Drosophila melanogaster lines that express polyQ-expanded Atxn3 with and without a functional UbS1. We found that mutating UbS1 markedly exacerbates the toxicity of pathogenic Atxn3. Additional studies indicated that UbS1 regulates the toxicity of Atxn3 not by affecting its aggregation or sub-cellular localization, but by impacting its role in ubiquitin processing. Our findings provide additional insights into the role of Atxn3’s domains in the pathogenicity of SCA3.Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) diseases that are caused by anomalous CAG triplet repeat expansions in several genes. SCA3 results from abnormal polyQ expansion in the deubiquitinase (DUB), ataxin-3 (Atxn3). To understand the role of the different domains of mutant Atxn3 on its pathogenicity, with the hope that they can be explored for therapeutic interventions, we have systematically studied their individual and collective effects on its toxicity. One such domain is ubiquitin-binding site 1 (UbS1) on the catalytic domain of Atxn3; UbS1 is necessary for the enzymatic activity of Atxn3. Here, we investigated the importance of UbS1 on the toxicity of pathogenic Atxn3. We generated transgenic  Polyglutamine (polyQ)-expansion diseases are inherited neurodegenerative disorders that result from the abnormal lengthening of a CAG triplet nucleotide repeat in specific genes, which is translated into a polyQ tract in corresponding proteins. Abnormal polyQ expansion leads to disease protein misfolding, formation of proteinaceous aggregates that include the offending protein, cellular malfunction, and death. The polyQ family consists of 9 clinically distinct disorders including Huntington’s Disease (HD), Dentatorubral–pallidoluysian atrophy, Kennedy’s Disease, and Spinocerebellar Ataxia Type (SCA) 1, 2, 3, 6, 7, and 17 .Spinocerebellar Ataxia Type 3  is a progressive, dominant ataxia. SCA3 patients experience coordination and balance issues accompanied by neurodegeneration in cerebellar pathways, substantia nigra, cranial motor nerve nuclei, dentate nuclei, and peripheral neurons. These symptoms typically appear in mid-adulthood; however, the time of their onset and severity is influenced by the length of the polyQ expansion within the disease protein, ataxin-3 (Atxn3). Atxn3 is a ubiquitously expressed deubiquitinating enzyme (DUB) that functions by binding poly-ubiquitin chains and cleaving them; it is involved in protein quality control, cytoskeletal regulation, DNA damage control, sensory organ development and function, and more . In miceLocated on the N-terminal half of Atxn3  is the JDrosophila melanogaster as the model organism.Over the past several years, our group has dissected the individual and combined effects of the various Atxn3 protein domains to understand their role in SCA3 and to explore their utility as therapeutic targets: The catalytic site, UbS2, the VBM, and the UIMs , 2022a. in vitro  causes a marked increase in the toxicity of polyQ-expanded Atxn3 in all tissues and stages tested. However, unlike with the mutation of other domains, UbS1 mutation enhances Atxn3 toxicity without seemingly altering its overall protein levels or aggregation. Instead, based on additional genetics and biochemical evidence, we propose that UbS1 regulates the pathogenicity of Atxn3 by impacting the processing of endogenous ubiquitin species.As a DUB, recombinant Atxn3 cleaves ubiquitin chains with preference for K63-linked species that are longer than 4 ubiquitin moieties long . Detailein vitro , but itsre toxic , we hypoATXN3 sequences used in previous publications  cDNA sequence was based on the human ications , 2022a. nsertion  into attnsertion .Anti-HA ; anti-ataxin-3 ; anti-tubulin ; anti-lamin ; anti-FLAG ; anti-ubiquitin ; peroxidase-conjugated secondary antibodies: Goat anti-mouse and goat anti-rabbit, 1:5000, were from Jackson ImmunoResearch.Drosophila Stock Center. UAS-DnaJ-1 lines were from FlyORF (#002258) and Bloomington Drosophila Stock Center (#30553). The following stocks were gifts: sqh-Gal4 , elav-GS-Gal4 , elav-Gal4 . All flies were heterozygous for driver and transgene(s). All transgenic Atxn3 lines used here were generated by us and were reported previously  was from the Bloomington eviously  or are bFor experiments measuring lifespan, adults were collected daily and secluded into groups of the same size  and gender to record deaths, unless otherwise noted. Flies were flipped into new food every other day. The number of adults tracked (N) is noted in figures. In cases where death tracking was focused on the developmental stage, flies were observed from the embryo stage through eclosure or adult death, and deaths at each stage were recorded multiple times each week.Western blots were performed with 5 adult flies or 5 dissected fly heads per biological sample (N), depending on the experiment and driver being used. Samples were physically homogenized in boiling lysis buffer , sonicated, boiled for 10 min, and centrifuged for 7 min at 13,300 rpm at room temperature. Samples were electrophoresed through pre-cast, 4–20% Tris/Glycine gels (Bio-Rad). ChemiDoc (Bio-Rad) was used to image Western blots, which were then quantified with ImageLab (Bio-Rad). Unless otherwise noted, quantification was done using the volume of whole lanes measuring the levels of each protein of interest and corrected for their respective background: Main band to top of gel for Atxn3 and whole lane for ubiquitin signal. Direct blue stains of total protein were performed by saturating the PVDF membrane for 10 min in 0.008% Direct Blue 71 (Sigma-Aldrich) in 40% ethanol and 10% acetic acid, and then rinsed with a solution of 40% ethanol/10% acetic acid before being air dried and imaged. Direct Blue signal was used as loading control, unless otherwise stated.g for 1 min at room temperature, and then diluted further by combining 100 μL lysate (supernatant only) with 400 μL PBS. 40 μL of this final lysate was added to a Bio-Dot apparatus (Bio-Rad) and vacuumed through a 0.45 μm nitrocellulose membrane (Schleicher and Schuell) that had been pre-incubated in 0.1% SDS in PBS. The membrane was then rinsed with 0.1% SDS/PBS twice, processed for Western blotting, and imaged with ChemiDoc.Three whole adult flies were used per sample. Samples were homogenized thoroughly in 200 μL NETN lysis buffer  to which protease inhibitor cocktail  was added. The lysates were then diluted with 200 μL 0.5% SDS in PBS. Diluted lysates were sonicated, centrifuged at 4,500 × Total RNA was isolated from 5 whole flies or 30 fly heads per sample using TRIzol reagent (Thermo-Fisher Scientific) and treated with TURBO DNAse (Thermo-Fisher Scientific) to remove genomic DNA. cDNA was generated with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR reactions were carried out on a StepOnePlus system (Applied Biosystems) using Fast SYBR™ Green Master Mix (Applied Biosystems). The PCR cycle threshold (Ct) was used for calculation of the relative mRNA expression level. Rp49 was used as internal control. Primers were: Atxn3-F: 5′-GAATGGCAGAAGGAGGAGTTACTA-3′; Atxn3-R: 5′-GACCCGTCAAGAGAGAATTCAAGT-3′; DnaJ-1-F: 5′-GTACAAGGAGGAGAAGGTGCTG-3′; DnaJ-1-R: 5′-CAGACTGATCTGGGCTGTATACTT-3′; Rp49-F: 5′-AGATCGTGAAGAAGCGCACCAAG-3′; Rp49-R: 5′-CA CCAGGAACTTCTTGAATCCGG-3′.g, 4°C, 10 min), supernatant was combined with bead-bound antibodies and tumbled at 4°C for 4 h. Beads were rinsed five times each with lysis buffer, and bead-bound complexes were eluted by adding Laemmli buffer (Bio-Rad) and boiling.Ten adult flies or 30 dissected adult fly heads, depending on experiment, were homogenized in 800 μL NETN or 1:1 NETN/PBS lysis buffer supplemented with PI. Samples were incubated at 4°C with agitation for 25 min. Following centrifugation of the incubated samples  using seven whole flies per group that were lysed in cytoplasmic extraction buffer (Bio-Rad) and processes as delineated by the supplier’s protocols.Statistical tests were conducted using Prism 9 (GraphPad) and are specified in figure legends. The number of biological replicates is noted on figures and corresponding legends.Drosophila, attP2. A single copy of the transgene is inserted in each case, in the same orientation. The expression system that we utilized to examine the effect of UbS1 mutation is the binary Gal4-UAS system, which enables the expression of targeted transgenes in a specific temporal and spatial manner -UbS1* , similarAs a result of: (1) previously published results that UbS1* renders Atxn3 catalytically dead , 2010, aTo explore these possibilities, we generated additional, transgenic fly lines that express Atxn3(Q80)-UbS1* either with a catalytic cysteine mutation (C14*), or with mutated UIMs  UIM*; Fi. We confUbiquitous expression leads to larval death in every line expressing Atxn3(Q80)-UbS1*, regardless of additional mutations ; we did We then examined whether the C14 and UIM mutations impact the UbS1* phenotype in all neurons. As we showed in Lastly, we compared the longevity of flies that expressed UbS1*-containing Atxn3(Q80) without additional mutations, or in addition to C14* or UIM*, only in adult neurons. We observed that mutating the catalytic cysteine slightly but significantly worsens the UbS1* phenotype . HoweverDrosophila. Alongside the other data presented here (The reduction in toxicity that we observed when the UIMs of Atxn3(Q80)-UbS1* are disrupted led us to examine whether UbS1-mutated Atxn3(Q80) interacts more strongly with ubiquitinated species in flies. We expressed Atxn3(Q80) with intact or mutated UbS1 in adult fly neurons for 24 h, IP-ed Atxn3, and then examined the levels of ubiquitin species that co-precipitate with it. We observed that UbS1 mutation leads to higher amounts of ubiquitinated species co-IP-ing with Atxn3(Q80)-UbS1* compared to Atxn3(Q80) . We obseted here , we propted here . But, asDrosophila melanogaster. These investigations have collectively increased the understanding of the disease-causing properties of Atxn3 and have identified potential therapeutic points of intervention.Protein-protein interactions play critical roles in the precise symptomatology of polyQ diseases . Toward in vitro  we in vitro ; (3) stuactivity ; and (4)activity . Thus, wDrosophila? The mechanism of this enhanced toxicity remains to be determined and will require a deeper understanding of the biological functions of wild-type Atxn3 and how those functions are impacted by polyQ expansions. Those examinations are outside the scope of this study and are confounded by the fact that the precise role of Atxn3 in intact organisms remains unclear. While various studies have provided significant evidence that this DUB is involved in endoplasmic reticulum-associated degradation and other types of protein quality control, DNA damage repair, response to heat stress, transcriptional regulation, sensory organ development and function, synaptic communication, etc. (Why does UbS1 mutation render pathogenic Atxn3 more toxic in on, etc. , 2022, ion, etc. . As withDrosophila and also help mediate Atxn3-Atxn3 interactions in the fly, which also occur in mammalian cells (What we propose based on this study and the investigations of other domains of Atxn3 , 2022a ian cells . Thus, sHowever, the above is likely not the only mechanism through which UbS1-mutated, pathogenic Atxn3 becomes more toxic. After all, UIM mutations do not fully reverse UbS1*-dependent toxicity in eyes and in developing neurons, and have no detectable modifying effect on UbS1-mutated Atxn3 expressed everywhere. Also, mutation of the catalytic cysteine of Atxn3 enhances UbS1*-dependent toxicity in adult neurons—although it does not noticeably impact it when tested in fly eyes or during development. Perhaps UbS1 also directly binds proteins other than ubiquitin and these interactions further impact Atxn3 toxicity. This idea is supported by the fact that the other ubiquitin-binding site on Atxn3’s catalytic domain, UbS2 directly interacts with both ubiquitin and the proteasome shuttle protein, Rad23; the latter interaction has critical outcomes for the degradation and pathogenicity of Atxn3 . This prDrosophila (To conclude, UbS1 mutations exacerbate the toxicity of Atxn3 in osophila , at leasThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MP: conceptualization, data curation, software, formal analysis, funding acquisition, validation, investigation, visualization, methodology, and writing and editing. KL: data curation, formal analysis, supervision, validation, investigation, and visualization. AH: data curation, formal analysis, validation, and visualization. W-LT: conceptualization, data curation, software, validation, investigation, visualization, methodology, and writing and editing. ST: conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, and writing and editing. All authors contributed to the article and approved the submitted version.


