARS-CoV-2 infection. To support the use of the TNF axis in COVID-19 is imperative to include the dynamics and functions of the TNFRs in the development of selective and efficient therapies. Results suggest that sTNFR1 has an essential role in COVID-19 severity and mortality reason by which must represent a focus of attention in this pathology and a potential therapeutic target in future clinical studies.


Objective: If a damaged submarine cannot be rescued in time, it is necessary to carry out a submarine escape by free ascent. Decompression illness is the greatest threat to the safety of submariners. The maximum depth at which a safe escape can be carried out is unknown. This study intends to explore the maximum safe escape depth by observing the effects of simulated submarine escape at different depths on animal models.Methods: We evaluated pulmonary function indexes, blood gas values, blood cell counts, the myocardial enzyme spectrum, coagulation parameters, and proinflammatory cytokine levels in rats, electrocardiographic activity in rabbits after simulated 150-m, 200-m, 220-m, and 250-m submarine escape by free ascent.Results: An escape depth of 150 m did not cause significant changes in the indicators. An escape depth of >200 m led to pulmonary ventilation and gas diffusion dysfunction, hypoxemia, myocardial ischemia, and activation of the fibrinolytic and inflammatory systems. The magnitudes of the changes in the indicators were proportional to escape depth.Conclusion: An escape depth of 150 m in animal models is safe, whereas escape at > 200 m can be harmful. If a submarine is unable to surface, the preferred option for the survivors is to await rescue within the distressed submarine (DISSUB). However, if environmental conditions within the DISSUB make waiting for rescue untenable then a submarine escape by free ascent is performed. The escape procedure involves rapid pressurization of one or two personnel at a time to the ambient sea pressure in the escape tower. When the tower pressure is equalized to the ambient sea pressure, the upper hatch opens, and the submariners ascend to the surface due to the buoyancy of the Submarine Escape Immersion Suit.It is anticipated that decompression illness (DCI) limits the maximum depth at which escape can be achieved, and that the risk of pulmonary barotrauma limits the pressurization and ascent rates. The Royal Navy has done a lot in the development of modern submarine escape by free ascent. The testing of human subjects is reported up to real escapes by subjects from submarines lying at 180 m . HoweverHere, we evaluated the effects of escape depth on electrocardiographic activity, pulmonary function indexes, blood gas values, blood cell counts, the myocardial enzyme spectrum, coagulation parameters, and proinflammatory cytokine levels in rat lung tissue.Interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Elabscience Biotechnology . Acetonitrile, methanol, and water were of high-performance liquid chromatography grade .n = 10 per group). Rabbits were randomly divided into 150-m, 200-m, and 220-m escape groups (n = 2 per group).All studies with animals were approved by the Animal Care and Use Committee of Naval Medical Center. Adult 8–9-week-old male Sprague Dawley rats were used. In addition, six male New Zealand white rabbits weighing 3 kg were used to assess electrocardiographic activity. Animals were housed in cages and acclimatized to the housing facility for 1 week before experiments. Rats were randomly divided into the control group and 150-m, 200-m, 220-m, and 250-m escape groups (1/4 index. Following a short hold at depth (4 s of bottom time), the chamber was decompressed to the surface at 3 m/s (0.03 MPa)  .After decompression, rats were removed from the chamber and pulmonary function tests were performed. At 30 min after decompression, rats were anesthetized with 3% sodium pentobarbital, and blood samples were collected. The control rats were placed in the chamber without compression and decompression.Electrocardiography (ECG) was performed on rabbits before compression and after decompression. New Zealand rabbits were weighed, and 30 g/L sodium pentobarbital was slowly injected through the ear vein at a dose of 30 mg/kg. After anesthesia, the rabbit was placed supine and fixed on the operating table, and needle-shaped electrodes were inserted under the skin of the limbs. Three standard limb leads  and three augmented limb leads  were recorded. The calibration voltage was adjusted to 1 mV = 10 mm, the paper speed was 25 mm/s, and the electrocardiogram was recorded under quiet conditions.The pulmonary function of rats was tested by whole-body plethysmograph . Awake rats were placed in four individual tracing boxes, the oxygen valve was opened, and the rats were accustomed to voluntary activities for 10 min. After adaptation, the basal inspiration time (Ti), expiration time (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), frequency (f), enhanced pause (Penh), midexpiratory flow (EF50), relaxation time (Tr), tidal volume, minute ventilation (MV), cumulative air volume (AV), difference between inspiratory/expiratory volume, and peak expiratory time ratio (Rpef) values were recorded over 3 min.Blood samples were obtained after 30 min of observation. Arterial blood (0.5 mL) was collected from the femoral artery, fixed in heparin sodium (10 U/mL) to avoid coagulation, and analyzed using a portable blood gas analyzer . Venous blood (6 mL) was taken from the right heart. Sample (1 mL) was injected into a vacutainer containing ethylene diamine tetraacetic acid as an anticoagulant  and analyzed using a blood counter (Abbott). Also, a 1-mL sample was used for assays of creatine kinase (CK), lactate dehydrogenase (LDH), LDH1, aspartate transaminase (AST), α-hydroxybutyrate dehydrogenase (α-HBDH), and myocardial-specific isoenzyme of creatine kinase (CK-MB) activities.Blood samples (2 mL) were dispensed into 3.2% sodium citrate. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), D-dimer concentration, and fibrinogen (FIB) concentration were measured by a dry-hematology analyzer .g at 4°C for 20 min and the supernatant was stored at −80°C. The levels of IL-1β, TNF-α, and IL-6 were evaluated by ELISA  according to the manufacturer’s instructions.Venous blood (2 mL) was drawn into tubes lacking anticoagulant and placed at 4°C for 2 h. The samples were centrifuged at 1000 × t-test and one-way analysis of variance (ANOVA) were performed to compare measurement data among groups. A p-value of < 0.05 was considered statistically significant.Statistical analysis was performed with Prism . Normally distributed data are presented as the means ± standard error of mean and non-normally distributed data as medians (minimum–maximum). Paired Rats in the 150-m escape group showed no symptoms, whereas those in the 200-m, 220-m, and 250-m escape groups had shortness of breath, laziness, and some had cyanotic symptoms. No other symptoms of DCI were observed.The ECG results changed with increasing depth of escape . The R wvs. 708 ± 56.88 U/L; p = 0.015; CK: 856.9 ± 65.28 U/L vs. 486.2 ± 26.26 U/L; p = 0.001; α-HBDH: 352.0 ± 44.25 U/L vs. 177.2 ± 14.87 U/L; p = 0.014). Moreover, the AST and CK-MB values of the 200-m and 250-m escape groups were significantly higher than the control group .Myocardial enzyme levels showed an increasing trend with escape depth . CompareThe 150-m escape did not significantly influence pulmonary function indicators . With invs. 7.38 ± 0.03; p = 0.019). The oxygen partial pressure (PO2) of the 150-m escape group was in the normal range (106.17 ± 8.27 mmHg) but decreased markedly in the 200-m (84.47 ± 14.10 mmHg), 220-m (76.38 ± 12.56 mmHg), and 250-m (76.5 ± 29.77 mmHg) escape groups; the PO2 values