Subcutaneous AT biopsies were collected from 59 nondiabetic (lean/overweight/obese) individuals. AT gene expression levels of dectin-1 and inflammatory markers were determined via real-time reverse transcriptase-quantitative polymerase chain reaction. Dectin-1 protein expression was assessed using immunohistochemistry. Plasma lipid profiles were measured by ELISA. AT dectin-1 transcripts and proteins were significantly elevated in obese as compared to lean individuals. AT dectin-1 transcripts correlated positively with body mass index and fat percentage . AT dectin-1 RNA levels correlated positively with clinical parameters, including plasma C-reactive protein and CCL5/RANTES, but negatively with that of adiponectin. The expression of dectin-1 transcripts was associated with that of various proinflammatory cytokines, chemokines, and their cognate receptors , but not with anti-inflammatory markers. Dectin-1 and members of the TLR signaling cascade were found to be significantly associated, suggesting an interplay between the two pathways. Dectin-1 expression was correlated with monocyte/macrophage markers, including CD16, CD68, CD86, and CD163, suggesting its monocytes/macrophage association in an adipose inflammatory microenvironment. Dectin-1 expression was independently predicted by CCR5, CCL20, TLR2, and MyD88. In conclusion, dectin-1 may be regarded as an AT biomarker of metabolic inflammation in obesity.In obesity, macrophage activation and infiltration in adipose tissue (AT) underlie chronic low-grade inflammation-induced insulin resistance. Although dectin-1 is primarily a pathogen recognition receptor and innate immune response modulator, its role in metabolic syndromes remains to be clarified. This study aimed to investigate the  The global prevalence of obesity has increased manifold over the last four decades ,2. WorldAdipose tissue (AT) is composed of multiple cells of different origins. Besides adipocytes, AT contains progenitor cells, residential leukocytes, and neuronal and vasculature cells ,16. CommκB signaling [antifungal response mechanisms [Dectin-1 belongs to the type II transmembrane family C-type lectin receptors (CLRs) expressed in myeloid cells  and antigen-presenting dendritic cells ,25 and iignaling ,27,28. Dchanisms ,30,31. Achanisms .The emerging roles of dectin-1 as a positive regulator of AT inflammation in high-fat diet (HFD)-fed MyD88 KO mice and as a biomarker for metabolic dysregulation in humans have recently been reported . Nonethe2 (m2)), the cohort was divided into 10 lean (BMI < 25 kg/m2), 20 overweight (25 ≤ BMI < 30 kg/m2), and 29 obese (BMI ≥ 30 kg/m2) individuals. For each category, the sample size was dependent on the sample availability and each participant’s decision to be engaged in the research study. Written informed consents were obtained from all study participants in accordance with the ethical guidelines stipulated in the Declaration of Helsinki and approved by the ethics committee of Dasman Diabetes Institute, Kuwait . Weights and heights were measured using calibrated electronic weighing scales and height-measuring bars. Waist circumferences were measured using constant-tension tapes. The IOI 353 Body Composition Analyzer  was used to determine the whole-body compositions .This study cohort included 59 nondiabetic individuals from both sexes who were recruited in the study at the Dasman Diabetes Institute, Kuwait. The exclusion criteria included pregnant women and individuals with diseases, such as lung, heart, kidney, or liver complications, immune dysfunction, diabetes, cancers, or hematologic disorders as previously described ,35,36. UAs previously described , human APeripheral blood was collected from fasted individuals and evaluated for metabolic and biochemical markers, as has been described previously ,40,41. FT) method, as has been described previously [GAPDH), and means ± standard error of the mean (SEM) are expressed as fold changes in the expression relative to controls as indicated [Human AT tissue total RNA was extracted using the RNeasy kit  as described in the manufacturer’s protocol. The first strand cDNA was synthesized from 500 ng total RNA using a High Capacity cDNA Reverse Transcription kit  as previously described . Real-tieviously . Resultsndicated .2O2 for 30 min and non-specific antibody binding was blocked with 5% nonfat milk for 1 hr, followed by 1% bovine serum albumin solution for 1 h. The slides were incubated at room temperature overnight with primary antibody ). After washing with PBS (0.5% Tween), the slides were incubated for 1 h with secondary antibody, namely goat anti-rabbit conjugated with horseradish peroxidase polymer chain DAKO EnVision Kit , and color was developed using a 3,3‘-diaminobenzidine (DAB) chromogen substrate. The specimens were washed, counterstained, dehydrated, cleared, and mounted as described elsewhere [Paraffin-embedded sections (4 μm thick) of subcutaneous AT were deparaffinized in xylene and rehydrated through descending grades of ethanol  to water. Antigen retrieval was then performed by placing slides in a target retrieval solution  in the pressure cooker boiling for 8 min and cooling for 15 min. After washing in PBS, endogenous peroxidase activity was blocked with 3% Hlsewhere . For anap < 0.05 was considered significant. Standard multivariable linear regression by the Enter method was used; variables that significantly correlated with dectin-1 were selected as predictor variables and were entered simultaneously to generate the model. The F-test was used to assess whether the set of entered independent variables collectively predicted the dependent variable. R-squared was used to determine how much variance in the dependent variable could be accounted for by the set of independent variables. The t test, p value, and beta coefficients  were used to determine the significance and the magnitude of prediction for each independent variable, respectively.Statistical analysis was performed using GraphPad Prism software  and SPSS for Windows version 19.01 . Unless otherwise indicated, data were shown as mean ± SD values. Since the sample size is small and the data are not normally distributed, non-parametric Mann-Whitney U test was used to compare means between groups. Spearman correlation and multivariable regression analysis were performed to determine associations between different variables. As has been previously described ,46, in aThe physiological and biochemical characteristics of the 59 individuals from both genders included in this study are summarized in The three groups had comparable levels of plasma cholesterol and LDL, unlike HDL, which, as expected, was significantly lower in overweight and obese individuals than in lean individuals . In thisDectin-1 has been shown to be elevated in mesenteric AT isolated from a small group of obese individuals [dectin-1 in other adipose tissues, we have examined the dectin-1 gene and protein expression profiles in the subcutaneous AT biopsies from healthy individuals with different BMIs. Relative to lean individuals, AT dectin-1 transcripts and protein expressions were significantly increased in obese individuals as assessed by RT-qPCR and immunohistochemistry analyses, respectively  and slightly positively correlated with IL-1β . Notably, dectin-1 transcripts were also correlated with that of IL-10, which is a regulatory cytokine that exerts both pro- and anti-inflammatory (pleiotropic) effects. No correlations between dectin-1 and IL-2, IL-6, or IL-33 transcripts were observed  and slightly positively correlated with that of CXCL9  and its associated IL-1R-associated kinase 1 (IRAK1)  expression. Importantly and as anticipated, gene expression levels of dectin-1 and IRF5 were also found to be positively correlated , CCR5 , TLR2 , and MyD88  were independently associated with dectin-1  AT of obese individuals [Dectin-1 is recognized as a pattern recognition receptor (PRR) expressed on myeloid cells  and antigen-presenting dendritic cells. Whereas the expression of PRR such as TLRs is well studied in obesity, changes in dectin-1 expression in the adipose tissue in obesity and its significance remain largely unclear. Herein, we show the elevated ividuals . Moreoveividuals ,50. TogeAT is an active endocrine organ that secretes several hormones, cytokines, and chemokines, collectively known as adipokines , which sLeukocyte activation and trafficking to inflammatory sites is known to be regulated by proinflammatory factors secreted within the AT. Among obese individuals that we studied, the upregulation of dectin-1 was found to correlate with expression of proinflammatory mediators, including several interleukins, CC chemokines, CXC chemokines, and TNF-α, but not with anti-inflammatory factors or pleiotropic cytokines.dectin-1 transcripts were detected in monocytes isolated from diabetic patients, which was also associated with defective production of IL-10 [The interplay between dectin-1 and inflammatory mediators has been reported. Elevated levels of of IL-10 , a potenof IL-10 ,54,55.dectin-1 expression and several proinflammatory mediators, especially the chemokines, indicate that these changes parallel the inflammatory cell infiltration into the AT, which might contribute to the pathogenesis of obesity, including the development of insulin resistance.The examined proinflammatory mediators have been associated with the pathogenesis of various metabolic disorders, such as diabetes, heart diseases, and atherosclerosis. Spiegelman et al. were the first to demonstrate the association of TNF-α with AT insulin resistance in obesity using animal models . It has CXCL9, CXCL10, and CXCL11 were found to be correlated with dectin-1. Whereas on one hand, these factors are sufficient to recruit and activate immune cells in the AT in obese individuals, on the other hand, their angiostatic effect might create a hypoxic tissue microenvironment, causing or exacerbating inflammation and metabolic dysfunction. Consistent with our study, at least in part, Hueso et al. observed an elevated gene expression of CXCL10 and CXCL11 in obese visceral AT, associated with the reduced neovascularization, suggesting that the impaired angiogenesis was most likely associated with increased expression of angiostatic chemokines [In obesity, white adipose tissue is expanded, including both hyperplasia and hypertrophy. The adipose tissue is both storage and an active metabolic organ. The increased metabolic activity within the expanding adipose tissue requires a consistent supply of oxygen as well as nutrients to suffice physiological needs and provide energy fuel. Therefore, adipose expression of the vascular endothelial growth factor (VEGF) triggers the process of angiogenesis to support neovascularization in the adipose tissue in obesity. These changes ensure a suitable microenvironment which is supportive of normal cellular metabolic activity and function. In our study, the angiostatic chemokines emokines .dectin-1, TLRs, and their related downstream signaling molecules. To this end, we found that elevated AT dectin-1 transcripts expression was positively associated with TLR2, TLR7, TLR8, and TLR10 gene expression in obese patients. In line with this finding, several studies show that TLRs expression in the AT is elevated in obese individuals, with/without type 2 diabetes, and these