In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development Adam10 deficiency (ecko)Adam10 in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of eckoAdam10 mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of eckoAdam10 mice was significantly reduced compared to wildtypes.Endothelial Collectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases. Atherosclerosis is a lipid-driven chronic inflammatory disease, which manifests in regions with disturbed flow in the medium- and large-sized arteries. The initiation of atherosclerosis is characterized by endothelial cell activation and dysfunction, leading to the disruption of the endothelial barrier function and the active recruitment of leukocytes into the vessel wall .Adam10 results in embryonic lethality at stage E9.5, when the vascular system develops  shedding. The A Disintegrin And Metalloproteinase (ADAM) family is involved in the shedding of numerous cell surface proteins, e.g., adhesion molecules, chemokines and cytokine receptors . One of develops . Indeed,develops  and to ldevelops –9. Additdevelops  and intrdevelops . ADAM10 develops , 13. Predevelops . In a cofibrosis . ADAM10 rization . HoweverAdam10  in murine aortic root atherosclerotic lesions of Western type diet (WTD) fed mice  challenged with oxidized low-density lipoprotein (oxLDL) resulted in a more severe pro-inflammatory, hence more pro-atherogenic, phenotype, in agreement with significantly reduced LOX-1 shedding.Here, we used the model of adeno-associated virus-mediated proprotein convertase subtilisin/kexin type 9 (PCSK9) gene transfer in mice deficient in endothelial Adam10 –17, to ifed mice . KnockdoTie2-Cre) Adam10 knockout (eckoAdam10) and wildtype (wtAdam10) littermate control mice on a mixed genetic background were previously described , and were performed in compliance with the Dutch government guidelines. Female endothelial specific , fixed overnight in 1% paraformaldehyde and embedded in paraffin. Cross-sections (4 μm) were cut and four sections (100 μm apart) were stained with Movat’s stain to visualize the elastic laminae. Pictures were taken using a Leica DM3000 light microscope and sections were analyzed in a blinded manner using computerized morphometry (Leica QWin V3). For each mouse, total vessel area , medial area , and lumen area  were measured at 100 μm distance from the aortic arch.Left carotid arteries were collected from female wildtype and eckoAdam10 mice  were rendered prone to atherosclerosis by a single intravenous injection of adeno-associated virus serotype 8 containing D377Y-murine PCSK9  interve0 weeks) . IPH has0 weeks) , featurec plaque , 44, 45,cko mice .eckoAdam10 mice. Angiogenesis, the sprouting of new blood vessels from the existing vasculature, is a tightly regulated process which is induced by various stimuli, including pro-angiogenic growth factors, like vascular endothelial growth factor (VEGF), hypoxia, metabolic stress and inflammation (Adam10 deficiency.The broad pattern regulatory function of ADAM10 makes it challenging to pinpoint a single responsible key mediator for the observed phenotype in ammation . ADAM10 ammation , 11, 47.ammation . Interesammation , suggestAdam10 deficiency involves ADAM10’s inflammation dampening functions. As highlighted in this and other studies (eckoAdam10 atherosclerotic plaques are more inflammatory, with an enrichment in inflammatory signaling, immune cell activation and cell adhesion, which could reflect both leukocyte adhesion and angiogenic processes. Previously, we have demonstrated that endothelial ADAM10 is responsible for the shedding of LOX-1, thereby increasing the surface expression of this pro-atherogenic receptor for oxLDL (Lox-1 expression results in an increased expression of ICAM-1 and VCAM (via ERK1/2 and p38 (ADAM10 deficiency indeed reduced LOX-1 shedding and enhanced endothelial proinflammatory responses to oxLDL, including p38 activation and chemokine secretion. Interestingly, the fact that ADAM17 appeared not to be involved in LOX-1 cleavage (Adam10 and Adam17 deficiency (A second important mechanism potentially contributing to the profound plaque phenotype of endothelial  studies , endothe studies . Our RNAor oxLDL , 35. It and VCAM  and also and p38 , 35, we ficiency .Taken together, this study demonstrates that endothelial ADAM10 is a protective factor in atherosclerosis, on the one hand by restraining plaque neovascularization and intraplaque hemorrhage, and on the other hand by limiting inflammation. While the ADAM10-Notch axis is a well-known master regulator of neovascularization, this study demonstrates a crucial role of ADAM10 in pro-inflammatory processes in endothelial cells, at least in part by regulating expression of leukocyte adhesion molecules and by controlling receptor availability for pro-atherogenic stimuli, as demonstrated for LOX-1. Although it remains to be determined to what extent endothelial ADAM10 can influence these vulnerable plaque features in humans, its overt expression in intraplaque microvessels in human plaques suggests an active regulatory function in humans as well .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Ethics Committee of Maastricht University, Netherlands.EV, EB, and MD: conceptualization. EV, KT, SM, and MD: methodology. EV, KT, SM, LP, HJ, TR, MG, MR, and YJ: formal analysis and investigation. EV and KT: writing—original draft preparation. EB and MD: writing—review and editing. EV and MD: funding acquisition. CW, ML, CL, DY, AL, and JB: resources. MD: supervision. All authors contributed to the article and approved the submitted version.


Both intervention approaches may preserve lean mass, while only combined interventions may lead to alterations in fat mass. However, further exercise and nutritional studies are needed to definitively understand their effects on body composition. As exercise and nutrition-related research continues in this understudied population, the knowledge gained will help guide supportive clinical treatments.Advanced and metastatic cancers significantly alter body composition, leading to decreased lean mass and variable effects on fat mass. These effects on body composition are associated with significant physical dysfunction and poor prognosis in patients with cancer. Whilst exercise and nutritional interventions are likely to be of benefit in counteracting these effects, relatively little is known about using such interventions in patients with advanced or metastatic cancer. Therefore, in this systematic review we examine the effect of exercise and combined exercise and nutritional interventions on lean mass and fat mass among patients diagnosed with advanced or metastatic cancer. Following PRISMA guidelines, we identified 20 articles from PubMed, EMBASE, CINAHL, Cochrane CENTRAL, PEDro, SPORTDiscus, and REHABDATA. Overall, advanced or metastatic cancer populations comprising of mixed cancer types were most commonly examined ( Patients with advanced and metastatic cancer are now living longer due to advancements in cancer therapy ,2,3. NevOne major change is loss of lean mass, mainly through skeletal muscle loss. Low lean mass has emerged as a driver of physical dysfunction and poor quality of life , and alsIn parallel with loss of lean mass, patients with advanced or metastatic cancer experience significant fat mass changes . Fat masExercise has emerged as an effective therapeutic adjunct to conventional cancer therapies and has been shown to improve the quality of life and physical function of patients with cancer . HoweverDespite the heightened interest in exercise and nutritional interventions in patients with more advanced disease, the evidence base is not clearly established. Previous reviews have provided preliminary evidence that exercise can improve quality of life and physical function in patients with advanced cancer ,56. AddiThis systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines . It is rThe search was performed in PubMed, Excerpta Medica Database (EMBASE), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Cochrane Central Register of Controlled Trials (CENTRAL), Physiotherapy Evidence Database (PEDro), SPORTDiscus, and National Rehabilitation Information Center Database (REHABDATA). Keywords were developed by initial idea gathering and searching a thesaurus for the following concepts: advanced/metastatic, malignancy/cancer, exercise/physical activity, and body composition. Further key words were then added through review of the titles and abstracts of relevant papers. For each database, specific medical subject headings were then added to the search terms. Databases were last searched on 23 February 2022. EMBASE search was limited to the English language, and studies indexed as “case report”, “review”, “meta-analysis” or “observational” were excluded. PEDro and Cochrane CENTRAL searches were limited to clinical trials only. Full search strategy for each database is presented in Inclusion and exclusion criteria are presented below, according to the PICOS framework . This review excluded studies that only reported undifferentiated body mass, body mass index, or bone mass outcomes. Study type: This review included all prospective randomized and non-randomized controlled trials, and therefore excluded retrospective and single-arm studies. Titles and abstracts from the database searches were imported into Covidence systematic review software . Each abstract was screened twice by a team of independent reviewers  with any conflict resolved through group discussion. The full texts were then also reviewed twice by a team of independent reviewers , with any conflict resolved through group discussion. OB screened every abstract and full text. If consensus could not be agreed by group discussion, the corresponding author (CDC) was available to aid decision making. Data from each included study were extracted using a predefined template in Covidence that included study details and background, participant characteristics, flow of participants through the trial, treatment of the intervention and comparator groups, body composition outcomes measured (including any quantitative non-significant and significant between- and within-group results reported), and adverse events.Risk of bias for randomized trials was independently assessed by two reviewers  within Covidence using the Cochrane risk-of-bias tool for randomized trials . Risk ofA total of 6092 records were identified from seven databases, leaving 4528 records after duplicate removal. Of these, 20 studies fit the inclusion criteria and were included in the systematic review . n = 4) [n = 3) [n = 3) [n = 3) [n = 2) [n = 2) [n = 2) [n = 1) [n = 1) [n = 1) [Of the twenty studies included in this systematic review, sixteen 80%) describe original results from randomized-controlled trials (RCTs) ,79,80,81% describn = 4) ,74,78,79 [n = 3) ,83,84, t [n = 3) ,81,85, A [n = 3) ,69,73, t [n = 2) , Switzer [n = 2) ,77, Norw [n = 2) ,85, Taiw [n = 1) , India ( [n = 1) , and the [n = 1) . In total, 1091 patients were allocated to the interventions of the included studies with an average age ranging from 44.0  to 76.9 n = 6; 30%) [n = 1; 5%) [n = 2; 10%) [During the respective study intervention periods, 12 studies (60%) examined patients who were actively receiving or about to receive treatment as dictated as part of their inclusion criteria. These treatments