of the 220-m and 250-m escape groups were lower than the normal value (80 mmHg). Compared to the control group, the PO2 of all escape groups decreased significantly, and the magnitude of decrease was proportional to the escape depth. The carbon dioxide pressure (PCO2) was within the normal range in all escape groups and did not differ significantly compared with the control group. The HCO3− results were consistent with those of PCO2—there was no significant change in the escape groups. The anion gap (AG) values of all escape groups were within the normal range, but that of the 250-m escape group was significantly higher than the control group . The actual base excess (AE) values of the escape group were also within the normal range, but the values of the 200-m, 220-m, and 250-m escape groups were significantly lower than that of the control group . The magnitude of decrease was consistent with the escape depth.The changes in acid–base status are shown in vs. 35.44 ± 1.21%; p = 0.76) than the control group. There were no significant changes in platelet, white blood cell, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration values.Compared to the control group, the hematocrit and hemoglobin levels and red blood cell count in the escape groups presented an increasing, albeit non-significant, trend . The ratvs. 0.42 ± 0.04 μg/mL; p < 0.05). The FIB values in all escape groups decreased non-significantly with increasing escape depth. The PT value increased with escape depth, and was significantly higher in the 250-m escape group than the control group . The TT value increased, albeit non-significantly, with escape depth. The aPTT value first decreased with the escape depth to a nadir at 220 m, and increased at 250 m, albeit non-significantly.The D-dimer value increased with increasing escape depth . Comparevs. 9.52 ± 1.04 pg/mL; p = 0.023; TNF-α: 121.30 ± 71.55 pg/mL vs. 43.03 ± 5.31 pg/mL; p < 0.05). The IL-1β level did not change significantly in any of the escape groups.The levels of IL-6 and TNF-α were similar in the 150-m escape group and the control group. At escape depths >200 m, the IL-6 and TNF-α levels increased with escape depth , and werSubmarine escape by free ascent enables survivor egress from a DISSUB. The theory of submarine escape by free ascent is that through rapid pressurization, a small amount of inhaled inert gas will not produce bubbles during rapid decompression. However, human physiology limits the depth of escape, principally as a result of DCI. The effect of depth escape and the safe depth range for escape are unknown.Prior animal studies have shown that decompression damage caused by submarine escape by free ascent is mainly to the heart and lungs, accompanied by changes in the coagulation and fibrinolysis system and subsequent activation of the inflammatory system. Therefore, we evaluated the effect of escape depth on ECG parameters, pulmonary function indexes, the myocardial enzyme spectrum, blood gas parameters, coagulation and fibrinolysis indicators, and inflammatory factors.At an escape depth of 150 m, there was no significant difference in the indexes compared to before escape. At an escape depth of 200 m, increased f and MV values indicated an increased respiratory rate and MV; significantly increased EF50 and Penh, as well as shortened Tr time, indicated increased airway resistance, and the decrease in Rpef indicated a small airway obstruction and decreased gas diffusion.2 and alkali remaining were significantly reduced at 200 m, and more so at 220 m and 250 m. Indicators of heart damage and changes in the coagulation and fibrinolysis system were also sensitive to escape depth. The myocardial enzyme spectrum and D-dimer values increased significantly at 220 m. Inflammatory factor, HCT, and hemoglobin levels and the red blood cell count increased with escape depth. Compared with the control group, the IL-6, TNF-α, and HCT levels in the 250-m escape group were significantly increased. The results suggest that 200 m is the threshold depth for safe escape in this animal model.Blood gas values were the earliest indicators of change. The POUnsafe submarine escapes cause more severe DCI than diving. DCI caused by unsafe submarine escape is mainly the result of cardiopulmonary injury, manifesting as pulmonary hypertension, acute pulmonary edema, and right heart failure. Indeed, pulmonary edema , hemorrhSevere DCI from diving is more commonly manifests as nervous system injury because air bubbles in the veins enter the arterial system through abnormal openings between the arteries and veins, such as the patent foramen ovale, leading to infarction of peripheral and central nervous tissue . The DCI2 values decreased with increasing escape depth. The PO2 value of the 220-m and 250-m escape groups were lower than normal, indicating hypoxia and acidemia. Elevated AG and decreased AE also suggested acidosis. Therefore, hypoxia is the earliest manifestation of decompression injury caused by submarine escape. The PO2 value was normal at an escape depth of 150 m, and decreased significantly at 200 m, indicating that escape is unsafe at depths >200 m.Pulmonary air embolism results in increased pulmonary vascular resistance, reduced cardiac output, systemic hypoxia, and hypercapnia . With inDecompression stress affects the blood coagulation system. Microbubbles affect clotting by activating coagulation and inducing platelet aggregation . The bubInflammatory response is an important manifestation of decompression injury. Air bubbles cause vascular endothelial damage, leading to adsorption of adhesion factors, activation of complement, and triggering of inflammation . The levAn increase in pulmonary artery (PA) pressure is the most prominent indicator of pulmonary air embolism. PA pressure rises more rapidly with increasing rate of air entering the pulmonary circulation. This is followed by a rapid drop in end-tidal carbon dioxide and oxygen saturation values. ECG can detect a variety of changes, including tachyarrhythmias, varying degrees of atrioventricular block, right ventricular strain, and ST-segment changes. Unfortunately, due to technical problems, PA pressure and right ventricular output were not measured in this study. The ECG parameters changed with increasing escape depth. Increased R waves appeared with increasing escape depth, suggesting ventricular hypervoltage. Small Q waves appeared at an escape depth of 200 m. At an escape depth of 220 m, pathological Q waves appeared in multiple leads, suggesting myocardial ischemia.2, increased PCO2, and an increase in the alveolar–arterial oxygen difference (Gas exchange abnormalities associated with pulmonary air embolism include decreased POfference . VentilaSafe at 150 m in this animal model, but there is a risk of harm at depths >200 m. The main manifestations are pulmonary ventilation and gas diffusion dysfunction, hypoxemia, myocardial ischemia, and activation of the fibrinolytic and inflammatory systems. Respiratory system changes show the greatest sensitivity to escape depth, and the performance of the right ventricle is a key determinant of prognosis.The limitation of this study is that the physiological structures of rats and humans are too different to be directly analogized. We also did a lot of goat experiments in the follow-up experiments. Compared with rats, goats are closer to humans in body weight, and body weight is an important factor affecting safe escape. The Royal Navy has used goats to define DCL risks following simulated submarine escape . Six goa