alterations can be directly associated with BMI, cytokines/chemokines expression, and insulin resistance [dectin-1 synergizes with the TLR pathway to induce the expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-12 [dectin-1 transcripts levels was associated with the TLR-downstream signaling molecules, including MyD88, IRAK1, and IRF5. MyD88 is a key adaptor protein involved in the signaling of all TLRs (except TLR3) and IL1R, linking them to the IRAK family kinases and downstream IRF factors [dectin-1 expression in AT residential macrophages isolated from MyD88 knockout mice, which correlated with the exacerbation of obesity and increased insulin resistance. Furthermore, they showed that treating mice with a dectin-1 inhibitor decreased the number of M1 macrophages in the AT and ameliorated insulin resistance [TLRs are well-characterized as the innate immune receptors and lately as the nutrient sensors that mediate metabolic inflammation through chronic expression of proinflammatory mediators, particularly in obese AT. TLR activation triggers a dynamic transduction cascade mediated by MyD88 and IRAK proteins, secreting proinflammatory cytokines . Herein,sistance ,40,45,61nd IL-12 ,63,64. I factors . Recentlsistance .IRF5 and dectin-1 in diabetic obese patients, but not in diabetic lean or overweight individuals [dectin-1 and IRF5. Consistent with our finding of an increased expression of the mentioned markers, similar changes in the AT expression of MyD88, IRAK1, and IRF5 have been reported in individuals with obesity and/or type-2 diabetes [We have previously observed a positive correlation between the expression levels of ividuals , which idiabetes ,45,66. Tdectin-1 transcripts expression and that of monocytes/macrophage markers, including CD16, CD68, CD86, and CD163. In obesity, monocytic extravasation into the AT occurs and fat cell necrosis initiates an M2 to M1 macrophage phenotypic shift in the adipose tissue, which is regarded as one of the causes of metabolic inflammation associated with the disease [Our data support a positive association between  disease . M2 macr disease . M1 macr disease . M1 macr disease ,72. A st disease , all of  disease .dectin-1 expression might be induced or modulated by TLR2, MyD88, CCL20, and/or CCR5, all of which have been identified as the independent predictors of dectin-1 adipose expression in obesity, as per our preliminary findings from this study.This study remains limited by certain caveats and concerns. First, the complexity of the intercellular crosstalk between adipocytes and resident immune cells may affect the correlative dynamics between dectin-1 and proinflammatory markers in the AT microenvironment. Second, we cannot exclude the confounding effects of the presence of other immunomodulatory factors/agents present in the adipose tissue that we could not herein study. Third, the existence of genetic polymorphisms within the human cohorts may also impact the outcome of such correlative investigations. In any case, further studies involving larger and more diverse cohorts will be required to investigate how the dectin-1 transcripts expression is positively associated with the gene expression of proinflammatory cytokines/chemokines, their cognate receptors, TLRs, and downstream signaling partners, as well as monocyte/macrophage markers (as described in the schematic AT


BRCA1 and BRCA2, which are associated with hereditary breast and ovarian cancer (HBOC). ATM, BARD1, CHEK2, PALB2, RAD51C, and RAD51D have also been recognized as CPGs with a high to moderate risk of BC. Primary and secondary cancer prevention strategies have been established for HBOC patients; however, optimal preventive strategies for most hereditary BCs have not yet been established. Most BC-associated CPGs participate in DNA damage repair pathways and cell cycle checkpoint mechanisms, and function jointly in such cascades; therefore, a fundamental understanding of the disease drivers in such cascades can facilitate the accurate estimation of the genetic risk of developing BC and the selection of appropriate preventive and therapeutic strategies to manage hereditary BCs. Herein, we review the functions of key BC-associated CPGs and strategies for the clinical management in individuals harboring the GPVs of such genes.Approximately 5–10% of all breast cancer (BC) cases are caused by germline pathogenic variants (GPVs) in various cancer predisposition genes (CPGs). The most common contributors to hereditary BC are  Breast cancer (BC) is the most common cancer affecting women globally ,2,3 and BRCA1 or BRCA2 (BRCA1/2) associated with hereditary breast and ovarian cancer (HBOC). BRCA1 is also known to be inactivated in sporadic breast cancer by somatic promoter hypermethylation [The most common cause of hereditary BC is a GPV in hylation , suggestBC is one of the most preventable types of cancer and the primary prevention strategies for BC include eliminating the causes of the disease through education and public health programs, risk-reducing surgery, and chemoprevention. Secondary prevention aims to reduce cancer mortality by early detection using appropriate screening methods. Considering the high risks of developing BCs, individuals harboring GPVs in the BC-associated CPGs are excellent candidates for risk minimization via primary prevention or the enhanced detection of certain cancers at their earliest and most treatable stages.ATM, BRCA1, BRCA2, BARD1, CHEK2, PALB2, RAD51C, and RAD51D, were observed to be significantly associated with a BC risk in at least one of the two studies [BRCA1 and BRCA2 are high-risk CPGs for BC. ATM, BARD1, CHEK2, PALB2, RAD51C, and RAD51D have also been recognized as CPGs with high to moderate risks for BC. Several BC-associated CPGs have been identified. Recently, the results of two large-scale case-control studies analyzing the association between GPVs in BC-associated CPGs and BC risk were reported. Dorling et al. analyzed a panel of 34 known or suspected BC-associated CPGs in 60,466 women with BC and 53,461 controls , whereas studies ,13  [This review outlines the functions of eight CPGs with high to moderate risks of BC, which collaborate in DNA damage repair pathways and/or cell cycle checkpoints, and the clinical management of individuals harboring GPVs in the genes. The r (HDGC) , Cowden r (HDGC) , Peutz–Jr (HDGC) , and Li–r (HDGC) , respectBRCA1, BRCA2, and PALB2 are the most widely known high-penetrance genes involved in severe BC risk. BRCA1- and BRCA2-associated HBOC accounts for approximately 50% of hereditary BC [itary BC   on chromosome 17 was identified in 1994 [BRCA1, BRCA2 (BReast-CAncer susceptibility gene 2), located on chromosome 13, was identified in 1995 [The first evidence of the existence of a BC susceptibility gene encoding a DNA repair enzyme on chromosome 17 was provided by linkage studies in 1990 , and BRC in 1994 . Followi in 1995  , which contributes to the maintenance of chromosome structure through homologous recombination (HR) repair, cell cycle checkpoint activation, and DNA replication fork protection [otection ,25,26.HR RepairThere are two major mechanisms of the repair of DSBs, which represent one of the most serious types of DNA damage and must be repaired to preserve chromosomal integrity: non-homologous end-joining (NHEJ) and HR. As NHEJ directly ligates broken ends without requiring extended homologies, DNA sequence errors are more likely to occur, and the repaired products often harbor sequence alterations. This repair pathway is active throughout the cell cycle, particularly in the G0/G1 phase when sister chromatids are absent. Contrastingly, HR is the main pathway used strictly in late S or G2 phase cells and can accurately repair the damage using intact homologous sister chromatids as a sequence homologous template for the DNA repair reaction ,27 Figua.After DSBs occur, the HR is sequentially organized. In mammals, the free DNA ends produced by DSBs are initially recognized by the MRE11-RAD50-NBS1 (MRN) protein complex. The MRN complex recruits and activates ATM, which phosphorylates several crucial proteins mediating the DNA damage response (DDR). Active ATM monomers phosphorylate H2AX in regions of the DSBs and create a platform to recruit BRCA1 . In the BRCA1/2 usually inherit a single variant copy in the germline. The “two-hit” paradigm of tumor suppression advocated by Knudson is widely supported and considered to lose its function when the second copy is inactivated by somatic variants or epigenetic events [BRCA1/2 GPVs leads to a loss of function (LOF) of these genes. Dysfunction of BRCA1/2 leads to cancer susceptibility via the induction of chromosomal instability and mutagenesis [BRCA1 and/or BRCA2 protect against such toxicity and suppress tumorigenesis [BRCA1/2 GPV carriers, with implications for public health [Carriers of GPVs in c events . A loss agenesis ,33. Seveigenesis ,35,36. Ic health  DNA Damage-induced Cell Cycle CheckpointsBRCA1 contributes to the activation of DNA damage checkpoints in the G1/S, intra-S, and G2/M phases, and various BRCA1 complexes regulate different phases of the cell cycle. Consequently, the LOF of BRCA1 causes checkpoint abnormalities [BRCA2 in the regulation of cell cycle checkpoints remains unclear. Notably, BRCA1 is essential for both the initial activation and G2/M checkpoint maintenance, whereas BRCA2 and PALB2 appear to be more important for its maintenance during DNA damage [Cell cycle checkpoints represent an important regulatory mechanism for cell survival, wherein the cell cycle process is inhibited in the presence of unresolved DNA damage; the cell cycle is inhibited until the damage is fully repaired ,26 Figub. In resmalities ; howeverA damage .R-loop Processing and TranscriptionDuring transcription, nascent RNA binds strongly to the template DNA strand, forming a unique RNA–DNA hybrid structure that displaces the non-template ssDNA . This thBRCA1 and BRCA2 play roles in R-loop turnover, working with several R-loop processing factors at the promoter-proximal pausing (PPP) sites or transcription termination (TT) sequences of expressed genes. In BRCA1/2-deficient cells, R-loop accumulation is a major cause of replication stress and DNA damage, resulting in chromosomal fragility. Consequently, the accumulation of R-loop associated with BRCA1/2 inactivation has been reported to be associated with BC development [BRCA1/2 GPV carriers. Differentiated luminal cells in the normal mammary gland express estrogen receptor (ER) and progesterone receptor (PR), whereas the ovarian tissue is ER-negative [Multiple proteins involved in transcription recognize the R-loop structure and subsequently resolve it, resulting in transcription-associated HR repair (TA-HRR)  Figure c. BRCA1 elopment ,40. It hnegative . Notablynegative .DNA Replication Fork ProtectionDNA replication is the process of producing two identical DNA replicas from an initially double-stranded DNA molecule during cell division. Proper control of DNA replication during each cell cycle stage is essential for genomic stability, and during DNA replication, a replication fork is formed by unraveling the double-helical DNA strands. BRCA1, BRCA2, RAD51, and components of the Fanconi anemia (FA) complex, such as FANCA, FANCD2, and FANCJ (BRIP1) [Replication is frequently arrested by various genotoxic stressors, including