included chemotherapy  ,81,82,85= 1; 5%) , and and 2; 10%) ,74, with 2; 10%) ,83,84. O 2; 10%) ,77,78,80 2; 10%) ,69, and  2; 10%) . The detn = 6; 30%) [n = 8; 40%) [n = 6; 30%) [Within this review, the included studies are categorized into three groups based on their intervention: exercise alone  ,74,75,78 8; 40%) ,83,84,86 6; 30%) ,80,81,85n = 7; 35%) [Regarding the exercise component, eight studies (40%) exclusively used supervised exercise ,78,83,84 7; 35%) ,83,84; h 7; 35%) ,82, depe 7; 35%) , or vari 7; 35%) ,77,81,85 7; 35%) ,74,76,80 7; 35%) . The int 7; 35%) ,73,81, r 7; 35%) ,82, or t 7; 35%) ,77,81. In terms of nutritional interventions, nutrition counselling occurred either at baseline only ,83,84,85Regarding the further components that studies included alongside exercise and nutritional interventions, several utilized psychological counselling/support ,79,81, wAdherence to supervised exercise sessions ranged from 54.0%  to 93.0%Body composition was identified as the primary outcome in six studies (30%) ,76,83,85Six exercise only studies reported a lean mass-related outcome ,74,75,782/m2) than the control group (−11.6 versus −15.6 cm2/m2) [2/m2) in the control group (−1.8 cm2/m2). Five studies examined the effect of a combined exercise and nutritional intervention on lean mass-related outcomes ,82,83,84 cm2/m2) . A separ cm2/m2)  reported cm2/m2)  reportedAmong the five exercise-only studies reporting fat mass-related outcomes, no significant effects were reported ,73,74,78The results of the risk of bias assessment for each included study are summarized in To the authors’ knowledge, this is the first systematic review to provide a comprehensive summary of the effects of exercise with or without nutritional interventions on lean mass and fat mass among patients with advanced or metastatic cancer. Twenty studies were considered eligible, and despite the inclusion of a heterogeneous sample, there was a trend for both exercise alone and combined exercise and nutritional interventions to preserve or improve lean mass-related outcomes. Moreover, only those studies utilizing combined exercise and nutritional interventions reported alterations to fat mass-related outcomes.Our systematic review indicates that both exercise and combined exercise and nutrition interventions have some impact on lean mass-related outcomes among patients with advanced or metastatic cancer, where 50% of studies measuring a lean mass-related outcome reported a significant intervention effect ,83,84,852 and encouraged a high volume of exercise  alongside the consumption of whole grains, fruits, and vegetables. Notably, Sheean et al. [Only two (17%) studies that reported a fat mass-related outcome noted a significant intervention effect ,80. Furtn et al. ,92. Our n et al. . Neverthn et al. . Therefon et al. . Future studies should continue to quantitatively assess the extent to which exercise, with or without a nutritional intervention or ‘other’ intervention components as detailed in A strength of this review is that it is the first to comprehensively examine exercise and combined exercise and nutritional interventions in patients with advanced and metastatic cancer beyond a single cancer type. Until now, nutrition as an additive component to exercise had not been addressed in a systematic review among patients with advanced or metastatic cancer. We acknowledge the additional benefit nutrition may have in an exercise intervention, with both components possibly acting in synergy and now recognized as critical in clinical practice ,52,53,54Another limitation, as noted by other reviews ,58, is tThis review provides a summary of the effects of exercise with and without nutritional interventions on body composition in patients with advanced and metastatic cancer. Our review indicates that exercise interventions with and without nutrition may preserve or improve lean mass-related outcomes among patients with advanced and metastatic cancer. Moreover, only combined exercise and nutrition interventions induced alterations in fat mass-related outcomes, although further studies are clearly needed. Whilst unable to definitively outline the extent to which exercise with or without nutritional interventions can alter lean and fat mass, our findings summarize the ongoing research in this field and suggest that future studies should investigate clear hypotheses about their intended effects on body composition. A better understanding of the role of body composition in advanced and metastatic cancer will benefit our understanding of cancer survivorship as a whole, particularly with respect to improved quality of life and tolerance of ongoing treatment. In turn, as exercise and nutrition-related research continues to develop in this understudied population, the inclusion of such strategies should be considered more frequently in oncologic care in line with emerging guidelines.


Differential diagnosis of CLDs has remained challenging due to the highly variable morphology features and immunophenotyping. Currently, the development of multiple-marker panel analyses by flow cytometry has opened a broad way for diagnosis of CLDs. We analyzed the peripheral blood and BM samples of 131 patients with B-cell CLDs  from April 2018 to April 2019, using a panel of specific markers by flow cytometry. p value less than 0.001 for both markers. In contrast to MCL patients, all the patients with atypical CLL, expressed kappa light chain. The IHC method used for cyclin D1 confirmed that the flow cytometry detection of kappa and lambda light chains could provide a new approach with high sensitivity (91%) and moderate specificity (50%) to distinguish MCL patients from atypical B-CLL. Our results indicated that the expression pattern of CD22, CD23, FMC-7, and CD5 allowed us to accurately and differentially diagnose the B-CLL, MCL, and CD5-/CD10- lymphoma, while it was not capable of differentiating MCL from atypical CLL. We, however, found that the expression patterns of CD38 and immunoglobulin light chain differed significantly between atypical B-CLL and MCL. CD38 and lambda light chain were remarkably expressed in MCL patients  compared to the atypical CLL (1.1% and 0% respectively), with the Expression of CD5, CD20 (bright), CD22, FMC-7, CD38, and lambda light chain with no expression of CD23 can accurately detect MCL and differentiate it from atypical B-CLL.  The flow cytometric analysis is nowadays considered as an accurate and a precise diagnostic tool for this purpose[4-7]. However, since no marker offers an absolute diagnostic value, applying a panel of MoAbs to create scoring systems are assumed to provide more beneficial diagnostic insight rather than single antigens.Chronic lymphoproliferative disorders are a group of heterogeneous diseases that require more specific targeted and therapeutic strategies. Novel technical advances have recently enabled clinicians to accurately diagnose such disorders. While clinical characteristics, morphologic features, and immunologic phenotypes provide reliable diagnosis in most cases of B-cell CLDs, a small subset of these malignancies shows a variable phenotype that makes the diagnosis challenging. Thus, the immunophenotypic characterization of cancer cells using MoAbs targeting specific CD markers, which are basically applied to characterize the chronic leukemia, has earned a crucial role in differential diagnosis of CLDs[2]. Although small lymphocytes with easily diagnosable clumped chromatin and specific immunophenotype make the diagnosis of classical B-CLL quite clear, the atypical B-CLL with its highly variable morphology and immunophenotype is faced with more challenges to be precisely diagnosed. In case of CLL, heterogeneous morphologic and immunologic characteristics have long been described. Moreover, its clinical features are highly variable, which are even observed within the same clinical stage. Based on the French-American-British classification and morphologic attributes, B-CLL is subdivided into typical composed of monomorphic lymphocytes and atypical (or B-CLL variant) with mixed cell types[10]. Morphologically, MCL patients show a monotonous proliferation of lymphocytes, which contain scant cytoplasm and slightly irregular nuclei. Immunophenotype of MCL cells is also characterized by the co-expression of CD5 and pan B-cell antigens , which are also detectable in CLL/small lymphocytic lymphoma. While some believe that CD23 might offer a distinguishing value, several contradictory results have been reported in this case[11]. Therefore, further accurate differential criteria are required for guiding the therapeutic options for these two types of malignancies. More recently, absolute quantification using flow cytometric assessment of several surface molecules have provided a potential diagnostic value in this context.MCL is characterized by the proliferation of B cells in the mantle zone of lymphoid folliclesTaken together, the combination of morphologic and immunophenotypic characteristics is essential to obtain an accurate diagnosis and to minimize interpretative variations. Therefore, we aimed to first assess 131 patients with various forms of CLDs by analyzing the most useful multi-marker panel in order to achieve a clearer insight into the distinctive diagnosis of such heterogeneous disorders based on immunologic criteria.Selection of patientsIn this study, 1,185 patients were referred to Takhte Tavous Pathobiology Laboratory, Tehran, Iran. BM and peripheral blood samples were taken from the patients from April 2018 to April 2019. Among these patients, 131 (with an age ranging from 37 to 90 years) were diagnosed with lymphoproliferative diseases based on analyzing the surface markers using flow cytometry. Patients with CLDs were new cases (with no history of previous treatment) diagnosed with a chronic lymphoproliferative disease with clinical presentation of CLDs such as leukocytosis, lymphadenopathy, or splenomegaly. The exclusion criteria were patients whose chronic lympho-proliferative disease have not been diagnosed. The patients with CLDs were divided into B-CLL (n = 91), atypical B-CLL (n = 15), MCL (n = 14), and CD5-negative/CD10-negative lymphoma (n = 11) based on the expression of B-cell surface markers, including HLA-DR, CD5, CD19, CD20, CD22, CD23, CD10, CD38, CD45, and kappa and lambda light chains. Classification of CLDs was performed according to the expression of CD5, CD1, and CD23 to distinguish CLL from MCL and other chronic B-cell disorders. Flow cytometry2-EDTA and gently homogenized several times. Samples were analyzed by multi-parametric flow cytometry  via MXP software using a combination of three surface markers of MoAbs in Takhte Tavous Pathobiology Laboratory. These three-color combination included (1) CD5 /CD19 /CD23 , (2) CD20 /CD22 /CD38 , (3) CD10 / CD19 /CD45 , (4) Kappa /Lambda /CD19 , and HLA-DR  in a single color tube. BM or blood cells were incubated with the aforementioned MoAbs at 4 °C for 30 min, and then erythrocytes were lysed using a standard lyse/wash technique. Finally, the samples were analyzed by a flow cytometer, and antigenic expression in blast cells was systematically analyzed by multi-parametric flow cytometry. The optimal cut-off for each marker was considered as 20% expression of the analyzed events for cell surface antigen expression. Fresh BM (5 ml) or blood sample was