Flexible work arrangements (FWAs) have been widely implemented during the COVID-19 pandemic. We aimed to assess the validity and reliability of the FWA perceived benefits and barriers (FWAPB) scale and subsequently, to determine the preference and perceived feasibility, perceived benefits and barriers, and readiness to implement FWA among healthcare workers.We conducted a cross-sectional study using a self-administered questionnaire in Miri Hospital. The questionnaire was administered via a web survey design (Google Forms). The convenience sampling method was applied to recruit respondents. All healthcare workers in Miri Hospital who could read and understand English were invited to participate in the study. Response process validation, exploratory factor analysis, reliability analyses and descriptive statistics were performed.A total of 339 respondents participated. All items had satisfactory response process indices. Exploratory factor analysis revealed a three-factor structure. Items of ‘perceived benefits-workplace management’, ‘perceived benefits-family life balance’ and ‘perceived barriers’ have high internal consistency reliability  and factor loadings. Flextime is preferred and perceived to be the most feasible work arrangement. Most agreed that FWA helps in improving social distancing among colleagues  and reduces their exposure to COVID-19 . A total of 44.0% of the respondents agreed Miri Hospital is ready to implement FWA.The FWAPB is valid and reliable. Almost half of the respondents were positive towards the implementation of FWA. These findings contribute to the understanding of FWA, and thus increase the readiness and acceptance of such an arrangement. Flexible work arrangement (FWA), is an organisational policy and practice that allows employees to choose, at least to some extent, when or where they work or to work different hours from the traditional working hours , which iThe WHO declared COVID-19 as a pandemic on 11th March 2020. Subsequently, the Malaysian government announced the first movement control order (MCO) on 18th March 2020, followed by a conditional movement control order (CMCO) and recovery movement control order (RMCO). One of the preventive measures under the MCO is to practise physical distancing to reduce exposure and slow COVID-19’s spread. Thus, many organisations and businesses are exploring adopting FWA as a new norm to practise physical distancing.The Malaysian government has adopted several FWA strategies among the civil service sectors, such as flexible working hours and shift work. During this pandemic, working from home (remote work) has been mooted as a new FWA mechanism. However, there has not been many studies to explore the acceptance and implementation of FWA among healthcare workers working in a hospital setting. Therefore, we aimed to: i) assess the validity and reliability of the FWA perceived benefits and barriers (FWAPB) scale; ii) explore the preference and perceived feasibility of FWA; iii) determine the perceived benefits and barriers of FWA and iv) determine the readiness to implement FWA among healthcare workers at Miri Hospital.We conducted a cross-sectional study between August 2020 and September 2020 at Miri Hospital, a major hospital in the northern zone of Sarawak, using a self-administered questionnaire administered via web survey design (Google Forms). The convenience sampling method was applied to recruit respondents. During the respondent recruitment period, an announcement about the study was made through various departmental communication groups. In addition, we distributed and placed the invitation notice with a QR code in all departments. All healthcare workers in Miri Hospital who could read and understand English were eligible to participate in the study. The respondents were requested to respond to each question at their convenience. Those who did not consent were excluded. To ensure the privacy of the respondents, the survey was strictly anonymous and did not ask for the identity of respondents. The choice of response would not affect their performance at work in any way.This study was divided into two parts. Firstly, there was an assessment of the validity and reliability of the FWAPB scale, which consisted of the development of the questionnaire, response process validation and internal structure evaluation. Secondly, the assessment of the implementation of FWA was made, including the preference for and perceived feasibility of FWA, its perceived benefits and barriers, and the readiness to implement FWA among healthcare workers in Miri Hospital. Details of the methods used are elaborated in the following sections.In the development stage, the first and second authors reviewed the relevant contents and information related to the topic. Subsequently, items were identified and generated based on the available guidelines, grey literature, and peer-reviewed FWA-related studies , 4–8 to Section A of the questionnaire assessed FWA options, in which the respondents were asked ‘In your current workplace, have you used the following types of FWA?’ The items assessing their preference and perceived feasibility of the FWA options were measured on a 5-point Likert rating scale ranging from ‘1 = most preferred’ to ‘5 = least preferred’ and ‘1 = most feasible’ to ‘5 = least feasible’, respectively. The FWA options in this study included: i) telecommuting, which refers to attending the office semi-regularly (e.g. 2 or 3 times per week), performing the job remotely for part of the time, using computers and technology; ii) remote working, which refers to not attending the office at all, performing the job entirely away from the office; iii) condensed work week, which refers to four 10-h working days to maintain a 40-h work week; iv) flextime, which refers to a schedule which involves flexible start and end times of the working day, and applies five 8-h working days to maintain a 40-h work week and v) shift work, which refers to a fixed 8-h shift schedule to maintain a 40-h work week.Section B consisted of FWAPB items, which explored the perceived benefits and barriers of FWA among the employees. The items were developed based on the literature review and expert opinions. The 21 items pertaining to the perceived benefits and barriers were then categorised into the identified preliminary domains, namely work-family balance, work-life benefits, workplace management and COVID-19 risk management. Section C elucidated the readiness to change. To assess this, the respondents were asked to read the following statements: ‘I will opt for FWA’; ‘I am confident I am able to adapt to FWA’; and ‘Miri Hospital is ready to implement FWA’. All items in Section B and C were framed using a 5-point response scale ranging from ‘1 = strongly disagree’ to ‘5 = strongly agree’. We reversed nine items in the scoring of the scale; higher scores indicated a positive perception on all items. Lastly, Section D contained 10 questions to gather respondents’ demographic information and their current work system.Upon receiving the completed questionnaires, a pre-processing and data cleaning step was applied to remove unconsented responses and incomplete or invalid data. The data were considered invalid when the responses contained the exact same answer to all items. After removing all the unconsented responses and incomplete or invalid data, we performed the data analysis using Microsoft Excel and SPSS version 23.0. For the assessment of the scale validity and reliability, we reviewed the response process validity using response process validity indices, whereas for the factor structure and reliability of the perceived benefits and barriers of FWA, we used exploratory factor analysis (EFA) and reliability analysis, as the section contained latent constructs.For the exploration of the implementation of a flexible work arrangement, descriptive statistics summarised the preference for and perceived feasibility of FWA based on their job scope, the perceived benefits