DNA damage. Stalled forks are unstable structures that cause genomic instability, which is a hallmark of tumorigenesis, if not properly processed; therefore, stalled forks are protected by various mechanisms, including replication fork reversal, until the reactivation of the paused forks. The reversed replication forks are protected from MRE11 nuclease degradation by  (BRIP1) ,44,45 , followed by the enzymatic religation of the broken strands to untangle the DNA in transcriptional regulatory sequences , and alsIn the process, TOP2 forms TOP2–DNA cleavage complex intermediates (TOP2ccs) but it frequently fails to complete the religation step, resulting in the formation of pathologically stable TOP2ccs as 5′ adducts. BRCA1 repairs pathological TOP2ccs by promoting the estrogen-induced recruitment of MRE11 to the TOP2cc sites.Similarly, TOP2β-mediated DSBs occur during AR transcription in the prostate, suggesting that BRCA1 dysfunction in ER- and AR-expressing organs contributes to the DNA repair defects that result in ER- and AR-associated tumorigenesis .R-loop Regulation of the Enhancer Region of ESR1 by BRCA1The mechanism of ESR1 transcriptional dysregulation due to BRCA1 dysfunction, resulting in basal-like BC has been previously reported  Figure .ESR1, which encodes the estrogen receptor (Erα), are involved in the differentiation of luminal progenitors into mature luminal cells. BRCA1-associated BCs have a high frequency of basal-like and triple-negative BC (TNBC) derived from luminal progenitor cells. BRCA1 mutation causes an elevated R-loop at a putative transcriptional enhancer upstream of the ESR1 gene and reduces the transcription of the corresponding neighboring genes, such as ESR1, CCDC170, and RMND1. The resulting abnormal expansion of ER-negative luminal progenitors may be a target for other oncogenic hits, leading to basal-like BRCA1-associated BC formation [During normal mammary development, ER-negative and PR-negative luminal progenitors differentiate into ER-positive mature luminal cells . Luminalormation ,50.BRCA1 were detected in 1.05% and 0.85% of patients with BC and 0.11% and 0.11% of the controls, respectively, whereas GPVs in BRCA2 were detected in 1.54% and 1.29% of patients with BC and 0.26% and 0.24% of the controls, respectively [In two large-scale case-control studies reported by Dorling et al. and Hu et al., GPVs in ectively ,13  than for ER-positive BC  (The odds ratios of ctively) .BRCA1 and BRCA2 GPV carriers, respectively [BRCA1/2 GPVs yielded a lifetime risk of approximately 50% at 80 years [BRCA1 and BRCA2 GPV carriers are also at high risk for the development of BC, with risk estimates of ~1–5% and 5–10%, respectively, compared with the general male population, with a lifetime risk of ~0.1% [The cumulative BC risk by 80 years was reported to be 72% and 69% for female ectively . Accordi80 years ,13, whicBRCA1/2 [BRCA1 and BRCA2 GPV carriers, respectively [It is estimated that 10–15% of patients with OC harbor GPVs in BRCA1/2 ,53, and ectively .BRCA1/2 GPV carriers have increased risks of developing prostate cancer, pancreatic cancer, and malignant melanoma. In a meta-analysis, the pooled relative risks (RRs) for prostate cancer were 4.35 and 1.18 for non-Ashkenazi European ancestry BRCA2 and BRCA1 GPV carriers, respectively [BRCA1 and BRCA2 GPV carriers, respectively, and the risks of developing pancreatic cancer by 75 years were reportedly 1–3% and 3–5% for BRCA1 and BRCA2 GPV carriers, respectively [In addition, ectively . The risectively ,56,57. Iectively .Biallelic variants in the genes involved in the FA/BRCA DNA repair pathway cause FA. FA is a genomic instability syndrome characterized by a predisposition to congenital abnormalities, early-onset bone marrow failure, and cancer. It is often an autosomal recessive genetic disorder .BRCA1/2 GPVs, the latest version of the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic, Version 2.2022  recommends BC awareness from an age of 18 years and clinical breast examination every 6–12 months from an age of 25 years. Between the ages of 25–29 years, annual breast magnetic resonance imaging (MRI) with contrast is recommended. Between the ages of 30–75 years, an annual mammogram with consideration of tomosynthesis and breast MRI with contrast is recommended. The management should be considered on an individual basis for BRCA1/2 GPV carriers aged > 75 years (For BC surveillance in women with 75 years .Risk-Reducing Mastectomy (RRM)BRCA1/2 GPVs reduces the risk of developing BC in the contralateral breast [Based on the results of meta-analyses, it is certain that contralateral RRM (CRRM) in BC patients with l breast ,61,62; hl breast ,61,62.BRCA1/2 GPV carriers without BC reduces the risk of developing BC in bilateral breasts; however, according to the results of some studies, BRRM is not significantly associated with improved survival [Furthermore, it is certain that bilateral RRM (BRRM) in survival ,63,64.Therefore, it is important to consider individual values and shared decision-making in the RRM options .Risk-Reducing Salpingo-Oophorectomy (RRSO)BRCA1/2 GPV carriers are at high risk of developing OC. In contrast to those of BC, the outcomes of advanced OC are poor, and there are considerable limitations to the effective early detection of OC. BRCA1/2 GPV carriers [Previous prospective cohort studies and meta-analyses have shown that RRSO is effective in preventing OC development and prolonging the overall survival (OS) of carriers .BRCA1 GPV carriers between the ages of 35 and 40 years, post-childbearing. As BRCA2 GPV carriers develop OC on an average 8–10 years later than BRCA1 GPV carriers, it is reasonable to postpone RRSO until the age of 40–45 years for BRCA2 GPV carriers unless family members are diagnosed at an earlier age [According to the NCCN guidelines, RRSO is usually advised for lier age .Currently, primary prevention is limited to risk-reducing surgeries for unaffected organs; therefore, effective non-surgical chemopreventive strategies need to be established.TamoxifenBRCA1/2 GPV carriers with primary unilateral BC  [BRCA1 GPV  and BRCA2 GPV  carriers [In a meta-analysis, tamoxifen was found to significantly reduce the incidence of CBC among R, 0.56) . Similarcarriers .BRCA1/2 GPV carriers, particularly in women who have also undergone oophorectomy, remains unclear.However, previous data are not consistent regarding the optimal dose and duration of treatment. In addition, its effect on OS in female Denosumab: Anti-RANK-L Monoclonal AntibodyBRCA1 GPV carriers have a high incidence of basal-like BC and basal-like BC is derived from ER-negative and PR-negative luminal progenitor cells. It has been reported that these luminal progenitor cells are stimulated by the paracrine action of RANK-L secreted by the surrounding mature luminal cells, and this proliferative signal leads to the development of BC. Denosumab, a RANK-L inhibitor, has the potential to prevent BRCA1-associated BC, and clinical trials on denosumab are ongoing [ ongoing ,68  regimens are used for BC treatment. Anthracyclines induce DSBs by inhibiting TOP2. In vitro data suggest that cells lacking the BRCA1 or BRCA2 functions are sensitive to agents such as anthracyclines, which cause DSBs and consequently increase apoptosis ,70,71.In contrast, taxanes are anti-microtubule agents that exert their effects by inhibiting mitotic spindle depolymerization and tubulin polymerization . PreclinBRCA1/2 GPV carriers and non-carriers. The BRCA1 GPV-positive status and ER-negative status in patients with BC are reportedly independently associated with an increased pCR rate [Neoadjuvant studies have investigated the pathological complete response (pCR) rate after AC-T in pCR rate ,74.Platinum-based Anticancer AgentsBRCA-associated BCs are more sensitive to platinum-based chemotherapy [BRCA1/2-deficient BC is expected to be particularly sensitive to platinum-based agents more than BRCA1/2 wild-type BC [otherapy ,23. The -type BC ,75,76.BRCA1 GPV to a neoadjuvant platinum agent [BRCA1 GPV, a higher rate of pCR (83%) was observed after treatment with cisplatin (75 mg/m2 every three weeks for four cycles) compared to that (22%) for AC (doxorubicin and cyclophosphamide) or FAC ; however, a randomized phase II study of neoadjuvant cisplatin versus AC in 118 HER2-negative BC patients with BRCA1/2 GPV (TBCRC 031) in 2020 demonstrated that the pCR or residual cancer burden (RCB) 0/1 was not significantly higher in the cisplatin-treated group than in the AC-treated group [BRCA1/2-associated TNBC patients  as compared to wild-type TNBC patients , although the difference was not significant [A retrospective study using the Poland registry in 2010 reported, for the first time, the sensitivity of patients with um agent . Among 1ed group . The pCRnificant .BRCA1/2 GPV [BRCA1/2-related advanced BC.The TNT and CBCSG006 trials examining the benefit of the first-line platinum agents in patients with metastatic TNBC have included those with A1/2 GPV ,80,81; hBRCA1/2 GPVs, carboplatin showed twice the overall response rate (ORR) of docetaxel . Progression-free survival (PFS) also favored the carboplatin (6.8 months vs. 4.4 months), but no difference was found in OS, probably because of the crossover design of the trial. The TNT trial compared first-line carboplatin with docetaxel . In the BRCA1/2 GPVs had a numerically higher ORR  and prolonged PFS  in the GP arm than in the GT arm.In the CBCSG006 trial, the cisplatin plus gemcitabine (GP) regimen showed an efficacy superior to that of the paclitaxel plus gemcitabine (GT) regimen (hazard ratio = 0.692) as the first-line treatment for metastatic TNBC . In an aCDK4/6 InhibitorsBRCA2 GPV carriers had poor outcomes following treatment with first-line CDK4/6 inhibitors plus endocrine therapy in 4640 patients with BC whose samples were subjected to germline and matched tumor tissue sequencing using MSK-IMPACT from April 2014 to May 2021. As RB1 and BRCA2 are located near the same chromosome, an RB1 loss associated with the LOH of BRCA2 is considered one of the mechanisms underlying CDK4/6 resistance [A study presented at the 2021 San Antonio Breast Cancer Symposium (SABCS) showed that sistance . BRCA1/2 (n = 10), ATM (n = 4), and CHEK2 (n = 1) GPVs, BRCA1/2-ATM-CHEK2 GPVs were suggested to be associated with poor outcomes in advanced BC treated with CDK4/6 inhibitors; however, there were no randomized controlled trials for CDK4/6 inhibitors in patients with GPVs in the HR genes [In retrospective real-world data with a total of 217 individuals including 15 patients with HR genes .PARP InhibitorsBRCA1/2-associated BC patients, in PARP inhibitors exhibit anticancer activities based on the theory of “synthetic lethality” between PARP inhibition and the LOF of BRCA1/2 due to pathogenic variants or depletion. Several PARP inhibitors with various PARP trapping activities have been developed. Here, we summarize the clinical studies of PARP inhibitors, including olaparib, talazoparib, veliparib, niraparib, and rucaparib, in BRCA1/2 GPVs have demonstrated a high sensitivity to Olaparib, based on the results of the OlympiAD and OlympiA trials, respectively [Olaparib is a PARP-1, -2, and -3 inhibitor. HER2-negative metastatic BC (MBC) and early BC with ectively ,85. In the OlympiAD trial, an olaparib monotherapy was clinically superior to the standard