collected into vacutainer tubes containing KImmunohistochemistryCyclin D1, a marker considered highly specific and sensitive for MCL was used to confirm the diagnosis of MCL. Four-micron sections of paraffin-embedded tissue blocks were stained using specific MoAb for human cyclin D1 . Slides were counterstained with Harris hematoxylin and examined by standard light microscopy. Samples were analyzed by using an Olympus BX51TF microscope, and pictures were taken using Olympus QColor3 and analyzed by using QCapture 2.60 software (QImaging). We considered cyclin D1 to be positive if more than of 10% of the cells showed nuclear positivity. Cytoplasmic staining was not considered positive.Statistical analysisAll statistical analyses were performed by SPSS software (version 21). We used one way ANOVA to compare the subtypes of CLDs. Correlation analysis between flow cytometric markers and hemoglobin, platelet count and leukocyte blood count were assessed by Pearson and Spearman test. P values of less than 0.05 were regarded as statistically significant.Characteristics of patients p = 0.000). The patients in the CD5-/CD10-lymphoma group had lower hemoglobin content compared with the other three patient groups. Platelet count was lower in the atypical B-CLL group compared with the B-CLL, MCL, and CD5-/CD10-lymphoma groups (p = 0.013).In this study, 131 patients were diagnosed with B-CLL (n = 91), atypical B-CLL (n = 15), MCL (n = 14) and CD5-/CD10 ymphoma (n = 11). There were 81 (61.8%) men and 50 (38.2%) women ranging from 37 to 90 years old . Characteristics of each group of patients, including gender, age, hemoglobin, platelet count, and leukocyte blood count, are summarized in Comparison of flow cytometry immunophenotyping markers between patient groupsp = 0.1 and p = 0.2, respectively), but in the case of CD22, all the patients, except B-CLL cases, expressed this marker (p = 0.000). Moreover, a high expression level of the CD5 marker was found in B-CLL and MCL patients , although atypical B-CLL patients expressed a lower level of this marker (45.5 ± 27.0). Remarkably, B-CLL patients and to a less extent, atypical B-CLL patients had CD23 expression (p = 0.000). However, three patients with MCL expressed CD23 nearly 20%. On the other hand, only MCL, CD5-negative, and CD10-negative patients expressed FMC-7 antigen (p = 0.000). Interestingly, the pattern of CD22, CD23, and FMC-7 antigens, along with the CD5 co-expression, permitted the accurate classification of all B-CLL, and CD5-/CD10-lymphoma using flow cytometry. The critical challenge is that these markers are unable to differentiate MCL from atypical B-CLL; however, CD38 is another marker that can be helpful for discrimination between these two groups. In this regard, all patients with atypical B-CLL, except for one case, were negative for CD38 expression, and all MCL patients, except for one case, were positive for CD38 expression (p = 0.000). According to these findings, the expression of CD38 allowed us to somehow distinguish MCL from the atypical B-CLL  expressed kappa immunoglobulin light chain. Besides, 12 out of 14 MCL patients (85%) had lambda chain, while two cases (15%) illustrated kappa light chain. In order to measure the specificity and sensitivity of flow cytometry assessment of light chain in detecting MCL, IHC method was conducted for cyclin D1 in MCL and atypical B-CLL patients on BM biopsy samples. In cases of atypical B-CLL, all patients showed negative results for cyclin D1. Although cyclin D1 was negative in one case with lambda light chain, it was positive in the other 11 patients with lambda light expression . Correlation between flow cytometric markers and hemoglobin content, platelet, and leukocyteblood countsp = 0.006 and r = -0.234, p = 0.007, respectively). Our results also indicated a strong negative association between CD22 , FMC-7 , and also CD38  with platelet count. Correlation studies showed a significant positive association between CD19  and CD20  with leukocyte blood count , a finding which is in agreement with a study performed by Bertoni et al.[17] who reported lambda light chain expression in 10 out of 12 subjects with MCL (83%). However, another study represented a kappa restriction in indolent MCL patients[18]. Overall, according to the guidelines for investigation of MCL, the classical MCL is lambda light chain-restricted, while the indolent MCL is mostly kappa-restricted[19].Currently, diagnosis of B-cell CLDs is mainly based on clinical findings, morphologic characteristics, and evaluation of the immunophenotyping markers, through which the diagnosis of a large group of B-cell CLDs can be made precisely. Nevertheless, few patients still require additional investigations for differential diagnosis. In this study, we assessed a total of 131 patients with different B-cell CLDs, including B-CLL, atypical B-CLL, MCL, and CD5-/CD10-lymphoma, for a panel of B-cell-specific markers viz CD5, CD20, CD22, CD23, and FMC-7. B-CLL and MCL, both known as CD5-positive B-cell malignancies, can be differentiated mainly via CD23, which is typically absent on the surface of MCL neoplastic B-cells but is highly expressed in B-CLL. Moreover, MCL cases are often positive for FMC-7, which is commonly not expressed in B-CLL. Generally, immunophenotyping can simply differentiate B-CLL from MCL, but the differentiation of atypical B-CLL from MCL, which are immunologically very similar, remained a big challenge. This study evaluated the immunophenotype of atypical CLL and MCL, to help in the differential diagnosis of CD5-positive monoclonal B cells. D’Arena et al.[20] displayed a significant negative correlation between CD20 expression and platelet counts. Furthermore, we found that the high expression of CD22, similar to CD20, was related to the low levelof both hemoglobin and platelet count. Our data also revealed a negative correlation between FMC-7 and hemoglobin, while CD5 and CD23 were positively correlated with hemoglobin content. Generally, our findings highlight the clinical importance of the quantitative expression of the immunophenotyping markers and their probable value as diagnostic markers in different subgroups of B-cell CLD patients. Altogether, expression of CD20 (bright), CD22, FMC-7, CD38, and lambda light chain, along with positivity in CD5 expression and negativity in expression of CD23, can confirm MCL detection and differentiate it from atypical B-CLL.Our results indicated that exploring the pattern of kappa and lambda immunoglobulin light chain expression by flow cytometry can distinguish MCL from atypical B-CLL with a high sensitivity (91%) and moderate specificity (50%); as all cases (100%) of atypical B-CLL expressed kappa light chain, and most cases (85%) with MCL had lambda light chain expression. The sensitivity and specificity were calculated in comparison with cyclin D1 expression using IHC method, as a specific and sensitive method for MCL diagnosis. We also assessed the correlation of hemoglobin, platelet, and leukocyte blood count with the immunophenotyping markers. Our results showed that CD20 had a negative correlation with hemoglobin level and platelet count, while it was positively correlated with leukocyte blood count. As a result, the higher CD20 expression is associated with the lower hemoglobin content and platelet number and also with the higher leukocyte blood count. In a recent study, Asnafi et al.We acknowledge the staff of Takhte Tavous Pathobiology Lab, Tehran, Iran for data collection and technical support.The above-mentioned sampling protocol was approved by the Medical Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.REC.1398.95). A written consent was obtained from each patient. The analyzed data sets generated during the study are available from the corresponding author on reasonable request.MM: evaluated the data and drafted the manuscript; MSHJ: re-evaluated the data and revised manuscript; LJ: revised manuscript; MM: evaluated the data; ESS: designed the study and collected the data. None declared.This study has received no financial support from any institute or organization.


In this article, we concisely and critically review the role of NAD+ in mechanisms promoting genome maintenance. Numerous NAD+-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD+ serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD+ levels.NAD In order to supply cells with “fresh” exogenous precursors, those can enter the cell during a passive process  or via membrane transporters—members of the solute carrier transporter family (for Trp and NA) or equilibrative nucleoside transporter (for NR and NAR). Furthermore, exogenous NMN can be taken up by cells, yet the cellular transport of this precursor molecule remains unclear  and intracellularly (iNAMPT) in the cytoplasm and nucleus, whereas its mitochondrial presence is disputed (+ by ATP-dependent nicotinamide mononucleotide adenylyl transferase (NMNAT). This enzyme in human tissues exists in three isoforms: the most ubiquitous nuclear NMNAT1, cytoplasmic NMNAT2 + in mammdisputed . Besidesdisputed , and cledisputed . In the c NMNAT2 , and mitc NMNAT2 , 27, 28.nsferase . In the n humans , 31. In lutamine .+ levels are maintained in the range of 100 μM in the cytoplasm and nucleus and 200 μM in mitochondria  and are variable in tissues with different metabolic roles. Similarities in nuclear and cytoplasmic levels are likely because of the passive diffusion of NAD+ and its precursors through nuclear pores , providing mechanistic insight how mitochondria are supplied with NAD+  are critical regulators of cellular bioenergetics, serving as cofactors in reactions of glucose metabolism, fatty acid β-oxidation, or the tricarboxylic acid cycle. For example, NAD+ is reduced to NADH during glycolysis by GAPDH. In turn, NADH is oxidized by lactate dehydrogenase, when pyruvate is converted to lactate under anaerobic conditions. NADH is a cofactor for fatty acid desaturases, and NAD+ is essential for alcohol dehydrogenase. NAD+/NADH-dependent malate dehydrogenase  and glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate shuttle) generate reducing equivalents transported across the membrane into the mitochondria. There, NAD+ is reduced by pyruvate dehydrogenase complex and the tricarboxylic acid cycle enzymes: malate dehydrogenase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase. NAD+ is also reduced by hydroxyacyl-CoA dehydrogenase in β-oxidation during fatty acid metabolism. Subsequently generated NADH is the major reducing factor in complex I mitochondrial electron transport chain, for the transfer of electrons in oxidative phosphorylation that produces ATP. Thus, the NAD+/NADH abundance reflects the energy status of the cell  in the cytosol (cNADK) and mitochondria (mNADK) phosphorylate NAD+ to NADP+, which together with its reduced form NADPH, is critical for the maintenance of cellular redox homeostasis. It regenerates molecules responsible for xenobiotic detoxification, for example, cytochrome P450, and eradication of reactive oxygen species (ROS), such as GSH, thioredoxin, or peroxiredoxin  signaling H couples serve as an electron carrier, NAD+ can be also irreversibly consumed (In addition to the numerous reactions where NAD(P)consumed . ADP-ribconsumed , 50, 51.consumed .Figure 3+ is also consumed by histone deacetylases known as sirtuins (SIRTs). They use NAD+ as