and barriers of FWA, and the readiness to implement FWA among healthcare workers in Miri Hospital. Normally distributed numerical data were expressed as mean ± standard deviation (SD), whereas ordinal and categorical data were summarised as frequency and percentage. The procedures are described in the next sections.In this study, the items assessing perceived benefits and barriers of FWA were pre-tested among the targeted population through response process validation. Response process validity is also known as face validity . ResponsThe Face Validity Index (FVI) is used to assess comprehensibility and clarity of each item by raters, to estimate the response process validity . The resThe raw scores were entered in Microsoft Excel to compute the item-level FVI (I-FVI) and scale-level FVI (S-FVI). We applied two methods for calculating S-FVI, namely: i) the average of the I-FVI scores (S-FVI/Ave) and ii) the proportion of items with a score of 3 or 4 by all raters (S-FVI/UA) . We recoThe validity evidence based on internal structure in this study was obtained based on the factor structure and reliability . We emplItem factor loadings and their significance were assessed to confirm the unidimensionality of the measure. Minimum acceptable factor loading was defined as 0.3 or higher, with a value of 0.5 and above indicating practical significance . UltimatFollowing that, the reliability analysis was applied to examine the internal consistency. The internal consistency of the tool was examined using Cronbach’s alpha. In this study, we adopted a minimum threshold of 0.7 for satisfactory internal consistency , 16.Using the 10:1 subject to item ratio, the minimum sample size for EFA was 210 respondents . The calA total of 485 responses were received during the recruitment period. We excluded 105 invalid responses, which contained the exact same answer to all items and 41 unconsented responses. Therefore, 339 usable responses were included in the final analysis. The final sample consisted of 208 nurses (61.4%), 43 doctors (12.7%), 17 administrators (5.0%) and 14 pharmacists (4.1%). The average age of the respondents was 34 years old and most of them were female (81.7%), married (65.5%) and were permanent staff (92.3%). Most respondents reported adopting shift work schedules (55.5%). The characteristics of the respondents are presented in In the response process validation, all items in FWAPB had satisfactory response process indices. We obtained the values of I-FVI, S-FVI/Ave and S-FVI/UA of 1.00 for all items. The finding met the acceptable FVI cut-off of at least 0.83 . As the The factor structure of FWAPB was examined in EFA using principal axis factoring extraction with Promax rotation method. KMO value was 0.898 and the Bartlett’s test of sphericity value was significant. Hence EFA was appropriate. In the factor analysis, all items met the minimum acceptable factor loadings of 0.3 . HoweverAs Factor 4 contained only two items which had larger loadings on the other factors, namely Factor 1 and Factor 2, we aimed to remove Factor 4 and retain three factors by fixing the number of factors to a three-factor solution. This was desirable to retain the factors with at least three items with loadings higher than 0.4 . The finIn addition, Cronbach’s alpha indicated good internal consistency for each factor and ranged between 0.852 and 0.884. The analysis also showed that Cronbach’s alpha would not increase with item removal. Hence, no item was deleted from the scale and no change made to the items. In the current study, we also attained the information on the types of FWA used in the respondents’ current workplace. The respondents may select all FWA options used. Our findings showed that shift work was most commonly used (61.4%), followed by flextime (34.5%), telecommuting (9.4%) and condensed work week (7.7%). The least frequently used FWA option was remote working (3.5%). The respondents preferred flextime and viewed it as the most feasible type of FWA. Meanwhile, remote working was the least preferred and viewed as the least feasible FWA option in their workplace .Most of the respondents agreed that FWA helps to improve social distancing among colleagues  and reduced their exposure to COVID-19 . Some viewed that FWA helped to reduce work-related stress levels .From the perspective of the perceived barriers, most respondents disagreed that FWA had negative effects on their personal well-being  and their family well-being . In addition, the respondents tended to disagree that FWA reduced their work productivity. The respondents perceived that the greatest barrier in FWA was the distraction associated with working out of the office .The mean scores for readiness statements ranged from 3.42 to 3.50 and 44.5% of the respondents viewed that they would opt for FWA at their workplace  whereas 45.7% of the respondents were confident to adapt to FWA and 44.0% of them believed that Miri Hospital was ready to implement FWA .The current study has developed the FWAPB, which can be used to assess perceived benefits and barriers of FWA. It is commonly understood that validity and reliability relate to the interpretation of psychometric instruments . A unitaOn the other hand, reliability and factor analysis are considered evidence of the internal structure validity . In thisIt is known that in self-administered surveys, it is common to observe insufficient effort and careless responding . Due to Identifying the underpinned factors related to the perceived benefits and barriers of the implementation of FWA in a hospital setting may help to identify employees’ concerns in adopting FWA, determine the employees’ participation, and hence may facilitate the implementation of FWA, especially among healthcare workers. In this study, most respondents perceived that FWA is beneficial in the risk management of COVID-19 in the workplace. The majority of the respondents were concerned in improving social distancing among colleagues and to reduce exposure to COVID-19. A study showed that there was a statistically significant association in social distancing measures with a decrease in the COVID-19 case growth rate . In addiFWAs were predominantly applied in the professional service and IT sector . PreviouWhile some authors argued that public sector employees were more likely to perceive greater benefits and exhibited fewer concerns . One stuTherefore, a one-size-fit-all approach is not workable as different individuals will choose different approaches of FWAs to meet their needs . As the A key strength of the present study was the sufficient sample recruited. In addition, the sample provided a comprehensive representation of the hospital population. However, there are several limitations in this study. Firstly, this is a single-centred study. Therefore, the result may not be generalisable to a population with different characteristics. Secondly, female healthcare members contributed disproportionately to the respondent data set, as compared to the male members. This is similar to some of the past studies, which asserted that a larger percentage of female respondents would return surveys than their male counterparts , 39. ThiIn conclusion, the FWAPB was found to have three factors and satisfies the response process validity, construct validity and reliability. Hence, it is a valid and reliable tool. Almost half of the respondents were positive towards FWA implementation. Yet, a high proportion of them remained neutral on its implementation. These findings contribute to the understanding of concerns that must be addressed to increase the readiness and acceptance of such arrangements. It is recommended that the management should look into different models of FWAs to suit different groups of healthcare workers.