chemotherapy of physician’s choice (TPC) in terms of both PFS (7.0 vs. 4.2 months) and ORR (59.9% vs. 28.8%). There was no statistically significant improvement in the OS . In the OlympiA trial for patients with high-risk BC after the local treatment and adjuvant chemotherapy, additional adjuvant olaparib for 12 months provided a significantly longer invasive or distant disease-free survival (DFS) than the placebo. The three-year invasive DFS was 85.9% in the olaparib group and 77.1% in the placebo group [BRCA1/2 GPVs showed that the PFS and ORR were significantly better in the talazoparib group than in the TPC group  [Talazoparib is a PARP-1 and -2 inhibitor with powerful PARP trapping. An EMBRACA trial comparing talazoparib and TPC in MBC patients with ctively) .BRCA1/2 GPVs [BRCA1/2 GPVs treated with paclitaxel alone, those treated with paclitaxel plus carboplatin, and those treated with paclitaxel plus carboplatin plus veliparib were 41%, 50%, and 57%, respectively.Veriparib is a PARP-1 and -2 inhibitor with the weakest PARP trapping activity, which has been essentially developed in combination with platinum-based chemotherapy. In the BrighTNess trial, the effects of the addition of carboplatin with and without veriparib to the standard neoadjuvant combination of paclitaxel followed by AC were evaluated in 634 TNBC patients including 92 patients with 1/2 GPVs . The strPALB2  was originally identified as a gene that produces a BRCA2-interacting protein [PALB2 GPVs result in an increased risk of BC, whereas bi-allelic PALB2 GPVs cause FA [ protein  and was cause FA ,100,101.PALB2, which is recruited to sites of DNA damage upon phosphorylation in response to DSBs, serves as a molecular scaffold for the formation of the BRCA complex (BRCA1-PALB2-BRCA2-RAD51), thereby facilitating RAD51-mediated strand invasion during HR ,100,102 BRCA1/2 in DNA replication fork protection  (PALB2 GPV carriers showed a strong association with developing ER-positive BC [PALB2 GPV carriers exceeded 30% [PALB2 GPV carriers with two or more first-degree relatives who developed BC under the age of 50 years [Approximately 0.5% of patients with BC harbor GPVs in the ctively) . Additioitive BC ,13. The eded 30% ,13, but 50 years .PALB2 has also been reported to be associated with increased risks of OC and pancreatic cancer [PALB2 GPV carriers, whereas that for pancreatic cancer is 5–10% [Recently, c cancer ,105. Theis 5–10% ; howeverPALB2 GPV carriers, the NCCN guidelines recommend annual mammograms with consideration of tomosynthesis and breast MRI with contrast from the age of 30. Similarly to that for BRCA1/2, the RRM option can be discussed. Evidence of RRSO in the management of OC risk is insufficient, and RRSO may be considered after menopause or earlier if there is a family history of OC [For BC screening in ry of OC   gene encodes a protein kinase at the peak of a signaling cascade that responds to DSBs and is required to coordinate the resulting cellular responses, such as DDR, cell cycle arrest, and/or apoptosis [TP53, and Chk2 encoded by CHEK2, which participate in different stages of the DDR [The poptosis ,113. Onc the DDR ,24. ATM  the DDR   [ATM, the odds ratio of ER-positive disease is higher than that of ER-negative disease [ATM may act in a dominant-negative manner to increase the risk of BC compared with truncating variants. Notably, carriers of c.7271T > G  are reportedly associated with a higher risk of invasive ductal BC (odds ratio = 3.76) [In two recent large studies, the prevalence of GPVs in ectively ,13 (Tablctively) ,13, withctively) ,116. For disease . Some ev = 3.76)  and haveATM GPV carriers are also associated with an increased risk of pancreatic cancer (odds ratio = 4.21), prostate cancer (odds ratio = 2.58), gastric cancer (odds ratio = 2.97), ovarian cancer (odds ratio = 1.57), colorectal cancer (odds ratio = 1.49), and melanoma (odds ratio = 1.46) [ = 1.46) .ATM GPVs cause a rare autosomal recessive disorder called A-T, characterized by progressive cerebellar ataxia, telangiectasia, oculomotor apraxia, immunodeficiency, cancer susceptibility, and radiation sensitivity [ATM GPV carriers against the risk of autosomal recessive conditions in their offspring is recommended [Homozygous or compound heterozygous sitivity ; therefoommended .ATM GPV carriers, annual mammograms with consideration of tomosynthesis beginning at 40 years and consideration of breast MRI with contrast are recommended by the NCCN guidelines. As there is insufficient evidence to recommend RRM and RRSO, the procedures are considered based on family history  treated with CDK4/6 inhibitors had poor outcomes [ATM GPVs.Recent studies have shown that the ATM-Chk2-Cdc25A pathway participates in resistance to CDK4/6 inhibitors ,121,122.outcomes ; howeverPARP InhibitorsBRCA1/2-associated tumors, a similar efficacy can be expected in BC patients with GPVs in ATM. Nevertheless, in an expanded phase II study of olaparib for MBC with GPVs in HR-related genes, no response was observed in patients with GPVs in either ATM or CHEK2 alone, although the reliability of the results is limited owing to the small sample size [Considering the efficacy of platinum-based agents and PARP inhibitors in treating ple size .ATM-deficient tumors [ATM-deficient tumors  [In a phase II clinical trial, olaparib combined with paclitaxel has shown a significant improvement in OS in patients with advanced gastric cancer, especially in those with t tumors ; howeverulation) .BRCA1, BRCA2, or ATM in tumors. The PROfound trial has shown that olaparib led to significantly longer PFS and OS than the physician’s choice of enzalutamide or abiraterone among men with metastatic castration-resistant prostate cancer who had at least one germline or somatic PV in BRCA1/2 GPVs derived the most benefit, and showed the hazard ratios for PFS and OS among patients with ATM GPVs (olaparib vs. control) were 1.04  and 0.93 , respectively [However, exploratory analyses suggest that patients with ectively ,126.Radiation TherapyATM GPV heterozygous carriers.The high sensitivity of A-T to radiation raises numerous concerns regarding the use of breast radiation therapy (RT) in ATM GPV carriers treated with RT after breast-conserving surgery (BCS), showed no significant differences in local recurrence or metastasis-free survival between the carriers and non-carriers [ATM variants with radiation-induced toxicity or the risk of secondary malignancy [ATM. The possible exceptions are patients with the c.5557G > A variant, in whom a small but increased risk for the development of both acute and late radiation effects has been identified. Furthermore, patients younger than 45 years with certain rare deleterious ATM variants may have an increased risk of developing CBC. Heterozygous ATM GPV should be recommended to not avoid RT [A study of 135 patients with BC, including 20 carriers . McDuff lignancy  and concavoid RT ,128.ATM GPV carriers are ongoing and remain controversial [ATM GPV carriers is of little benefit because mammogram rarely detects BC missed on MRI [ATM GPV carriers when screening with MRI [Mammogram screening benefits and harms for young oversial ,129. Mamd on MRI , and couCHEK2 is a tumor suppressor gene that encodes protein Chk2 (checkpoint kinase 2), a serine/threonine kinase [TP53 and BRCA1, which are involved in various cellular pathways that activate checkpoint-mediated cell cycle arrest, DNA repair, and apoptosis [e kinase . Chk2 ispoptosis  . The CHEK2:c.1100delC variant showed a higher relative risk (odds ratio = 2.66) than the other truncating and missense GPVs [CHEK2, the odds ratio for ER-positive disease is higher than that for ER-negative disease [In two recent large studies, the prevalence of ectively ,11. CHEKnse GPVs ,13. The nse GPVs ,135. For disease .CHEK2 GPVs, particularly c.1100delC and p.I157T, are also associated with increased risks of other types of cancer, such as colorectal, prostate, kidney, and thyroid cancer [d cancer ,136.CHEK2, the management should be based on the best estimates of the carcinogenic risk for a particular GPV. For CHEK2 GPV carriers, an annual mammogram with consideration of tomosynthesis starting at 40 years and breast MRI with contrast is recommended by the NCCN guidelines. Since sufficient data are not available on the benefits of RRM, prophylactic surgery is considered based on family history  [In exploratory analyses of the PROfound trial evaluating the efficacy of olaparib compared with the treatment of physician’s choice in patients with metastatic castration-resistant prostate cancer who had disease progression while receiving a new hormonal agent, the hazard ratio for PFS among the patients with to 4.13) .CHEK2 GPVs is unclear, and the utility of CHEK2 as a companion test for predicting treatment sensitivity has not been established.The sensitivity to specific treatment regimens in BC patients with BARD1 (BRCA1 associated RING domain 1) encodes a ubiquitin ligase that interacts with the N-terminal region of BRCA1. BRCA1-BARD1 heterodimerization is required for their mutual stability, HR function, and tumor suppressor activity [activity ,139,140 BARD1 were detected in 0.13% and 0.06% of BC patients and controls, respectively  [BARD1 were associated with a moderate risk of ER-negative BC and TNBC, but not ER-positive BC [In a recent population-based study, protein-truncating GPVs in 0.00098)   [In a recent population-based study, GPVs in ctively)  , than with ER-positive BC  [The estimated lifetime absolute risks of BC for e 15–40% ,13,14. Nctively) .RAD51C and RAD51D are also associated with an increased risk of developing OC  [RAD51C and RAD51D GPV carriers [GPVs in ctively) . The lifcarriers ,145,146.Owing to insufficient evidence, BC screening is based on family history. In contrast, the NCCN guidelines recommend that RRSO should be considered beginning at 45–50 years of age or earlier, based on a specific family history of an earlier onset of OC  Table 2Table 2.BRCA1, BRCA2, PALB2, ATM, BARD1, CHEK2, RAD51C, and RAD51D, as well as the clinical management of individuals harboring GPVs in these genes. This review has summarized the molecular functions of key BC-associated CPGs, namely, BRCA1/2 convey the highest genetic risk of BC and data on the prevention and treatment of BCs associated with GPVs in BRCA1/2 are accumulating, optimal clinical strategies for BCs associated with GPVs in CPGs other than BRCA1/2 have not yet been established. Most BC-associated CPGs participate in the DNA damage repair pathways and cell cycle checkpoint mechanisms and work together in such cascades; therefore, a fundamental understanding of the pathogenic cascade caused by GPVs in BC-associated CPGs may enable us to accurately estimate the genetic risk of developing BC and to select the appropriate preventive and therapeutic strategies for treating hereditary BCs. The penetrance of GPVs in each gene varies, reflecting the distinct risks of BC susceptibility. Although GPVs in In addition, a refinement of the risk models using polygenic risk scores may enable a better definition of the personalized risks for hereditary BCs , with anFor rare genes where there is limited data for targeted therapeutics in each trial, sharing data across trials is the only way to enable an effective analysis of the therapeutic response, and is key to future trial development.


Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection. Ubiquitination is a post-translational modification that involves the covalent binding of one or more ubiquitin (Ub) molecules to a target protein, which leads to changes in its function, location, activity, or stability . Ub conjUbiquitination is a dynamic process that is reversible due to deubiquitinating enzymes (DUBs), which hydrolyze poly-Ub chains, remove Ub from labeled proteins, and process Ub precursor proteins. The human genome encodes nearly 100 DUBs, which are classified into seven families according to their structure and mechanism of catalysis. Six are cysteine proteases, and the seventh family includes metalloenzymes. Ubiquitin-specific proteases (USPs) are the largest and most diverse family, with 56 members, followed by ovarian tumor proteases (OTUs), with 17 members, and the JAMM family , with 12 members. The MINDY family, Machado Joseph disease protease family (MDJ), ubiquitin terminal carboxyl hydrolases (UCHs), and the newly discovered ZUFSP/Mug105 family are less common .Fundamental cellular processes, such as cell cycle progression, endocytosis, intracellular trafficking, DNA repair, transcription, signaling, and antigen presentation, are regulated through ubiquitination, deubiquitination, and the UPS system . VariousMultiple studies have shown that some alphaviruses, flaviviruses, retroviruses, coronaviruses, and herpes viruses require ubiquitination and proteasome activity in the early stages of infection (entry and uncoating) or for other crucial processes, such as viral genome synthesis, viral protein translation, assembly, maturation, and the release of viral progeny ,7,8,9.Analysis of the changes in the cellular proteome caused by infection with chikungunya virus (CHIKV) suggests that the UPS system is essential during infection in both cell cultures and murine models ,11,12. TTogaviridae and the causative agent of a febrile illness accompanied by rash, headache, myalgia, and severe joint pain [CHIKV is an alphavirus of the family int pain . Arthritint pain ,17. Duriint pain  but alsoint pain .No vaccine or specific drugs have been approved for the treatment of CHIKV. In a search for new antiviral strategies against CHIKV, the requirement for DUB activity during CHIKV infection, which is yet unknown, was analyzed by evaluating the effect of selective inhibitors of DUBs on viral replication.® CRL-11268G-1™), Vero clone E6 (ATCC® CRL-1586™), Huh-7 (JCRB0403) and BHK-21 (ATCC® CCL-10™)  were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C and 5% CO2. The CHIKV isolate used in this study, COL7624, was collected in Colombia in 2015 and passaged four times in Vero cells before titration (GenBank ID: MH329298.1). The characterization of this viral isolate, including its ability to infect and replicate in HEK293 and Huh-7 cells, has already been reported by Archila et al., 2022 [The adherent cell lines HEK293 (ATCCThe DUB inhibitors PR-619 (Cat#662141) and WP1130 (Cat#681685) were purchased from Calbiochem, San Diego, CA, USA and were used at the concentrations indicated in the different experiments. Mouse polyclonal antibody directed against the E1 protein was provided by Anne Claire Brehin , and rat polyclonal serum against nsP1 was obtained after injection of a recombinant protein produced in our laboratory. For Western blotting and flow cytometry, the following antibodies were used: mouse monoclonal anti-CHIKV virus antibody , anti-actin mAb , and the DUB Antibody Sampler Kit including anti-rabbit mAbs against HAUSP, UCHL1, A20/TNFAIP3, USP10, STAMBP, CYLD, and pCYLD proteins . Horseradish peroxidase (HRP)-conjugated anti-mouse (12-349), anti-rat (12-349), and anti-rabbit (12-348) secondary antibodies were purchased from Sigma–Aldrich.Cells were seeded in 96-well plates, and after overnight incubation, the medium was removed and replaced with 100 µL of fresh medium containing either 0.1% DMSO or a DUB inhibitor at the indicated concentration and incubated for 24 h. Then, resazurin solution (44 µM) was added, and the plate was incubated at 37 °C for 2 h. Finally, the fluorescent signal was read using a Tecan Infinite 200 fluorometric reader  at excitation and emission wavelengths of 535 and 595 nm, respectively.HEK293, Vero-E6, and Huh-7 cells were seeded at densities of 100.000–200.000 cells per well and incubated overnight at 37 °C. The next day, monolayers were incubated with PR-619, WP1130, or 0.1% DMSO for 1 h. Treated cells were then infected with CHIKV at MOI 1 or 0.5 for 1 h. Finally, infected cell monolayers were incubated in DMEM containing 2% FBS and inhibitor for 24 h at 37 °C. Supernatants were collected for titration, and cells were fixed for antigen detection using flow cytometry.For the post-infection assay, cell monolayers were infected with CHIKV for 1 h at 37 °C. Then, the inoculum was removed, and the cells were incubated in DMEM with 2% FBS, followed by the addition of DUB inhibitor. Finally, at 24 h post-infection (hpi), supernatants were collected for titration, cells were collected for antigen detection using flow cytometry, RNA was prepared for viral RNA quantification using RT-qPCR, and viral proteins were analyzed using Western blotting .Cells were permeabilized using Cytofix/Cytoperm , incubated with anti-E1 antibody (Abcam) diluted 1:50 in Perm/Wash buffer (BD Biosciences), and then stained with secondary FITC-conjugated anti-mouse Ab (Sigma). Flow cytometry was performed using a BD Accuri™ C6 cytometer (BD Biosciences). Data were analyzed using FlowJo™ Software (BD Biosciences).Cells were lysed in RIPA buffer containing a protease inhibitor cocktail for 30 min at 4 °C. Protein samples were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes . Membranes were blocked with 5% non-fat milk in TBST and incubated with the appropriate primary antibody for 1 h. For DUB detection, the membranes were blocked with 3% bovine serum albumin in TBST. After extensive washing, the membrane was incubated with each antibody and then with anti-mouse, anti-rat, or anti-rabbit secondary antibodies coupled to horseradish peroxidase (HRP). The blots were developed using West Pico Blotting Detection Reagent , and images were captured using a Gel Doc system .® Probe system  and the following primers targeting the nsp4, E1, and actin as a reference gene for normalization: nsp4F, TCACTCCCTGTTGGACTTGATAGA; nsp4R, TTGACGAACAGAGTTAGGAACATACC; nsp4P Texas Red/AGGTACGCGCTTCAAGTTCGGCG/BHQ2; E1F, AAGCTYCGCGTCCTTTACCAAG; E1R, CCAAATTGTCCYGGTCTTCCT, E1P FAM/CCAATGTCYTCMGCCTGGACACCTTT/BHQ-1; actin F, GGATGCAGAAGGAGATCACTG; actin R, CGATCCACACGGAGTACTTG; and actin HEX/CCCTGGCACCCAGCACAATG/BHQ1. Each reaction (20 μL) contained 10 μL of 2× master mix, 200 nM primers, 200 nM probes, and 60 ng of RNA. Reactions were performed in a CFX96 real-time PCR cycler (BioRad) under the following cycling conditions: 15 min of reverse transcription at 55 °C, followed by a denaturation step at 95 °C for 3 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The fold changes in the inhibitor-treated samples, when compared to the levels in the untreated samples, were calculated using Pfaffl’s (2001) method [Total RNA was extracted from infected cells using the PureDireX Total RNA Isolation Kit  according to the manufacturer’s instructions. The eluted RNAs were quantified, normalized to 20 ng/mL, and stored at −80 °C until use. Samples were assayed with a one-step RT-qPCR protocol using the Luna) method .3) (Sigma-Aldrich), supplemented with FBS and antibiotic was added; cells were incubated for 72 h. Then, cells were fixed and stained with 1% crystal violet (Sigma-Aldrich). The number of plaques was counted and is expressed as plaque-forming units per milliliter (PFU/mL).BHK-21 cells were seeded at a density of 80.000 cells/well in 24 well plates. The next day, confluent monolayers were infected with 10-fold dilutions of the samples prepared in 2% media. After one hour of incubation, media containing DMEM , 3% CMC, 7.5% sodium bicarbonate  followed by Dunnett’s post-hoc test was performed to determine the significance of differences between the experimental groups and the controls using GraphPad Prism 7. A This study investigated the requirement of deubiquitinating activity during CHIKV infection using specific inhibitors that selectively modulate DUB activities with a relatively low range of toxicity and off-target activities. PR-619 is selective for USP and UCH families over other cysteine proteases, including phospholipase A, calpain 1, cathepsin B, cathepsin D, matrix metallopeptidase 13, and trypsin . While, To assess the effect of DUB inhibition on productive virus infection, cells were treated with the inhibitor, infected with the virus, and kept in the presence of the inhibitor for a further 24 h. Then, the infectious viral progeny released into the supernatant was quantified using plaque assays in BHK-21 cells. Treatment with WP1130 caused a significant decrease in viral progeny in all the tested cell types. Similar behavior in Huh-7 and HEK293T cells was evidenced since the viral titer fell by 1 log unit (~10 times lower) and 1.1 log units (~12.5 times lower), respectively. In contrast, Vero cells displayed the most significant reduction of 1.4 log units (~25 times lower) in viral yield. Treatment with PR-619 had different effects. In HEK293T and Huh-7 cells, no significant changes in viral titer were observed, whereas in Vero cells, viral titer was significantly reduced by 2.5 logarithmic units  or 4 h after virus inoculation (post-infection), and viral antigen was detected at 24 hpi A. Cell lTo assess whether the decrease in the infective viral progeny was due to a defect in viral protein synthesis or genome replication events in the early stage of the viral replication cycle; we evaluated the levels of both structural proteins, which are indispensable for virion formation, and nonstructural proteins, which are essential for genome replication, in cells treated with the inhibitors. In infected cells treated with DMSO, antibodies recognized a 49 kDa band corresponding to E1 and a 60 kDa band corresponding to nsP1 A. ExposuE1 and E2 are glycoproteins located in the virus envelope that allow interaction with the host cell at different phases of viral entry, such as target cell adhesion, internalization, intracellular trafficking, and fusion of membrane proteins . In contTo analyze the effect of DUB inhibition on the synthesis of viral RNA (vRNA), vRNA levels were quantified using RT-qPCR. Beta-actin was used as a reference gene, and cells treated with DMSO as a sample control. There was a significant reduction in vRNA levels in inhibitor-treated cells. A substantial decrease was observed in all the cell lines treated with WP1130. Exposure to PR619 resulted in a 5-fold reduction in vRNA levels in Vero cells B. These Several studies have shown the overexpression or downregulation of some DUBs during viral infection ,27,28,29In the last decade, CHIKV has re-emerged as a major public health threat due to frequent infections, which result in high morbidity and economic costs . CurrentDUBs cleave multiple