an acceptor during the transfer of acetyl (Ac) and other groups from target protein, which is a common post-translational modification, implemented in numerous cellular pathways, including those involved in genomic maintenance (+ consuming enzymes are glycohydrolases (NADases), such as CD73, CD38, or sterile α and TIR motif–containing 1. Apart from generating ADPR, this enzyme exhibits ADP-ribosyl cyclase activity, which produces cyclic ADPR, a second messenger in Ca2+ signaling. For instance, CD38 is a major extracellular NAD+ consumer, and its activity has a wide range of implications in the context of infection, metabolic dysfunction, aging, or tumor biology , and TNKS2 (tankyrase 2). Those PARPs share NAD+ as a substrate with the SIRTs, of which some family members, such as SIRT1, SIRT6, and SIRT7, are directly involved in genome maintenance  covalently attached to the target molecule. Thereby, ARTDs modify protein residues  or terminal phosphates at DNA breaks (+ is cleaved and NAM is released. The elongation of PAR chains involves the formation of the glycosidic bond between two riboses: 2′–1′′ for linear chains and 1′–2′′ for branched molecules and can involve up to 200 ADPR units (A). Although PARylation is reversible, NAD+ is not restored—PAR chains are degraded by poly(ADP-ribose) glycohydrolase to free PAR and/or ADPR molecules because of the cleavage of O-glycosidic bond. The terminal protein-ADPR moiety is cleaved by ADP-ribosyl hydrolases, the macrodomain containing proteins MacroD1/2, or the terminal ADP-ribose protein glycohydrolase 1 (TARG1) (ARTDs (aka PARPs) are the major consumers of cellular NADA breaks , 62. DurPR units A. Althou (TARG1) . The hig (TARG1) , 50. Fur (TARG1) , 64.+ pool  ,   or to transcription enhancers   , homologBRCA1]),  or NHEJ NA-PKcs)  (more exNA-PKcs) . PARP1 iNA-PKcs) , 75. PARNA-PKcs) . Moreoveprotein) , 78. PARprotein) . Other Aprotein) . For a dprotein) , 82, 83,protein)  and tranprotein) , 86. Morprotein) , 88, 89,protein) .Table 1N+ levels. ATP-dependent NAD+ re-synthesis can lead to an energy crisis and metabolic shifts  which cause cellular damage and subsequent cell death via necrosis (D993A) resulting in hypo-PARylation activity and the formation of hypobranched PAR, revealed compromised genotoxic stress response during replication and led to a shift of the cell fate of mouse embryonic fibroblasts after genotoxic treatment away from DNA repair towards cell death and senescence  for major PARPs are similar to or higher than the concentration of free NAD+, which indicates that NAD+ levels in different subcellular compartments can significantly affect the activity of PARP1 and other PARP family members  PARP1 activity, but upon DNA damage, PARP1 interaction with DNA strand breaks leads to allosteric changes that allow full NAD+ access to its catalytic site and promotes activation of the enzyme , a potential tumor suppressor, which inhibits PARP1 activity and leads to DNA damage accumulation  and deacetylated protein (B) (SIRTs (SITRs) are NADhologies , 107. SI protein B  , and thetein (B) , 111. Whtein (B) , 113. Owtein (B) , 115, intein (B) , circaditein (B) , redox htein (B) , autophatein (B) , apoptostein (B) , as welltein (B) , 54, andAnalogously to PARPs, nuclear SIRTs contribute to genome integrity and DNA repair at numerous levels . SIRT1 , 122, SI+ availability , or NMN (from NAD+). Upon binding to nicked DNA, the AMP moiety is transferred from the Lys to the 5′-phosphate end. Subsequently, the hydroxyl group at 3′ end (3′-OH) attacks the adenylated 5′-phosphate (5′-PO4) to establish the new phosphodiester bond between the two ends, which releases AMP and finalizes the ligation (C)  . Mammalition (C) , 172. Prn (C)  (Besides direct reactions mediated by NADSWI/SNF) , 177, whSWI/SNF) , kinasesSWI/SNF) , 180, orSWI/SNF) . LikewisSWI/SNF) , 183.+-dependent energy and redox homeostasis with genome maintenance is GAPDH. When oxidized by ROS, it acquires nonglycolytic functions and translocates to the nucleus, where it contributes to the maintenance of DNA integrity, transcriptional regulation, and tRNA export , impair the activity of molecules regulating DNA damage response, DNA repair, and other nuclear processes, and influence the activity of transcription factors, modulating gene expression. Such a wide array of ROS-induced alterations affects genomic integrity, contribute to mutagenesis, and cell death , 186. AnA export .+-derived cyclic ADPR regulates Ca2+ signaling  is a disorder associated with decreased ability to repair DNA via nucleotide excision repair. NR treatment ameliorated numerous endpoints in mitochondrial dysfunction in XPA protein–deficient cells and rescued the life span defect in XPA-1–deficient Caenorhabditis elegans  DNA helicase  number of mechanisms relying on NADnditions . In labodiseases , 214. Siexercise , 106. Hoexercise . The rea+ levels .+ involves the risk of fueling cancer cells. Increased metabolic demands of rapidly growing cancer cells require more NAD+    may imparomotion . Numeroue, NAMPT , 220, nie, NAMPT , 222, NAe, NAMPT , which mf tumors , 224.] Bg effect , 225.+ in nucleic acid maintenance has significantly expanded during recent years, the complexity of NAD+ cellular functions makes this topic of relevance in future studies. Many aspects of NAD+ metabolism remain unresolved, such as cell type–specific differences or subcellular compartmentalization. Topics, such as the role of NAD+ in mitochondrial DNA repair, or NAD+ capping, are yet poorly studied and deserve further exploration. Equally important is an understanding and evaluation of the risk–benefit ratio associated with elevating NAD+ levels, which may act as a double-edged sword in the context of maintaining DNA integrity and overall NAD+-dependent cellular homeostasis. It will be exciting to see how future studies will provide a deeper understanding of these mechanisms.Although our understanding of the role of NADThe authors declare that they have no conflicts of interest with the contents of this article.


SMAD4 and SNAI2 as the most putative target genes of NSHL. But pathogenic and deleterious non-synonymous single nucleotide polymorphisms discovered within SMAD4 is anticipated to have an impact on NSHL progression. Additionally, the identified deleterious variants in the functional domains of SMAD4 added a supportive clue for further study. Thus, the identified deleterious variant i.e., rs377767367 (G491V) in SMAD4 needs further clinical validation. The present outcomes would provide insights into the genetics of NSHL progression.Non-syndromic hearing loss (NSHL) is a common hereditary disorder. Both clinical and genetic heterogeneity has created many obstacles to understanding the causes of NSHL. The present study has attempted to ravel the genetic aetiology in NSHL progression and to screen out potential target genes using computational approaches. The reported NSHL target genes (2009‒2020) have been studied by analyzing different biochemical and signaling pathways, interpretation of their functional association network, and discovery of important regulatory interactions with three previously established miRNAs in the human inner ear as well as in NSHL such as miR-183, miR-182, and miR-96. This study has identified  Hearing impairment is the most common neurosensory disorder  in humanGJB2, GJB3, GJB6, SLC26A4, KCNQ4, DFNA5, SLC26A5, MYO1A, MYO7A, MYH15A, and CDH23 has been identified with respect to the progression of congenital NSHL. Among these, GJB2 is established as an imperative target of NSHL [The association of several important genes such as  of NSHL . In addi of NSHL ,11 whichGenerally, identification of potential biomarkers through the integration of multiple complementary approaches  is bettehttps://pubmed.ncbi.nlm.nih.gov/), Science Direct (https://www.sciencedirect.com/), Cochrane Library (www.cochranelibrary.com/), and JSTOR (www.jstor.org/) databases using MeSH terminologies such as “Non syndromic hearing loss” , non-syndromic hearing loss* (MeSH), non-syndromic hearing loss (MeSH)) and “genetics” (gene* (MeSH), genetics* (MeSH)), AND “epidemiology & pathogenesis” (epidemiology* (MeSH), pathogenesis*(MeSH)). The articles were collected from the last decade up to December 2020. Following inclusion and exclusion criteria were followed to select the appropriate literature for reference.A systematic review of literature was carried out by following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The scientific articles on NSHL were searched in PubMed (The following criteria were followed for selecting the articles:(1) Literatures providing information about target genes involved in NSHL.(2) The articles published in the English language.(3) The full-length article available from period 2009-2020.All available full-length original research articles and case reports were included.The rest of the literatures were excluded based on the following exclusion criteria:(1) The review articles, abstracts, articles written in other languages, letters to the editor, short reports, and correspondences.(2) The articles are based on syndromic HL and the adult population.(3) Articles without any genetic information.The NSHL target genes were selected from shortlisted literatures and subjected for removal of duplicate genes. The unique NSHL target gene list was prepared for further analysis.https://www.genome.jp/kegg/mapper.html).All of the selected genes were employed to discover their association in different biochemical and signaling pathways involved in the development and progression of NSHL using KEGG (Kyoto Encyclopedia of Genes and Genomes) Mapper web server (https://string-db.org/) web server in order to represent a functional network between them.The pathway associated NSHL target proteins were analyzed using STRING (http://miRTarBase.cuhk.edu.cn/) web server to analyze their regulatory interaction with three previously reported miRNAs . The identified miRNA-gene interaction hubs were validated and graphically represented through miRNet (https://www.mirnet.ca/) tool.Functionally associated target genes were subjected to miRTarBase (https://www.ncbi.nlm.nih.gov/snp/). Validation of SNPs were performed using SIFT (https://sift.bii.a-star.edu.sg/) [https://loschmidt.chemi.muni.cz//predictsnp1/), and PredictSNP2 (https://loschmidt.chemi.muni.cz/predictsnp2/) algorithms [Effective SNPs were predicted from NSHL target genes to identify their functional variants. SNPs were searched from dbSNP database (edu.sg/) , Predictgorithms .The schematic representation of overall procedures is depicted in Total 14,215 numbers of articles were selected on the basis of PRISMA guidelines from different scientific repositories such as PubMed , JSTOR (209), Science Direct , and Cochrane (7). Among these, a total of 787 studies were satisfied with all of the inclusion criteria and considered for this study , whereasSNAI2, ACTB, ACTG1, FGFR1, MET, TJP2, CLDN14, CLDN9, MARVELD2, MYH9, NEDD4, NF2, RDX, IFNLR1, IL13,NOTCH1, RBPJ, LRP5, SMAD4, RAF1, ADCY1, GJA1,  and PTPN11 were anticipated as potential NSHL targets. Afterward, functional association of these 23 NSHL targets was studied using the STRING tool with the high level of confidence parameter (score 0.007). The resulting protein-protein network showed a strong interaction between 16 nodes (genes)   with a spairment .SMAD4 and SNAI2 