Tumour necrosis factor-α (TNF) is a multifunctional cytokine. First recognized as an endogenous soluble factor that induces necrosis of solid tumours, TNF became increasingly important as pro-inflammatory cytokine being involved in the immunopathogenesis of several autoimmune diseases. In the liver, TNF induces numerous biological responses such as hepatocyte apoptosis and necroptosis, liver inflammation and regeneration, and autoimmunity, but also progression to hepatocellular carcinoma. Considering these multiple functions of TNF in the liver, we propose anti-TNF therapies that specifically target TNF signalling at the level of its specific receptors. TNF is a pro-inflammatory cytokine critically involved in autoimmune diseases. Therefore, TNF inhibitory approaches successfully entered the clinic for therapy of e.g. rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease (IBD) . UnfortuIn the liver, TNF mediates hepatocellular death and seems to be involved in sterile inflammation, viral hepatitis, AIH, and hepatocellular carcinoma (HCC) –7. The mL), most of them interacting with signalling molecules of the death signalling pathway [TNF is produced as a 26-kDa transmembrane precursor protein (mTNF) that is proteolytically cleaved by TNF-converting enzyme  to generate a soluble 17-kDa protein (sTNF). Both sTNF and mTNF are bioactive and signal as homotrimers via two structurally distinct membrane receptors, p55 TNFR1 and p75 TNFR2, which are trimerized upon ligand binding . While T pathway .Fig. 1TNThe death-inducing signalling complex (complex II) is formed upon recruitment of Fas-associated death domain (FADD) to TRADD, which in turn recruits pro-caspase 8 to finally execute the proapoptotic downstream pathway of TNFR1 , 8. Actid-galactosamine , a transcriptional inhibitor, sensitizes wild-type mice to lethal toxicity of LPS and TNF [d-GalN-sensitized mice caused fulminant hepatitis [d-GalN is primarily metabolized in hepatocytes where it depletes uridine nucleotides and thereby functions as transcriptional inhibitor [Tnfr1−/− mice but fully inducible in Tnfr2−/− mice following combined activation of both TNF receptors in the murine liver . APAP iiewed in . Sterileiewed in , which riewed in ). DAMPS iewed in . Althougiewed in  and miceiewed in . These fiewed in .Table 1MALD is one of the most common causes of chronic liver disease worldwide that may progress from alcoholic steatohepatitis to fibrosis, cirrhosis, and HCC . Metabolratios of primary to secondary BA levels underscore the impact of microbial alterations in the gut of NASH patients [Due to the growing incidence of obesity worldwide, NAFLD is a major cause of chronic liver disease with increasing prevalence. NAFLD is characterized by hepatic steatosis which may progress to non-alcoholic steatohepatitis (NASH) and development of fibrosis, cirrhosis, and HCC . Co-morbpatients . In addipatients . Interespatients ). Therefpatients , 58, 61.TNF is a mediator of insulin resistance in obesity, where it is overexpressed in adipose tissue and induces the release of the pro-inflammatory lipokine leptin from adipocytes , 63. In + T cells also release TNF and other cytokines upon antigen recognition which facilitate viral control [principle, a sustained antiviral immune response mediates the immunopathogenesis of chronic viral hepatitis, as the viruses themselves are not cytopathic. With respect to HBV infection, polymorphisms in the TNF promoter region affect the susceptibility towards chronicity and disease pathology of hepatitis B (summarized in [Infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) elicits an antiviral immune response characterized by an innate type I interferon response and an innate (NK cells) and adaptive cellular immune response (T cells). The cellular response eliminates infected hepatocytes by induction of apoptosis, elicited either by members of the TNF superfamily such as Fas ligand or TNF-related apoptosis-inducing ligand (TRAIL) or by granzyme B and perforin. However, activated effector CD8 control . Althougrized in ).L. mRNA transcripts of these anti-apoptotic proteins were also significantly reduced in liver tissue of patients with chronic hepatitis C compared to non-viral hepatitis. Transfection experiments revealed that TNF sensitisation of HCV-infected Huh-7.5 cells was probably mediated by the core, non-structural HCV proteins (NS)4B and NS5B [Since HBV and HCV do not infect murine hepatocytes in vivo, several cellular studies analysed the role of viral proteins for sensitisation towards TNF-induced hepatocyte killing. For example, transfection of the HBV proteins HBx or HBV core protein (HBc) sensitized cell lines towards TNF-induced apoptosis , 73. Besand NS5B .Tnfr1−/− mice displayed less hepatic apoptosis and necrosis, and reduced neutrophil infiltration into the infected liver as well as prolonged survival [d-GalN [Several studies analysed molecular and immunological mechanisms of TNF signalling during viral hepatitis in mouse infection models. TNF has been shown to promote hepatitis in murine hepatitis virus (MHV) infection as survival . The hep [d-GalN .Tnfr1flox/flox mice. In these animals, TNF challenge failed to induce liver damage. In TNFR1 expressing hepatocytes, TNF induced cleavage of caspases 8, 9, and 3. However, administration of the Cre-recombinase encoding adenovirus to Casp8flox/flox mice failed to attenuate liver damage upon TNF injection, indicating that the observed mode of cell death was independent of caspase 8. Notably, the authors neither observed necroptosis, pyroptosis, ferroptosis, nor oxeiptosis upon TNF injection to adenovirus-infected mice. However, inhibitor studies and studies using genetically modified mice or calcium scavengers as well as determination of the mitochondrial membrane potential provided evidence for a reactive oxygen species (ROS) and calcium-dependent deterioration of the mitochondrial membrane potential of infected hepatocytes exposed to TNF in vivo, indicating that