types of poly-UB chains, thereby modulating critical processes occurring inside the cell that are regulated by protein ubiquitination. In the present study, we evaluated the role of deubiquitination during CHIKV infection using two DUB inhibitors (PR-619 and WP1130). PR-619 is a reversible inhibitor that induces endoplasmic reticulum (ER) stress and related apoptosis and is a broad-spectrum DUB inhibitor that targets nine members of the USP family (2/4/5/7/8/15/20/28/47) and three of the four members of the UCHL family (1/3/5) in human cells. In contrast, WP1130 is a specific inhibitor of USP5, 9X, 14, 37, and UCHL5. It is currently in preclinical studies as a drug against melanoma and glioblastoma [The experiments were performed in three cell lines with different susceptibilities to infection. HEK293T cells reached moderate infection percentages of 25%, whereas Vero and Huh-7 cells reached infection percentages of 80–90% at 24 hpi. Decreasing deubiquitinating activity by treating cells with WP1130 led to significant reductions in infected cells and infective progeny in all the cell lines tested. In contrast, exposure to PR-619 did not affect viral replication in HEK293T and Huh-7 cells. These results indicate that DUB activity is fundamental to the CHIKV replicative cycle. However, these data suggest differences between the analyzed cell types in either the active DUBs and those involved in viral replication. The blocking activity of WP1130 is restricted to five DUBs; however, two of these enzymes, USP5 and UCHL5, are also targets of PR-619, which did not affect productive infection. Another target of WP1130 is USP14, which can be blocked by a specific inhibitor called IU1, which has previously been shown to have no effect on CHIKV replication in HEK293T cells, even at concentrations of 75–100 µM . TherefoSome authors have previously reported a requirement for DUBs during viral infection. Nag and Finley showed that IU1 interferes with the replication of some flaviviruses, such as West Nile (WNV), yellow fever virus (YFV), and dengue (DENV), with the most potent effect on DENV . Perry eOur findings demonstrate that DUB activity is essential during the early phase of CHIKV infection. However, DUB activity could also modulate late events in the CHIKV replication cycle . ProcessFinally, the expression of six DUBs was evaluated during CHIKV infection. USP7, USP10 and CYLD, members of the USP family; UCHL1, which belongs to the UCHL family; STAMBP, which is a JAMM family metalloprotease; and otubain A20. These enzymes are involved in signaling, DNA repair, transcription, and endocytosis, and some of them, such as A20 and CYLD, contribute to establishing the antiviral status of cells . During This work demonstrates for the first time that cellular DUBs are required for optimal CHIKV infection in mammalian cells. DUB inhibitors impair the synthesis of viral RNA and viral proteins. However, the mechanisms by which DUBs are involved in these processes remain to be defined.


Neuronal growth and synaptic function are dependent on precise protein production and turnover at the synapse. AMPK-activated protein kinase (AMPK) represents a metabolic node involved in energy sensing and in regulating synaptic protein homeostasis. However, there is ambiguity surrounding the role of AMPK in regulating neuronal growth and health. This study examined the effect of chronic AMPK activation on markers of synaptic function and growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 nM) for 1, 3, or 5 days before AMPK, mTORC1, and markers for synapse function were examined. Cell morphology, neuronal marker content, and location were quantified after 5 days of treatment. AMPK phosphorylation was maintained throughout all 5 days of treatment with A-769662 and resulted in chronic mTORC1 inhibition. Lower total, soma, and neuritic neuronal marker contents were observed following 5 d of AMPK activation. Neurite protein abundance and distribution was lower following 5 days of A-769662 treatment. Our data suggest that chronic AMPK activation impacts synaptic protein content and reduces neurite protein abundance and distribution. These results highlight a distinct role that metabolism plays on markers of synapse health and function. Alzheimer’s disease (AD) is a neurodegenerative disease defined by amyloid-airments ,2. The dairments ,4. Recenairments ,6,7. Theairments ,9,10,11.AMPK is a serine/threonine kinase that is activated by AMP and plays a major role in regulating metabolic homeostasis ,13. By bn tissue . Previoun tissue ,19,20, rn tissue , and impn tissue ,19.AMPK activation also restricts anabolic processes such as cellular proliferation/growth and protein synthesis in an effort to preserve cellular ATP content through the inhibition of a major downstream target of AMPK, the mechanistic target of the rapamycin complex (mTORC1) . mTORC1 Considering the importance of metabolic homeostasis in protein synthesis and cellular growth, the AMPK-mTORC1 signaling cascade has gained attention in some areas of AD research ,24. AMPKv/v) FBS , 5% non-essential amino acid , and 1% Penicillin/Streptomycin . Differentiation was accomplished with retinoic acid (1 SH-SY5Y human neuroblastoma cells were cultured using high glucose Dulbecco’s Modified Eagles Media (DMEM Sigma D6429), containing 10% or 1% . Cells were then fixed using 4% paraformaldehyde and permeabilized using 20% triton X-100 before being incubated in buffer containing the appropriate primary antibodies followed by the Alexa Fluor secondary antibodies . Bassoon and Homer-1 (as pre- and post-synaptic markers) and synaptophysin were imaged. Images were captured using a Biotek Cytation5 cell imaging reader. Immunofluorescent (IF) images were captured in a 5 × 5 image grid (10×) where images were analyzed using DAPI  and phalloidin-actin . DAPI was used to determine cell number and a phalloidin actin stain was used to determine soma size, total cell area, and projection length. Neurite length was defined by fluorescent signals measured 7–70 p < 0.05 was considered significant. Western blot data are reported as mean ± SEM with results consisting of n = 3–6 independent experiments with 3 technical replicates per condition. IF results are presented as an average of the number of cells analyzed across three technical replicates per condition (1119–1903 cells). Brown–Forsythe homogeneity tests and Bartlett’s test were used to assess normality.Differences in protein content/phosphorylation status, cell size, neurite length, and fluorescence levels were determined using one-way ANOVA followed by a Tukey’s post hoc test. A value of To assess the validity of CC and A76 in modulating AMPK phosphorylation status in RA differentiation of SH-SY5Y cells, multiple, previously reported doses of both compounds ,37,38,39 of T172 . Cells w0 nM A76 A and 10 30 nM CC B. For bo30 nM CC , with no30 nM CC C; 30 nM Interestingly, while neither A76 dose decreased cell viability following 7 days of treatment, both doses caused an increase in viability of which we are unsure as to why. A76 has been reported to decrease cell viability; however, these studies report usage at much greater concentrations than were used in this study, and were not performed in the SH-SY5Y cell line ,44. SH-SY5Y cells treated with 100  control A–C. Trea control A–C. Give control D–F, wher control E,F. To c control G–I. OverWe next examined if chronic AMPK activation or inhibition had an effect on synaptic protein content. Synaptophysin, Homer-1, and BDNF are key markers of neuronal health and plasticity that are decreased in AD and can be regulated by the AMPK signaling cascade ,49,50,51No changes in synaptophysin, Homer-1, or BDNF protein levels were observed following 24 h or 3 days of treatment of SH-SY5Y cells with CC or A76 A,B; howeAs AMPK signaling has been implicated in neuron growth and neurite polarization ,31, we eell body . Bassoonell body ,53, invoell body , and whoell body . Reductiell body . Treatment with 30 controls D. With Acontrols A–C was ocontrols A–C. Follcontrols D. AMPK plays a large role in a number of metabolic diseases and has therefore become an important therapeutic target ,23,55,56function ,58,59. Ffunction . Howeverfunction ,34,61. Wfunction ,63, the In this work, we investigated the effect of acute (24 h) and chronic (3–5 days) AMPK activation on markers’ neuronal growth. Our results demonstrate that chronic activation (5 d) of AMPK and mTORC1 inhibition via raptor phosphorylation negatively impacted cell morphology and reduced key markers of synaptic plasticity and neuronal growth in SH-SY5Y neuroblastoma cells. Specifically, we demonstrate that 5 days of AMPK activation reduced total content of a ubiquitous synaptic marker, synaptophysin, marker of post-synaptic stability Homer-1, and the major neurotrophic factor BDNF. Further, neurite distribution of synaptic marker content (defined by the content of a given protein within a neurite) was also reduced after 5 days of AMPK activation and was consistent with the reductions in total cell size, area, and projection length. These results were only seen with 5 days of AMPK activation, highlighting a distinct role for AMPK activity to affect synaptic markers in a time-dependent manner. The ability to maintain and respond to stressors is paramount to maintaining healthy brain homeostasis, especially through a major regulator such as AMPK ,56,64. H buildup . More so buildup ,11,25. R buildup . While iIt is important to note that, though it has not been shown to occur specifically in SH-SY5Y neuroblastomas cells, both CC and A76 have been shown to have AMPK-independent actions ,68. Desprylation C. SimilaOur results demonstrate an AMPK time-dependent action on synaptic markers that are linked to proper activity-dependent synapse formation, stability, and transmission ,51,70,71The results of this study provide a basis for future work examining the connection between AMPK activity and synaptic markers. Here, we utilized the SH-SY5Y cell line, as it has been used widely in experimental neurological studies, including analysis of neuronal differentiation, metabolism, and function related to neurodegenerative processes, neurotoxicity, and neuroprotection ,76,77,78This study provides novel evidence for the role of chronic AMPK activation in regulating the expression and localization of markers directly related to synaptic stability and neuronal health. Importantly, this study showed significant impairments in these markers after chronic activation of AMPK (5 days), but not acutely (24 h–3 d). These results highlight a role for a timeframe of AMPK activation that plays a major role in regulating proper neuronal health. Coupled with reductions in cell morphology and synaptic machinery, our data provide further support for the role of metabolism and synapse health. As this study focused on the chronic modulation of AMPK under non-metabolically strenuous conditions, the impact of AMPK modulation should be further explored under metabolically strenuous conditions in order to further elucidate how metabolism can influence synaptic function.