with miR-182 and miR-183, whereas miR-96 regulates only SNAI2  confirmed from previous analysis. The prediction was resulted total 178 rsIDs of pathogenic variants for SMAD4 gene with clinical significance from dbSNP  was identified 18 rsIDs (out of 178 rsIDs) in SMAD4 with deleterious effect (SMAD4 (Non-synonymous SNPs (nsSNPs) were predicted for two important NSHL targets  with three established miRNAs  was obtained which have added a worthy value to this current study. The expressions of these three miRNAs in the human inner ear and their involvement in NSHL progression [SMAD4 expression in human bladder, prostate cancer, and ovarian cancer [ATP2B4, BTG1, EZRIN, GNG5, KCNJ14, NCS1, NEFL, PEX19, PPP2CA, SLC6A6, TREK-1, ZCCHC3 [CaV1.2, MITF, RDX [MYRIP, AQP5, CaBP1, CaV1, CELSR1, CELSR2, GRID1, KCC2, MITF, RDX, RYK, and TFCP2L3 [SNAI2 gene by miR-96 [Afterward, participation of 16 genes (out of 23) in different biological processes such as inner ear development, homeostatic process, chemical homeostasis, signal transduction, inner ear auditory receptor cell differentiation, and regulation of response to external stimulus related to hearing impairment and NSHL progression was elucidated through study of their PPI network -41. Thergression -18 have n cancer -44. AparITF, RDX ,18. Simi TFCP2L3 ,18. At ty miR-96 .SMAD4 and SNAI2 through miR-183, miR-182, and miR-96 in human NSHL is still not clear, further investigation may provide proper direction into it.As, the regulation of SMAD4 gene. SMAD4 is a multifunctional modulator, which regulates cell proliferation, differentiation, autoimmunity, pluripotency, plasticity, cell growth, apoptosis, autophagy, invasion, and metastasis. Deregulation of SMAD4 is also associated with embryonic developmental diseases [SMAD4 variants G352E [SMAD4 protein has emphasized their importance for further study. MH2 domain is a multifunctional region that mediates differential interaction with other proteins, transcription factors, co-activators, and co-repressors. These interactions provide specificity and selectivity to SMAD4 functions [Here, total of 13 deleterious nsSNPs  were obtained within the functional region i.e., MH2 domain of the diseases . It has diseases . The SMAts G352E , G352R [ts G352E , C363R [ts G352E , and L53SMAD4 was discovered and is not reported in the NCBI ClinVar database for any disease conditions. Thus, present findings will not only provide insights into the genetics of NSHL but also help in further genetic counselling of NSHL after subsequent investigation.Interestingly, the association of a deleterious variant i.e., rs377767367 (G491V) in NSHL target SMAD4 as the most potential target gene in the HL development pathways. The discovery of important miRNAs, miR-183, and miR-182 in associated with the regulation of SMAD4 has also supported the above hypothesis. Identification of pathogenic and deleterious nsSNPs within its functional domain is also added an additional clue to this study. Outcomes of the present investigation may be useful in understanding the genetics of NSHL and also throw light on the diagnosis modalities for NSHL. All of these findings are needed to experiment with for further validation.NSHL is a critical hereditary disorder. Complexity in disease diagnosis increases due to its clinical and genetic heterogeneity. The present study has reported


Nitric oxide (NO), as a ubiquitous gas signaling molecule, modulates various physiological and biochemical processes and stress responses in plants. In our study, the NO donor nitrosoglutathione (GSNO) significantly promoted tomato seedling growth under NaCl stress, whereas NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) treatment reversed the positive effect of NO, indicating that NO plays an essential role in enhancing salt stress resistance. To explore the mechanism of NO-alleviated salt stress, the transcriptome of tomato leaves was analyzed. A total of 739 differentially expressed genes (DEGs) were identified and classified into different metabolic pathways, especially photosynthesis, plant hormone signal transduction, and carbon metabolism. Of these, approximately 16 and 9 DEGs involved in plant signal transduction and photosynthesis, respectively, were further studied. We found that GSNO increased the endogenous indoleacetic acid (IAA) and salicylic acid (SA) levels but decreased abscisic acid (ABA) and ethylene (ETH) levels under salt stress conditions. Additionally, GSNO induced increases in photosynthesis pigment content and chlorophyll fluorescence parameters under NaCl stress, thereby enhancing the photosynthetic capacity of tomato seedlings. Moreover, the effects of NO mentioned above were reversed by cPTIO. Together, the results of this study revealed that NO regulates the expression of genes related to phytohormone signal transduction and photosynthesis antenna proteins and, therefore, regulates endogenous hormonal equilibrium and enhances photosynthetic capacity, alleviating salt toxicity in tomato seedlings. Meanwhisistance . Salt stsistance . Additiosistance . It intesistance ,5. As a sistance . ConsequRecently, the signaling of gases, such as hydrogen sulfide, nitric oxide (NO), carbon monoxide, and hydrogen, has been shown to play a vitally important role in various physiological processes in plants, ,8,9. AmoS-nitrosylation in response to heavy metal stress [Previous studies have shown that NO treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of certain endogenous hormones . In varil stress . These fl stress ,22. Photl stress . Thus, iTo explore the effect of NO on the growth and development of tomato seedlings under salt stress, the NO donor GSNO and the NO scavenger cPTIO were used to treat tomato seedlings under salt stress. As shown in p-value less than 0.05”, a total of 739 DEGs were obtained through RNA-seq , which induce IAA production, were upregulated by GSNO (SRK2I) were significantly downregulated by GSNO. Meanwhile, of the two PP2C (protein phosphatase 2C) genes, PP2C 51-like was downregulated, while PP2C 75 was upregulated in the presence of GSNO. The EBF (EIN3-binding F-box protein) genes EBF2 and EBF1 as well as the ERF (ethylene response factor) gene ERF-C3 were downregulated after adding GSNO under salt stress. Additionally, some DEGs involved in the SA, GA, and CTK signaling pathways were expressed in upregulated patterns (LOC101265854) or downregulated patterns   . In addi by GSNO . In the d ORR26) .GH3.1, IAA35, and GH3-9), two ABA-related genes (SRK2I and PP2C 51-like), two ETH-related genes (ERF-C3 and EBF2), and two SA-related genes (LOC101265854 and EIL1). Compared with NaCl, the expression levels of GH3.1, GH3-9, and LOC101265854 exhibited a significantly increased trend in the NaCl + GSNO treatment, while the expression levels of IAA35, SRK2I, PP2C 51-like, ERF-C3, EBF2, and EIL1 were significantly decreased in the NaCl + GSNO treatment. These results show that all the expression patterns analyzed by qRT-PCR were consistent with our transcriptome analysis, confirming the reliability of the RNA-seq data.To determine the reliability of our transcriptome data, the expression of 9 DEGs related to plant hormone transduction during NO alleviation of salt stress was conducted . These Dchlorophyll a-b binding protein 1C (Cab-1C), Cab-3C, Cab-1A, and Cab-1D), LHcb3 (CAB13), and LHcb2 (CAB5). In addition, these six DEGs annotated as LHCIIs of PSII were significantly upregulated by GSNO + NaCl treatment compared with NaCl treatment alone. Furthermore, three DEGs, light-harvesting complex I, chlorophyll a/b binding protein 1 Lhca1 (LOC101252151), and LHca4 (CAB11 and CAB12), enriched in light-harvesting chlorophyll complexes of photosystem I (LHCI), were significantly upregulated after adding GSNO under stress.According to the KEGG results, nine significantly enriched DEGs in the photosynthesis antenna proteins pathway (sly00196)  were invCAB13, Cab-3C, LOC101252151, and CAB11 were decreased under NaCl stress compared with the control. Meanwhile, the expression of these genes under NaCl + GSNO treatment increased, showing about a one-fold increase in comparison with NaCl treatment alone. However, this increase was inhibited by cPTIO under salt stress compared with NaCl + GSNO treatment. In addition, cPTIO treatment alone also slightly decreased the expression level of these photosynthesis-related genes, implying that NO probably alleviated salt stress by regulating photosynthesis.To verify the expression of DEGs, four photosynthesis-related genes were selected. As shown in Since 16 DEGs were related to “hormone signal transduction”, most of the DEGs were involved in the IAA, ABA, ETH, and SA signal transduction pathways. In order to determine whether NO could also affect plant endogenous hormones in response to salt stress, the content changes of four plant hormones in tomato seedlings, including IAA (the most common auxin), ABA, ETH, and SA, were determined . In compTo explain the stimulatory roles of NO in the alleviation of salt stress involving photosynthesis, the photosynthesis pigments and chlorophyll fluorescence parameters in tomato seedlings were determined in this study . As showNO, a ubiquitous bioactive gas, plays an important role in plant growth and development and the adaptation of plants to stress conditions. Increasing evidence shows that the supplementation of exogenous signaling molecules can enhance the resistance of plants to salt toxicity by stimulating biochemical and physiological defense systems in plants ,11,24. NCH3.1, CH3-4, and CH3-9 genes were upregulated, and IAA35 and SAUR50 were downregulated by NO under salt stress, implying that NO might mediate the degradation of Aux/IAA repressors to induce auxin-regulated responses. GH3 genes can maintain endogenous AUX homeostasis by conjugating excess AUX with amino acids. Singh et al. [SAUR75 was downregulated in wheat roots under salt stress, and the overexpression of SAUR75 increased drought and salt tolerance in Arabidopsis [PP2C 75, and downregulated PP2C 51-like and the serine/threonine-protein kinase gene SRK2I (The major hormones in plants are auxins (AUXs), cytokinins (CTKs), abscisic acids (ABAs), gibberellins (GAs), ethylene (ETH), salicylic acid (SA), jasmonates (JAs), brassinosteroids (BRs), and strigolactones. Among them, ABA, JA, SA, and ETH are indicated to play major roles in the response to and plant defense against abiotic stress ,31. Geneh et al.  indicatebidopsis . These rbidopsis , implyinbidopsis , which fne SRK2I . This mane SRK2I . Interesne SRK2I , furtherIt is well known that photosynthesis in plants begins with the absorption of light energy by photosynthetic pigments, and the photosynthetic capacity of plants can be indirectly reflected by the content of photosynthetic pigment in plant leaves . Additio−2 s−1 photon irradiance) at 26 °C and 8 h dark at 20 °C in 60% relative humidity.The tomato “Micro-Tom” was used as the material in our study. The seeds were disinfected with 1% of NaClO and transferred to 1/2 Hoagland solution for 7 days after germination, then continued cultivation in Hoagland solution for another 21 days. The uniform seedlings were chosen for treatment with control (distilled water), NaCl (150 mM), NaCl + nitrosoglutathione GSNO (10 μM) (a NO donor), NaCl + NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) (10 μM), and cPTIO after adding