TNF-induced cell death in adenovirus-infected mice was characterized as a caspase 8-independent, MPT-dependent, non-canonical type of hepatocyte apoptosis.In a recent study, Lampl and co-workers  providedTnfr1−/− mice showed increased numbers of virus-specific memory CD8+ T cells upon infection with the hepatotropic lymphatic choriomeningitis virus (LCMV), compared to Tnfr2−/− mice [+ T cell numbers. TNF receptor deficiency did not affect proliferation but rather reduced apoptosis of LCMV-specific CD8+ T cells, pointing to a role of TNF for T cell contraction during chronic viral infection. In another study, LCMV-specific T cells from chronically infected mice displayed increased expression of programmed cell death (PD)-1 and other co-inhibitory molecules, indicating enhanced T cell exhaustion [+ and CD8+ T cells, a concomitant reduction of PD-1 expression, and enhanced antigen-specific cytotoxicity of CD8+ T cells along with an improved control of viral infection. Interestingly, neutralization of TNF resulted in elevated levels of serum transaminases which was probably due to T cell–mediated hepatitis. The relevance of TNFR signalling for T cell immunity was analysed by adoptive transfer of CD4+ and CD8+ T cells from acutely infected wild-type or TNFR1/TNFR2 double knockout mice into chronically infected animals. Analysis of the transferred T cells demonstrated significantly increased frequencies of LCMV-specific TNFR-deficient T cells compared to wild-type T cells, decreased frequencies of PD-1+ T cells, and a concomitantly enhanced ability of TNFR-deficient T cells to clear the virus. Using transgenic mice, the authors demonstrated that continuous NFκB activation in LCMV-specific CD4+ T cells resulted in increased PD-1 expression and impaired helper function in infected mice, which resulted in substantially lower frequencies and reduced effector functions of LCMV-specific CD8+ T cells. Accordingly, the authors discovered NFκB responsive elements in the PDCD1 locus. Together this study indicates that continuous NFκB activation in CD4+ T cells may provide a molecular link between TNF and T cell exhaustion during chronic viral infection. However, blockade of TNF during viral hepatitis might be contraindicated not only because of the well-documented side effects of reactivation of quiescent infections but also because of restricted elimination of infected hepatocytes.Although TNF seems to control viral hepatitis by induction of cell death in virus-infected hepatocytes, TNF signalling might also favour viral persistence by impairment of the antiviral T cell response, either by induction of T cell apoptosis  or by en−/− mice . Intereshaustion . TreatmehighCD127low exhausted CD8+ T cells [In an immunocompetent mouse model of chronic HBV infection, using hydrodynamic injection of a HBV plasmid to wild-type and cIAP1 and 2–deficient mice, it was shown that cIAPs, which are activated in the TNFR-dependent NFκB pathway, attenuated TNF-induced cell death of HBV-infected hepatocytes, thereby allowing viral persistence . Accordi T cells . AlthougTnfr1−/− and Tnfr2−/− mice provided considerable evidence for a pathogenic role of TNF in acute T cell–dependent liver injury [+ T cells were detectable in peripheral blood and liver biopsy specimens compared to healthy controls. As the majority of TNF-producing CD4+ T cells also produced IFNγ, the authors concluded that these cells are pathogenically activated Th1 cells. Indeed, antigen-specific activation of T cells resulted in significant TNF production by CD4+ T cells from AIH patients who developed the corresponding antigen-specific antibody response [+ T cells, which is probably mediated by reverse signalling via mTNF, that can function as a receptor of the antibody  loci, emphasizing a critical role of adaptive T cell responses in the immunopathogenesis of AILD , 87. Prer injury , 88, 89.r injury , 91. In response . In addiresponse . Intriguresponse . Since a+ T cells to TNF. TNF, via TNFR1, pre-dominantly activates the innate immune response including maturation of dendritic cells, thereby priming CD4+ T cells [+ T cells to TNF. In contrast to the ubiquitous expression of TNFR1, TNFR2 expression is restricted to certain cell types including lymphocytes. It is well documented that TNFR2 is preferentially expressed by human and mouse CD4+Foxp3+ Tregs [Foxp3 promoter, and prevents pathogenic conversion of Tregs under inflammatory conditions [Tnfr1−/− mice were protected whereas Tnfr2−/− mice displayed disease exacerbation. Accordingly, treatment with either TNFR1 antagonists or TNFR2 agonists ameliorated the disease , suggesting that T cell–dependent adaptive immune responses contribute to the immunopathogenesis of PSC [+ T cell apoptosis [PSC is a chronic cholestatic liver disease characterized by biliary inflammation and progressive fibrosis, end-stage liver disease, and a high risk of malignancy. Current medical treatment of PSC is of limited effectiveness. More than 80% of PSC patients have IBD with a unique clinical phenotype . Alterats of PSC . Recentlpoptosis  and redupoptosis . In addipoptosis . Indeed,poptosis , 106. Mopoptosis .