To investigate the effect of multidisciplinary team (MDT) continuous nursing on glucose and lipid metabolism, pregnancy outcome, and neonatal immune function in gestational diabetes mellitus (GDM).  A total of 90 patients with gestational diabetes mellitus (GDM) from January 2018 to December 2019 were recruited and assigned to receive routine care (routine group) or MDT continuous care (study group) according to different nursing methods. Outcome measures included glucose and lipid metabolism, pregnancy outcomes, and neonatal immune function.  There were no significant differences in glucose and lipid metabolism indices and self-rating anxiety scale (SAS) scores, before nursing. After nursing, MDT continuous care resulted in significantly lower levels of fasting blood glucose (FBG), 2 h postprandial blood glucose (2hPBG), glycosylated hemoglobin (HbAlc), triglyceride (TG), and homeostasis model insulin resistance index (HOMA-IR) versus routine care. After nursing, the SAS scores in the two groups were significantly decreased, with lower results in the study group. Patients in the study group showed better compliance than those in the routine group. MDT continuous care was associated with a significantly lower incidence of premature rupture of fetal membranes, cesarean section, premature delivery, macrosomia, and hypoglycemia versus routine nursing. There were no significant differences in immunoglobulin (Ig) A and IgM levels. Patients in the study group showed a higher IgG level and lower CD3, CD4, CD8, and CD4/CD8 levels than those in the routine group.  MDT continuous nursing could effectively regulate glucose and lipid metabolism and improve pregnancy outcomes and neonatal immune function in patients with GDM. Gestational diabetes mellitus (GDM) refers to impaired glucose tolerance or diabetes that first occurs during pregnancy . AccordiP > 0.05)  . The resInclusion criteria: pregnant women who were diagnosed with GDM as per the 2010 IADPSG diagnostic criteria for gestational diabetes , with siThe diagnostic criteria for hypertensive disorders during pregnancy, excessive amniotic fluid, premature rupture of membranes, postpartum hemorrhage, and fetal macrosomia  are as fExclusion criteria: patients with underlying diseases such as hypertension and heart disease, with withdrawal of consent, and with severe mental diseases were excluded.The routine group received routine nursing. Routine nursing included regular prenatal care after diagnosis, routine pregnancy guidance and education, assessment of fetal conditions, body mass management, self-monitoring guidance, psychological counseling during pregnancy, and dietary guidance. The fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPBG), and glycosylated hemoglobin (HbAlc) were measured at 32 and 37 weeks of pregnancy.The study group received MDT continuous nursing: ① an MDT team composed of endocrinologists, dietitians, mother-infant specialist nurses, midwives, diabetes specialist nurses, psychological specialist nurses, and rehabilitation instructors was established. ② Nursing issues and solutions: nursing issues such as inadequate health knowledge education and poor self-management ability of patients were jointly discussed and analyzed by the MDT members to enhance the compliance and pregnancy outcomes of patients. ③ Health education : the patAn AU5800 automatic biochemical analyzer was used to determine the levels of FBG, 2hPBG, HbAlc, fasting insulin (FINS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and homeostasis model insulin resistance index (HOMA-IR).The SAS includes 20 items, with each item being scored as 0-4 points. A score of <50 indicates no anxiety, 50-59 indicates mild anxiety, 60-69 indicates moderate anxiety, and ≥70 indicates severe anxiety.The self-made questionnaire of our hospital was used for compliance assessment. Dietary compliance: patients adhering to the diet for 6 days or more per week were given 4 points, 3 points for 5 days, 2 points for 3-4 days, and 0 points for less than 3 days. Exercise compliance: patients who exercised 5 times or more per week were given 4 points, 3 points for 4 times, 2 points for 3 times, and 0 points for less than 3 times. Blood glucose monitoring compliance: patients who performed blood glucose monitoring 4 times or more per week were given 4 points, 3 points for 3 times, 2 points for 2 times, and 0 points for less than 2 times. A score of ≥3 points indicates good compliance and <3 points indicate poor compliance.Hyperhydramnios, fetal membrane, cesarean section, neonatal distress, premature delivery, macrosomia, and hypoglycemia were recorded.The levels of immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) in peripheral blood of neonates were determined using immunoturbidimetry. The levels of CD3, CD4, and CD8 in T cells were determined using flow cytometry, and the CD4/CD8 values were calculated.The pregnant women had no obvious hunger, with a fasting glucose value of 3.3-5.3 mmol/L, 2-hour postprandial glucose value of 4.4-6.7 mmol/L, and glycated hemoglobin < 5.5%. Venous blood was collected from pregnant women at 32 and 37 weeks of gestation in the outpatient laboratory to determine FBG, HbAlc, and 2hPBG levels. The values of FBG, 2hPBG, and HbAlc that were normal in two tests were considered satisfactory blood glucose control.n (%)] and measurement data (mean ± SD) were analyzed by chi-square and t-test, respectively. Differences were considered statistically significant at P < 0.05.SPSS 22.0 software was used for data analyses, and GraphPad Prism 8 was used to plot the graphics. Enumeration data [P > 0.05). MDT continuous care resulted in significantly lower levels of FBG, 2hPBG, HbAlc, TG, and HOMA-IR versus routine care (P < 0.05) (There were no significant differences in glucose and lipid metabolism indices before nursing ( < 0.05) .P > 0.05). After nursing, the SAS scores in the two groups were significantly decreased, with lower results in the study group (P < 0.05) (There were no significant differences in SAS scores before intervention ( < 0.05) .P < 0.05)  .P < 0.05)  .P > 0.05). Patients in the study group showed a higher IgG level and lower CD3, CD4, CD8, and CD4/CD8 levels than those in the routine group (P < 0.05) (There were no significant differences in IgA and IgM levels ( < 0.05) .Gestational diabetes mellitus (GDM) is a common complication of pregnancy , which rMDT continuous nursing effectively regulates glucose and lipid metabolism in patients with GDM and improves pregnancy outcomes and neonatal immune function, so it is worthy of clinical promotion. The innovation of this study is the use of patient-centered MDT continuous nursing, which relies on a multidisciplinary team to develop an optimal comprehensive treatment plan that is standardized, personalized, and continuous, thereby enhancing the quality of life of patients. However, the limitation of this study lies in the absence of detailed studies on the intelligence and development of the children, which will be investigated in future studies.