Hoagland solution for 7 days. The concentrations of NaCl, GSNO, and cPTIO were based on the results of a preliminary experiment. The experimental environment was kept at 16 h light  on whole single plants. Plant height was obtained by a Vernier caliper using the straight-line distance from the stem base to the apex of the shoot apical meristem. After removing the aboveground part of the seedlings, the image of the roots was scanned by a root scanner , and then the total root length of each plant was obtained by the root analysis software WinRHIZO 5.0 . The mean values of plant height, leaf area, and total root length were measured for five tomato seedlings in each replication.w/v) TTC and 7 mL phosphate buffer (pH 7.5) at 37 °C, and then 4 mL of H2 SO4 (1 M) was added. Roots were dried with filter paper and extracted with ethyl acetate. The absorbance was measured at 485 nm.The fresh weight (FW) and dry weight (DW) were determined using the aboveground part of the tomato seedlings. The FW was measured using an electronic balance , and after the plant samples were dried at 80 °C for 48 h, which was when they reached a constant weight, the DW was measured. Then, the root activity was measured by the triphenyl tetrazolium chloride (TTC) method as described by Yan et al.  with minp-value < 0.05. GO enrichment and KEGG pathway annotation were detected using BLAST2GO software and KOBAS software, respectively [Plant samples treated with 150 mM NaCl and 150 mM NaCl + 10 μM GSNO were used for RNA transcriptome sequencing analysis. Total RNA was extracted using the TaKaRa MiniBEST plant RNA extraction kit , and more than 1 μg of RNA samples were collected for RNA-seq library construction according to the instructions of the Illumina Standard mRNAseq library preparation kit . The RNA-seq libraries were sequenced on the Illumina HiSeq platform to generate 150 nt paired-end reads. Then, raw data were filtered with Cutadapt software, and clean reads were mapped to the reference genome sequence of tomato SL2.40 (ITAG2.3) by use of TopHat. The values of fragments per kilobase of transcript per million reads for each biological replicate were determined using Cufflinks. The expression level of each gene was normalized by the value of fragments per kilobase of transcript per million fragments mapped (FPKM). The DEGs between different treatments were employed and analyzed using the DESeqR package. DEGs were defined on the basis of an adjusted absolute log 2 (fold change) > 0 and a ectively ,53.To validate the RNA-seq results, 9 DEGs involved in the phytohormone signal transduction pathway and 4 DEGs involved in photosynthesis antenna proteins were selected, and qRT-PCR assays were performed. Total RNA extraction, cDNA synthesis, and qRT-PCR were conducted as described by Gao et al. . All thev/v) methanol solution. After incubation for 12 h at 4 °C, the extracts were collected after centrifugation for 15 min under refrigerated conditions at 4 °C. The residues were suspended in 2.5 mL of extraction solution and stored at 4 °C for 1 h, then centrifuged again at 3059× g for 15 min at 4 °C. The extraction was repeated twice, the supernatant was merged, and the volume was increased to 10 mL with 80% pure methanol. Two milliliters of the supernatant were evaporated in a rotary vacuum at 38 °C for 4 h and then dissolved with 2 mL of 80% cold pure methanol. Finally, to establish liquid chromatograph detection, the extract was filtered with a 0.22 μm filter. The contents of IAA and ABA were detected by Quaternary gradient ultra-fast liquid chromatograph using Waters Acquity ARC 600-2998 equipped with the Symmetry-C18 column . The SA content was measured by Liquid chromatography–mass spectrometry equipped with a C-18 column  at a flow rate of 0.3 mL min−1.The contents of endogenous plant hormones were measured according to the study previously reported by Qi et al. . Tomato ETH production was measured according to the method of Wang et al.  with minThe chlorophyll fluorescence parameters of tomato leaves were measured using an FMS-2 pulse-modulated fluorometer . The FS under light adaptation, maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency (φPS II), non-photochemical quenching (NPQ), and photochemical quenching (qP) were obtained under dark adaptation . The conp < 0.05 level.Each treatment contained 3 replicates, with each replicate consisting of 5 tomato seedlings. The experiment was completely randomized. The data were analyzed using SPSS 22.0 . The values were expressed as the means ± SE of three independent experiments with 3 replicated measurements. The different treatments were analyzed by Duncan’s multiple range test at a In conclusion, this study clarifies the vital role of NO in alleviating salt stress in tomato seedlings. Moreover, the transcriptome analysis identified a large number of candidate genes involved in the alleviation of salt stress through pairwise comparisons. The results indicate a mechanism of NO-relieved of salt toxicity that involves two pathways: the signal transduction and synthesis of plant hormones, especially IAA, ETH, ABA, and SA, and photosynthesis. Collectively, these findings provide new insights into the biological functions of NO in plant growth and stress response. However, the mechanisms underlying NO and abiotic stress are quite complex, and future works should be established to characterize the functional roles of these DEGs.


Reactive Oxygen Species (ROS) produced by the non-catalyzed partial reduction of O2 were highly toxic eliminating many species. Survivors developed different strategies to cope with ROS toxicity. At the same time, using O2 as the final acceptor in respiratory chains increased ATP production manifold. Thus, both O2 and ROS were strong drivers of evolution, as species optimized aerobic metabolism while developing ROS-neutralizing mechanisms. The first line of defense is preventing ROS overproduction and two mechanisms were developed in parallel: 1) Physiological uncoupling systems (PUS), which increase the rate of electron fluxes in respiratory systems. 2) Avoidance of excess [O2]. However, it seems that as avoidance efficiency improved, PUSs became less efficient. PUS includes branched respiratory chains and proton sinks, which may be proton specific, the mitochondrial uncoupling proteins (UCPs) or unspecific, the mitochondrial permeability transition pore (PTP). High [O2] avoidance also involved different strategies: 1) Cell association, as in biofilms or in multi-cellularity allowed gas-permeable organisms (oxyconformers) from bacterial to arthropods to exclude O2. 2) Motility, to migrate from hypoxic niches. 3) Oxyregulator organisms: as early as in fish, and O2-impermeable epithelium excluded all gases and only exact amounts entered through specialized respiratory systems. Here we follow the parallel evolution of PUS and O2-avoidance, PUS became less critical and lost efficiency. In regard, to proton sinks, there is fewer evidence on their evolution, although UCPs have indeed drifted in function while in some species it is not clear whether PTPs exist.From 2.5 to 2.0 billion years ago, atmospheric oxygen concentration [O These aea level  (Rosas-Lea level . Intereseathing) .2-avoidance efficiency rendered alternative redox enzymes superfluous and thus these were lost  increases the respiratory rate 6–10 times while lowering pO2 to less than 0.03 mm Hg+   . RC consins (RC) . Then teins (RC) . In aerooxidases . As an adases   .Bacillus cereus, which during sporulation, dormancy, or germination expresses different RC activities depending on its menaquinone concentration , peroxynitrite, sulfide, ammonia, and cyanide. This is most likely why harmful bacteria have so much cytochrome bd. Because these enzymes are not found in eukaryotes, they are particularly appealing as prospective targets for novel antibacterial drugs  plus one internal NDH2 (Ndi1)  . In S. cve NDH2s . NDH2s mve NDH2s .2 substituting the cytochrome pathway  and alternative (Km = 10–20 µmol) pathways enable COX to sustain OxPhos while AOX lowers [O2]  and an AOX coexist with Complexes I to IV , enhancing survival abilities in participating species . In this2] gradients or using secondary metabolite concentration gradients to find a niche within the community (Thermodesulfovibrio) and a strictly aerobic species (Phenylobacterium) coexisted  is present in hypoxia-tolerant invertebrates from the phylum Porifera, Cnidaria, Nematoda, Annelida, Mollusca, and Echinodermata . In thesr stress .Crassostrea virginica. Under normal settings, AOXA has greater expression than AOXB, but under hypoxia, AOXB is the dominant form, and suggesting that it might be a stress-adaptation  and the permeability transition pore (PTP) are both immersed in the inner mitochondrial membrane .UCPs belong to the six-transmembrane helix family of mitochondrial transporters . UCPs heDrosophila melanogaster, DmUCP5 is involved in metabolic homeostasis  . PTP seetrocytes . In isoltrocytes .2+ and prevents ROS overproduction, which prevents tissue damage    may remarequired . When threstored . A speci cations . Mitochog stress .2 consumption is traditionally known as known as state-3 respiration and results from a partial, reversible decrease in ΔΨm and these conditions decrease ROS production similarly to PUS activation , catalase  and the glutathione recycling system made by glutathione peroxidase , glutathione reductase , and glutathione S-transferase . In addition, there are molecules, that sequester ROS, such as glutathione, carotenoids, vitamin C, vitamin A, and vitamin E . ROS detClostridium acetobutylicum, a strict anaerobe organism that contains Fe- and Mn-SODs, heme- and Mn-catalases and in addition 1Fe or 2Fe-SORs that reduce the superoxide anion to H2O2 (Antioxidant systems exist in all kingdoms . These a to H2O2 .2 and its toxic derivatives are very strong drivers of evolution. All O2-exposed organisms are at risk of being damaged, age and eventually die because of the deleterious actions of ROS. Many systems have been designed to counteract toxicity while using the advantages of aerobic metabolism. However, the two most important mechanisms to prevent ROS toxicity evolved in opposite sense, as avoiding O2 by exclusion became optimal, branching of RC decreased and eventually disappeared. That is, as oxyregulators optimized O2-exclusion, they enhanced their ATP producing efficiency, and slowly lost stress-response ability in their RCs. It remains to be analyzed whether proton sinks those first appeared in eukaryotes have also lost efficiency or not. Substrate channeling as described above is another ROS overproduction prevention system that seems to exist only in eukaryotes. A second line of defense, which was considered only briefly here, are the ROS detoxifying system that play vital roles during crisis such as ischemia/reperfusion and anoxic periods.O