+ T cells of PSC patients has been established, in which single cell sequencing and trajectory analysis provided evidence for expansion of tissue resident CD4+ T cells exhibiting a phenotype of naïve T cells that are prone to polarize towards effector Th17 cells [RORA, as well as pro-inflammatory cells, characterized by expression of high levels of TNF [Emerging evidence from clinical and preclinical studies indicates the development gut dysbiosis in PSC patients  and in m17 cells . Amongsts of TNF . Hence, s of TNF , 114. Its of TNF .Mdr2−/− mouse model, bearing a targeted disruption of the multidrug resistance gene that corresponds to the human orthologue MDR-3 [Mdr2−/− mice and a good response to antibiotic treatment in DDC-fed animals [Mdr2−/− mice [Two well-accepted murine models serve as animal models for sclerosing cholangitis. These are (i) the ue MDR-3  and (ii)ue MDR-3 . Liver h animals . Moreove−/− mice ) or Th17−/− mice ).Mdr2−/− mice compared to wild-type mice [Mdr2−/− mouse model. Using Mdr2−/− × Tnfr1−/− double knockout mice, we observed an aggravated disease pathology of sclerosing cholangitis associated with an increased Th17 response in Mdr2−/− × Tnfr1−/− mice compared to Mdr2−/− mice [Ccr6 and Cxcr6) and expression of Ccl2 and Cx3cl1, which are critically involved in the recruitment of inflammatory monocytes, were also elevated. Accordingly, we observed increased frequencies of CD11b+CX3CR1+ monocytes/macrophages in livers of Mdr2−/− × Tnfr1−/− mice compared to Mdr2−/− mice which changed their phenotype by inappropriately expressing Ripk3. CX3CR1+ RIPK3+ macrophages have been recently shown to produce the cytokines IL-1β and IL-23 which promote Th17 responses [Enhanced levels of TNF have also been detected in ype mice . A preclype mice . We wond−/− mice . Moreoveesponses .Mdr2−/− mice are a mouse model of inflammation-induced tumour development, and IL-17 has been closely associated with strong induction of regeneration and angiogenesis in the tumour microenvironment [Mdr2−/− × Tnfr1−/− compared to control mice. We observed a significant increase of the expression of Pcna (gene of proliferating cell nuclear antigen), Ccna2 (gene of cyclin A2), Cdk1 (gene of cyclin-dependent kinase 1), Ssp1 , and Afp (gene of α-feto protein) in the double knockout mice, indicating that they might be more susceptible to development of HCC than Mdr2−/− mice. As mentioned earlier, activation of TNFR1 induces cell survival and liver regeneration by activation of NFκB. Interestingly, targeted disruption of NFκB signalling specifically in intestinal epithelial cells in the gut resulted in intestinal barrier disruption and chronic colitis, which did not develop under germ-free conditions [Mdr2−/− mice [Since ironment , we analnditions . Moreove−/− mice . TherefoMdr2−/− mice resulted in apoptosis of transformed hepatocytes and lack of progression to HCC [Mdr2−/− mice, indicated by a reduced expression of proliferation and tumour markers [Functional evidence for a role of TNF in HCC was provided by studies in mice. Deficiency of the TNFR-dependent anti-apoptotic NFκB signalling pathway was shown to be essential for induction of compensatory proliferation of live hepatocytes in response to hepatocyte death which results in development of HCC . Mice lan to HCC . Hence,  markers , while a markers .By activation of the ubiquitously expressed TNFR1, TNF provides signals for eradication of pathogens and tissue damage such as inflammation and cell death, but also induces cell survival, proliferation, and tissue regeneration, thereby contributing to resolution of inflammation and tissue repair. Therefore, it is not surprising that anti-TNF therapy has major beneficial clinical effects in several autoimmune diseases such as RA and IBD, but also induces adverse effects such as increased susceptibility to infection and more unexpectedly exacerbation of pre-existing autoimmune disease (e.g. multiple sclerosis) or even new development of autoimmune-like symptoms in different organs , 8. In t+ Tregs could suppress the antigen-specific T cell response against the tumour [While the role of TNFR1 for liver inflammation, induction of several forms of hepatocyte death, cell survival, liver regeneration, hepatocyte proliferation, and tumorigenesis is well documented, the role of TNFR2 for acute and chronic liver injury is scarcely described. A few reports suppose that TNFR2, although devoid of a death domain and primarily activating NFκB, might facilitate TNFR1-induced liver cell death , 40, a me tumour . Taken t


Thiothrix are filamentous, sulfur-oxidizing bacteria found in flowing waters with counter-oriented sulfide and oxygen gradients. They were first described at the end of the 19th century, but the first pure cultures of this species only became available 100 years later. An increase in the number of described Thiothrix species at the beginning of the 21st century shows that the classical phylogenetic marker, 16S rRNA gene, is not informative for species differentiation, which is possible based on genome analysis. Pangenome analysis of the genus Thiothrix showed that the core genome includes genes for dissimilatory sulfur metabolism and central metabolic pathways, namely the Krebs cycle, Embden–Meyerhof–Parnas pathway, glyoxylate cycle, Calvin–Benson–Bassham cycle, and genes for phosphorus metabolism and amination. The shell part of the pangenome includes genes for dissimilatory nitrogen metabolism and nitrogen fixation, for respiration with thiosulfate. The dispensable genome comprises genes predicted to encode mainly hypothetical proteins, transporters, transcription regulators, methyltransferases, transposases, and toxin–antitoxin systems.Representatives of the genus  Thiothrix were made by Rabenhorst as early as 1865, when he described the first member of this genus as Beggiatoa nivea -hydrogenase, as well as hypABCDEF genes involved in hydrogenase maturation, which indicates the potential for lithotrophic growth in the presence of molecular hydrogen.The core genome contains genes Thiothrix can assimilate carbon dioxide through the Calvin–Benson–Bassham cycle. The genes responsible for the regeneration and reduction of the substrate in the Calvin–Benson–Bassham cycle were found in the core genome. RuBisCO in the genus Thiothrix is represented by three types: IAq, IAc, and II. Previous pangenome analysis has shown that RuBisCO of the IAq type is common to all species, in contrast to IAc and II types [Thiothrix winogradskyi’ CT3T into the pangenome analysis excluded type IAq RuBisCO from the core genome, since its genome does not contain this type of RuBisCO. All species, except for ‘Ca. Thiothrix anitrata’ A52, encode RuBisCO of type II, and all species except T. fructosivorans QT, T. lacustris BLT, and MAG of Thiothrix sp. 207 encode type IAc , glucokinase (glk), dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex (pdhD), aconitate hydratase (acnB), and 6-phosphogluconolactonase (pgl), the level of similarity of which is below 70%. The genes of the first, second, and third complexes of the electron transport chain, as well as ATPase (complex V), are present in the core genome. The genes of the fourth respiratory chain complex were found in the core genome . Genes cyoE, cox11, coxBACD, cydABCDX, and ccoN are also present in all species but fell into the auxiliary genome due to higher sequence divergence. The cytochrome ubiquinol oxidase is present in all Thiothrix species except for ‘Thiothrix winogradskyi’ CT3T.As for phosphoribulokinase in the genus Thiothrix revealed that variable part of the genome that comprises genes responsible for anaerobic respiration in the presence of nitrogen compounds and nitrogen fixation. The distribution of genes responsible for different stages of nitrate reduction is irregular  was found in all members of the genus, except for ‘Ca. Thiothrix anitrata’ A52.Pangenome analysis of the genus rregular . The genrG genes . The T. Ca. Thiothrix anitrata’ A52. The gene encoding dissimilatory nitrite reductase (nirS), involved in the reduction of nitrite to NO, was not found in ‘Ca. Thiothrix anitrata’ A52, ‘Ca. Thiothrix sulfatifontis’ KT, T. nivea JP2T, Ca. Thiothrix moscovensis RT, ‘Thiothrix subterranea’ Ku-5T, or T. lacustris BLT. Nitric oxide reductase NorBC catalyses the reduction of NO to N2O. ‘Ca. Thiothrix anitrata’ A52, ‘Ca. Thiothrix sulfatifontis’ KT, T. nivea JP2T, ‘Thiothrix subterranea’ Ku-5T, and T. lacustris BLT lacked these genes. Analysis of the MAG of Thiothrix sp. 207 revealed the nosZ gene encoding nitrous oxide reductase, which enables the reduction of N2O to molecular nitrogen. This gene was most likely acquired via horizontal transfer from other gammaproteobacteria. Thiothrix sp. 207 therefore could be capable of complete denitrification.Nitrite could be reduced to ammonium by the assimilatory nitrite reductase NirBD, which is encoded in the genomes of all species, with the exception of ‘glnA), glutamate synthase (gltBD), and aspartate aminotransferase (aspB) in the core genome.Classical amination is represented by the genes for glutamine synthetase (nifASUBNXX2YB2ENQVWMHDKZTO) was found in T. litoralis AST, T. caldifontis G1T, T. unzii A1T, the MAG of Thiothrix sp. 207, Ca. Thiothrix moscovensis RT, ‘Thiothrix subterranea’ Ku-5T, and T. nivea JP2T  most likely indicates the loss of the phs genes in Ca. Thiothrix moscovensis RT and ‘Thiothrix winogradskyi’ CT3 [Previously, respiration in the presence of thiosulfate as a terminal electron acceptor was not described for any e family . Verifickyi’ CT3 .Thiothrix differ significantly in their ability for anaerobic respiration in the presence of various electron acceptors  allowed a comparative genome-based analysis of the main metabolic pathways.The genera hiothrix , initialThiolinea, such as Thiothrix, are capable of autotrophic carbon fixation through the Calvin cycle. Their genomes contain genes encoding RuBisCO types IAq and II [Thiofilum flexile lacks the RuBisCO genes and therefore is capable of only lithoheterotrophic but not lithoautotrophic growth.First of all, members of all three genera have the same enzyme systems for the oxidation of sulfur and hydrogen sulfide under aerobic conditions. Members of the genus q and II . The phyThiothrix species, the genomes of Thiolinea and Thiofilum encode different sets of genes to reduce nitrogen compounds. In the genomes of Thiofilum flexile and Thiolinea disciformis, genes for denitrification systems  were not found, while the ‘Thiolinea eikelboomii’ genome contains only genes for dissimilatory Nar-type nitrate reductase. Unlike most Thiothrix species, none of the members of Thiolinea and Thiofilum have genes of the nif-cluster, which enables nitrogen fixation.Like different Thiothrix is the presence of a FAD-dependent membrane-bound malate:quinone oxidoreductase instead of the typical NAD-dependent cytoplasmic malate dehydrogenase. The mqo genes are also present in the genomes of Thiolinea and Thiofilum flexile, but the latter also contains the mdh gene. It is likely that the acquisition of mqo occurred in a common ancestor of all three genera, and mdh was subsequently lost in the Thiolinea/Thiothrix lineage.As noted above, one of the features of the genus Thiothrix revealed that the genes of dissimilatory sulfur metabolism are the most conserved part of the genome. The ability to carry out the interconversions of reduced sulfur compounds is probably a key characteristic for these bacteria, which once again indicates their lithotrophic type of metabolism.A pangenome analysis of the genus Thiothrix thrives as a surface-attached organism in running water, which contains H2S and O2 in fairly high concentrations. In such habitats, Thiothrix filaments exist under conditions in which the concentrations of these substances (oxygen and hydrogen sulfide) could change significantly in a short period of time. As a rule, the O2 concentration in Thiothrix growth zones can vary from full to 10% of saturation. In such frequently changing environmental conditions, bacteria have developed an adaptation strategy through changes at the genomic level.phsABC thiosulfate reductase genes, and the discovery of new species-specific genes (nosZ), indicate a dynamic process of gene acquisition via horizontal transfer and lineage-specific gene losses that allows bacteria to adapt to variable environmental conditions. Thus, thiosulfate and oxidized forms of nitrogen, which can be used as electron acceptors during anaerobic respiration, are not always available in different ecosystems and can be interchanged; the presence of various RuBisCO forms can be explained by the adaptation to different concentrations of CO2 and O2, which vary in a wide range.Significant differences in the inventory of genes for nitrogen metabolism, RuBisCO gene profiles , and phs