Fasn, Scd1, and Srebp-1c significantly in the HFD + HE group compared with those in the HFD + normoxia group; after replacing a HFD with a ND, hypoxia treatment achieved more significant changes in the measured variables. In addition, the protein expression of HIF-1α was increased only in the ND + HE group but not in the HFD + HE group. Even though hypoxia exposure did not affect hepatic TG and NEFA levels, at the genetic level, the intervention had significant effects on hepatic metabolic indices of fatty acid synthesis, especially in the ND + HE group, while HFD suppressed the beneficial effect of hypoxia on hepatic lipid metabolism in male ApoE KO mice. The dietary intervention of shifting HFD to ND could be more effective in reducing hepatic lipid accumulation than hypoxia intervention.Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with a characteristic of abnormal lipid metabolism. In the present study, we employed apolipoprotein E knockout (ApoE KO) mice to investigate the effects of hypoxia exposure on hepatic fatty acid metabolism and to test whether a high-fat diet (HFD) would suppress the beneficial effect caused by hypoxia treatment. ApoE KO mice were fed a HFD for 12 weeks, and then were forwarded into a six-week experiment with four groups: HFD + normoxia, normal diet (ND) + normoxia, HFD + hypoxia exposure (HE), and ND + HE. The C57BL/6J wild type (WT) mice were fed a ND for 18 weeks as the baseline control. The hypoxia exposure was performed in daytime with normobaric hypoxia . Body weight, food and energy intake, plasma lipid profiles, hepatic lipid contents, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and molecular/biochemical makers and regulators of the fatty acid synthesis and oxidation in the liver were measured at the end of interventions. Six weeks of hypoxia exposure decreased plasma triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) contents but did not change hepatic TG and non-esterified fatty acid (NEFA) levels in ApoE KO mice fed a HFD or ND. Furthermore, hypoxia exposure decreased the mRNA expression of  Nowadays, nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide ,2 and inMcad) and carnitine palmitoyl transferase 1/2 (Cpt1 and Cpt2), are targeted by PPARα protein and have crucial roles in fatty acid catabolism [Acc), ATP-citrate lyase (Acly), fatty acid synthase (Fasn), and stearoyl-CoA desaturase 1 (Scd1) [Abnormal lipid metabolism is one of the major characteristics of NAFLD. Peroxisome proliferator-activated receptor α (PPARα) is expressed at high levels in the liver and is known to be involved in mitochondrial fatty acid β-oxidation ,8. The gtabolism ,10. Unde1 (Scd1) . A previ1 (Scd1) .AMP-activated protein kinase (AMPK) plays a central role in controlling lipid metabolism. The phosphorylation of ACC (at Ser79) by AMPK could inactivate ACC, which might ultimately decrease the level of malonyl-CoA and is beneficial for the recovery of CPT-1 activity and fatty acid oxidation ,14. The Apolipoprotein E (ApoE), primarily produced by the liver, works with other lipoproteins to mediate the lipid transport in the circulation . ApoE knSeveral lines of studies have shown that moderate hypoxia protocols had beneficial effects on metabolism, including reduced body weight, blood glucose, and cholesterol levels, and improved insulin sensitivity ,22. HypoIn this study, after 12 weeks of feeding of a HFD in ApoE KO mice, we focused on the effects of hypoxia exposure on fatty acid metabolism in the liver of the mice, and whether it would be more effective when shifting a HFD to a normal diet in this special animal model. We hypothesized that the intervention of hypoxia exposure would present beneficial impacts on hepatic fatty acid metabolism and HFD would suppress the beneficial effect in ApoE KO fed HFD.n = 24, aged 10 weeks) and male C57BL/6J wild type (WT) mice  were purchased from Charles River Development, Inc. . All mice were housed indoors under a temperature of 22 ± 2 °C, humidity of 50–70%, and 12-h light/dark cycles.The protocol of this study was approved by the Animal Care and Use Committee of Beijing Sport University (2019100A). Male C57BL/6J ApoE KO mice (w/w) fat, 43% (w/w) carbohydrate without sucrose, and 1.5% (w/w) cholesterol, 4.554 kcal/g  for 12 weeks. In the food protocol of for the first seven days, a HFD was gradually added into a normal diet until the feeding proportion was totally a HFD. After 12 weeks of the HFD feeding, all ApoE KO mice were forwarded into a six-week interventional experiment and randomly allocated to two groups: normoxia and hypoxia exposure (HE); then they were further divided into HFD and normal diet (ND) groups, respectively (w/w) fat, 50–60% carbohydrate, and no added cholesterol and sucrose, 3.420 kcal/g . WT mice were fed a ND for 18 weeks as a baseline control.The ApoE KO mice were fed HFD, containing 21%  with an oxygen concentration of 11.2%  together with the cage [Mice had the cage . The chaTo avoid acute effects of the last hypoxia treatment session, the mice were placed in normoxia at least 48 h prior to tissue collection. The mice were fasted overnight and were anesthetized  and the blood samples were collected by the percutaneous cardiac puncture. The liver samples from the left lateral lobe were removed, cleaned, and quick-frozen in liquid nitrogen, and then stored at −80 °C.The mice were weighed weekly using an analytical scale. Any remaining food was replaced, and food intake was measured daily. The energy intake was calculated by energy content per gram of food × food intake.Plasma triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured following the methods and assay kits of our previous study . Plasma The supernatant of tissue homogenate was used to determine TG, TC, and NEFA in hepatic tissue by enzymatic methods according to the commercial kits . Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercially available detection kits . Changes in absorbance were determined with Bio Tek Synergy H1  at 510, 510, 546, 510, and 510 nm, respectively.Mcad (QT00111244) and 18S ribosomal RNA  commercial primers from Qiagen  were used. The primer sequences of Cpt1a, Cpt2, Acly, Acc1, Fasn, Scd1, and Srebp-1c were listed in −ΔΔCt method, as described previously [Total RNA was isolated from about 50 mg of liver tissue, reverse-transcribed to cDNA and performed real-time quantitative PCR analysis as previously described . Mcad , PPARα , SREBP-1 (2A4) , DEC1 (s-8) , AMPKα1/2 , Thr172-p-AMPKα1/2 , and β-actin ; the above-mentioned antibodies were all from Santa Cruz Biotechnology, Dallas, TX, USA. ACC  and Ser79-p-ACC . The density of protein bands was analyzed using Bio-Rad imaging software . The individual values were originally expressed as a ratio of a standard (β-actin content) and then expressed as a fold change of the control group value.+. NADPH had a characteristic absorption peak at 340 nm. The activity of FASN was calculated by measuring the decreasing rate of absorbance at 340 nm . ACC catalyzed acetyl CoA, NaHCO3, and ATP in the production of malonyl CoA, ADP, and inorganic phosphorus. Molybdenum blue and phosphate generated substances with characteristic absorption peaks at 660 nm . The increase of inorganic phosphorus was measured by the ammonium molybdate phosphorus determination method to reflect the ACC activity.The activities of FASN and ACC in the liver tissue were determined using commercially available kits from Gene Lab  and Solarbio , respectively. FASN catalyzed malonyl coenzyme A, acetyl coenzyme A, and NADPH to produce long chain fatty acids and NADPp < 0.05.All values are reported as means ± SD. Statistical calculations were performed using SPSS Statistical software V 19.0 . Data were analyzed using a two-way ANOVA (ND × HE), and simple effect analysis with the least significant difference (LSD) post hoc test was performed to identify significant mean differences between groups. Significance was set at p < 0.05 or p < 0.01) of plasma TG, TC, NEFA, LDL-C, HDL-C, and ALT than those of WT mice fed a ND (p < 0.05 or p < 0.01)  D–F.There were no significant differences in body weight between the HFD and ND groups with or without HE during the six-week interventions (from the end of week 12 to the end of week 18) A. HoweveHE strongly decreased plasma TG, TC, LDL-C, and ALT levels in the HFD + HE group compared with those of the HFD + normoxia group, and also significantly reduced plasma TG, TC, LDL-C, ALT, and hepatic TC levels in the ND + HE group compared with those of the ND + normoxia group. Under normoxia, ND strongly decreased plasma TG, TC, NEFA, LDL-C, ALT, and hepatic TG, TC, and NEFA levels in the ND + normoxia group compared with those of the HFD + normoxia group. Under hypoxia, ND also strongly decreased their levels in the ND + HE group compared with those of the HFD + HE group A–D,F,H–JCpt1a, Cpt2, and Mcad, between the HFD and ND groups with or without HE /AMPKα, p-ACC(Ser79)/ACCα in the liver were measured. Immunoblotting revealed that there was an increasing trend in the hepatic p-AMPKα (The172)/AMPKα and p-ACC(Ser79)/ACCα ratios of the HFD + HE group or ND + HE group compared with the HFD + normoxia group or ND + normoxia group, respectively, but not significant between them A,B.Srebp-1c in the liver of HFD + HE mice, than that of the HFD + normoxia group  in the liver, which suggests that HIF-1 prevents lipid synthesis [Srebp-1c and its target genes involved in fatty acid synthesis including Acc1, Fasn, and Scd1, as well as ACC activity  is a process by which lipids are endogenously synthesized from dietary sources, such as carbohydrates. SREBP has three isoforms: SREBP-1a, SREBP-1c, and SREBP-2. The liver predominantly expresses the SREBP-1c isoform together with SREBP-2. SREBP-1c is mainly involved in de novo FA and TG synthesis by inducing the expression of lipogenic genes such as Fasn, Acc, and Scd1, whereas SREBP-2 controls cholesterol homeostasis [Scd1, which can convert saturated fatty acids to monounsaturated fatty acids. Meanwhile, the protein expression of hepatic p-AMPKα(Thr172)/AMPKα and PPARα, the mRNA expression of PPARα’s target genes, such as Cpt1a, Cpt2, and Mcad, were not significantly different between the HFD and ND groups. Altogether, the evidence indicated that the dietary intervention with a ND could regulate hepatic fatty acid metabolism by inhibiting lipid synthesis.eostasis . On the eostasis . As expeThere are no reports about hypoxia exposure improving hepatic fatty acid metabolism in humans with NAFLD in the current literature. However, in animal studies, chronic intermittent hypobaric hypoxia has beneficial effects on the body of rats ,59. ThisThe present study has some limitations. While focusing on the changes of fatty acid metabolism in the liver, we did not assess other variables of lipid metabolism, for example, cholesterol metabolism. Future study may expand on the findings of cholesterol metabolism by addressing the effects and its mechanism. In addition, the liver histological observations were not performed, such as Oil Red O staining of samples in the study. This information would add more comprehensive insights. Because female mice and male mice have different hormone levels and physical conditions, the present study only selected male mice for the experiment to avoid the sex differences. The current results are not applicable to female animals.Six weeks of hypoxia exposure did not change hepatic TG and NEFA levels in male ApoE KO mice. However, at the genetic level, there were significant effects on hepatic metabolic indices of fatty acid synthesis, especially in the ND + HE group, while HFD might suppress the beneficial effect of hypoxia on hepatic lipid metabolism in male ApoE KO mice. Comparatively, the dietary intervention of shifting HFD to ND was more effective in reducing hepatic lipid accumulation than hypoxia intervention.