The results revealed viral positivity and negativity detection rates of 88% (74/84) and 12% (10/84), respectively. Of the 21 targeted pathogens, human rhinovirus/enterovirus (30%), respiratory syncytial virus , and severe acute respiratory syndrome coronavirus 2 (24%) were mostly detected, with other viruses being 20% and a co-infection rate of 64.2% (54/84). Generally, RSV-positive samples had lower Ct values, and fewer viruses were detected during the third wave. Changes in the circulation patterns of respiratory viruses with total absence of influenza virus could be attributed to measures against COVID-19 transmission, which may result in waned immunity, thereby increasing susceptibility to severe infections in the following season. High viral co-infection rate, as detected, may complicate diagnosis. Nonetheless, accurate identification of the pathogens may guide treatment decisions and infection control.Severe acute respiratory infections (SARI) contribute to mortality in children ≤5 years. Their microbiological aetiologies are often unknown and may be exacerbated in light of coronavirus disease 19 (COVID-19). This study reports on respiratory pathogens in children ≤5 years (n = 84) admitted with SARI during and between the second and third waves of COVID-19 infection in South Africa. Nasopharyngeal/oropharyngeal swabs collected were subjected to viral detection using QIAstat-Dx The World Health Organization (WHO) defines patients with severe acute respiratory infections (SARI) as anyone with acute respiratory infection (ARI) with symptoms such as cough and measured fever of ≥38 °C within 10 days of presentation and requiring hospitalisation ,3 and a Viral and bacterial aetiologies of SARI cannot be distinguished based on clinical presentations, and this necessitates specific laboratory diagnoses which may not be available/not routinely performed in some settings. As such, aetiologies of respiratory tract infections (RTIs) are often unknown ,12,13. AOf note, in the absence of resources for testing, clinicians may be inclined to choose early empirical antibiotic treatment in SARI patients before identifying the causative pathogen(s) . AlthougFurthermore, the traditional circulating pattern of viral respiratory pathogens since the onset of COVID-19 has drastically changed from previous years due to nonpharmaceutical interventions (NPIs) taken against COVID-19 ,28,29,30This cross-sectional study was conducted at Botshabelo District Hospital, National District Hospital, and Pelonomi Regional Hospital, Free State Province, South Africa, between December 2020 and August 2021. Sampling and participant enrolment took place during the second wave, the period between (time between the second and third waves), and during the third wave of the COVID-19 infection .The waves were defined as the period during which COVID-19 weekly incidence in the Free State was greater than 30 cases per 100,000 persons until the weekly incidence was equal to/or below 30 cases per 100,000 persons  35]..35].Between December 2020 and September 2021, a total of 149 children with SARI were recruited. Of these, a total of 84 children (n = 28 per waves and period between) whose samples were collected during the weeks of the COVID-19 waves (second and third waves) and period in between the waves were included in this study. These participants were selected to have an even distribution during the periods. The WHO case definitions for SARI were implemented as children admitted with ARI, with a measured fever ≥38 °C and cough, with onset within the last 10 days. Before admission, a structured questionnaire was used to collect patient demographics and specific clinical signs/symptoms.Upon admission, nasopharyngeal and oropharyngeal swabs  were collected per child by qualified medical personnel. The swabs were inserted in a viral transport media (VTM) . Samples were labelled and transported to the University of the Free State-Next Generation Sequencing (UFS-NGS) Unit, Division of Virology, Bloemfontein, Free State, South Africa, via cold chain transportation. Samples were registered and given a unique identification number before storage at −80 °C until processing.Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae [Pathogen detection was performed using QIAstat-Dx Respiratory SARS-CoV-2 Panel . The panel detects, generates cycle threshold (Ct) value, and differentiates nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the following organism types and subtypes: adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, SARS-CoV-2, human metapneumovirus A+B (HMPV), influenza A, influenza A H1, influenza A H3, influenza A H1N1/pdm09, influenza B, PIV 1, PIV 2, PIV 3, PIV 4, rhinovirus/enterovirus, RSV A+B, eumoniae . The panRaw data were calculated using Microsoft Excel 365 version 2206, and results are presented as simple percentages, mean, and standard deviations of replicate determinations.Ethical approval was received from the University of the Free State (UFS) Health Science Research Ethics Committee (UFS-HSREC) with the ethical approval number UFS-HSD2019/1129/2910. Further approval to perform the study was received from the Free State Department of Health, Bloemfontein, South Africa. An information document with a clear description of the study in the language of choice was provided for the parent/guardian. Permission and consent in the language of choice was also requested from the parent/guardian for their children to participate in the study.Of the participants enrolled, a total of n = 28 were recruited during each of the three periods investigated. Demographically, 34 (40.4%) were females, while 50 (59.6%) were males. The age range of participants was from 0 to 60 months, with most patients  aged 0–24 months. Of the participants, 35 (41.6%) required oxygenation, and 17 (20%) had human immunodeficiency virus (HIV), 24 (28.5%) had chest indrawing, 9 (10.7%) had feeding difficulty, 3 (3.5%) were asthmatic, and 0 (0%) required the need for intensive care unit.Of the 21 pathogens targeted by the QIAstat-Dx Respiratory SARS-CoV-2 Panel, across all participants, 8/21 viral pathogens were detected. Of the 84 participants recruited, n = 74 (88%) tested positive for at least one viral pathogen, and n = 10 (12%) cases had no targeted pathogens detected. Of the detected viral pathogens, HRV/enterovirus (EVs) (n = 46 (30%)), RSV A+B (n = 40 (26%)), and SARS-CoV-2 (n = 36 (23.3%)) were the most detected pathogens . Other vIn the 20 non-co-infected samples, detected pathogens were RSV (n = 8), SARS-CoV-2 (n = 5), HRV/EVs (n = 5), Adv (n = 1), and HMPV (n = 1) . Of the The severity of additional clinical symptoms is not directly associated with higher number of co-infecting viruses, as shown in Of the 28 children included during each period , a total of n = 26, n = 26, and n = 22 children tested positive for at least one viral pathogen, respectively, and no targeted pathogens were detected in n = 2, n = 2, and n = 6 children, respectively. The profile of pathogens detected during each period investigated are depicted in Globally, viral aetiology of SARI varies between 2 and 80% ,40,41,42Similar to other studies having cases of SARI/ARI with no targeted pathogens detected ,40,41,44The unprecedented COVID-19 disease, caused by SARS-CoV-2, has become a global pandemic with severe health consequences . SeveralNotably, fewer viral pathogens were detected during the third wave of the COVID-19 infection, and this could be due to stricter adherence to the implemented COVID-19 prevention strategies (NPIs) during the period between second and third waves, which may have resulted in low pockets of viral circulation during the third wave of infection. The unchanged detection rate for HRV is not surprising, as HRV is often detected throughout the year in South Africa ,52,53,54Whilst children have been proposed to be less susceptible to COVID-19 infection, it does not negate that they can still become infected. This notion was substantiated in the present study, with the SARS-CoV-2 virus detected in 36/84 patients (31 presented as co-infections and 5 presented as single infections), all of whom required hospitalisation. Moreover, some studies have reported possible transmission from children ,66. In eAs previously shown, mRT-PCR methods enable sensitive detection of viral co-infections ,67,68. IFurthermore, children with HIV have been reported to have a higher frequency and worse prognosis with viral respiratory infections ,78. NotaSome limitations of this study should be acknowledged. Firstly, due to this study’s restriction to nasopharyngeal/oropharyngeal samples, it is possible that it overlooked a relationship between lower airway carriage of bacteria and SARI hospitalisation. Another limitation is that the length of hospital stay and details of death, if any, were not recorded for this study. Therefore, the consequences of co-infection, the effect of asthma, the requirement for oxygenation, or HIV infection on the clinical course/outcome could not be established. This study was also limited by sample size due to the limited number of available QIAstat-Dx cartridge for diagnosis. However, it has provided baseline data on the spectrum of viruses circulating in children ≤5 years during the different waves of COVID-19 infection in the country while simultaneously highlighting viruses as a significant contributor to SARI morbidity in the study setting.It is becoming more crucial to make a prompt and accurate identification of respiratory infections in clinical settings in order to prevent the spread of virulent or resistant microorganisms ,86, espe


Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology with diverse clinical and laboratory manifestations, including thrombocytopenia. About 25% of patients with SLE may be affected by thrombocytopenia, many of whom are asymptomatic. Some patients, however, experience platelet counts that drop quite low and predispose them to bleeding. Thrombotic thrombocytopenic purpura (TTP) is defined with a classic pentad of clinical features, such as thrombocytopenia, microangiopathic hemolytic anemia, neurological symptoms and signs, renal symptoms and signs, and fever. The association of TTP and SLE has been sporadically reported in the literature.We describe a 16-year-old girl with SLE and immune thrombocytopenia, in whom TTP was diagnosed.She was treated with pulse methylprednisolone, whose platelet counts normalized after therapy with plasmapheresis and an anti-CD20 monoclonal antibody (rituximab).A pediatric patient with SLE and immune thrombocytopenia in whom TTP developed was treated with plasmapheresis and rituximab therapy successfully, though the patient experienced a disease relapsed after 18 months, which was controlled by the same management. Congenital TTP is caused by an inherited deficiency of ADAMTS13 and acquired TTP is caused by autoantibodies against ADAMTS13. Uncleaved, large, VWF multimers formed due to a defect in ADAMTS13 leads to spontaneous platelet aggregates in the microvasculature of the brain, heart, and kidneys. Acquired TTP, also called as immune-mediated TTP, can occur without any precipitating cause, or secondary to other autoimmune diseases including systemic lupus erythematosus (SLE).Thrombotic thrombocytopenic (TTP) is a rare and life-threatening disorder characterized by microangiopathic hemolytic anemia with thrombocytopenia, fever, renal changes, and neurological symptoms.,2 While es TTP.3, Congenit Clinical manifestations derive from the involved organs, including the central nervous and gastrointestinal systems. Neurologic symptoms include difficulty speaking, numbness, weakness, seizure, and mental state changes, whereas gastrointestinal symptoms include abdominal pain, diarrhea, nausea, and vomiting. With such clinical features, it is important to confirm a defect in ADAMTS13 activity in order to diagnose TTP.The identification of microangiopathic hemolytic anemia and thrombocytopenia without alternative etiology are essential in the diagnosis of TTP.,7 Clinic TTP rarely develops in patients with SLE, though, and outcomes are known to be similar to those of primary immune TTP.SLE is an autoimmune disease in which organs, tissues, and cells undergo damage mediated by tissue-binding autoantibodies and immune complexes. Hematologic manifestations are common in SLE; leukopenia occurs in 50% to 60%, and thrombocytopenia occurs in 25% of patients with SLE.,10 TTP rmune TTP.,12We describe a pediatric patient with SLE who manifested TTP, was treated successfully with plasmapheresis and rituximab, and experienced a TTP relapse after 18 months that remitted after the same management.2A 16-year-old girl was admitted for whole body petechiae that began 2 days prior to admission, and headache, dizziness, and left arm and leg weakness that began that day. Relevant past medical history included presenting to the pediatric outpatient clinic with whole body petechiae and thrombocytopenia one year prior in September 2018. She was referred to rheumatology for leukopenia (2500/μL), thrombocytopenia , positive anti-nuclear antibody , a positive anti-double stranded (ds)DNA antibody (25.8 IU/mL), hypocomplementemia , alopecia, and malar rash. She was diagnosed with SLE. After treatment with hydroxychloroquine and a low dose steroid (6 mg methylprednisolone), her symptoms resolved, but whole body petechiae and menorrhagia recurred in April 2019, and severe thrombocytopenia (8000/μ) developed without anemia (Hb 12.4 g/dL) or leukopenia (3500/μL). A bone marrow biopsy was done and showed normocellular marrow. She recovered with high dose steroid and intravenous immunoglobulin treatments.On the admission day (June 2019), the patient was acutely ill-looking but had no fever. She was normotensive (BP 102/52 mm Hg) and her pulse rate was 99/minute. Bilateral leg petechiae was noted. There was no cervical lymphadenopathy or hepatosplenomegaly. She did not have joint pains, oral ulcers, malar rash, or photosensitivity. Laboratory studies at this time showed a hemoglobin level of 7.9 g/dL, a white blood cell count of 10,800/uL, a platelet count of 8000/uL, erythrocyte sedimentation rate of 29 mm/h, C-reactive protein 0.13 mg/dL, and total bilirubin of 4.8 mg/dL. Urinalysis revealed one red blood cell per high-power field and negative proteinuria. C3 and C4 levels were 82 mg/dL and 6 mg/dL, respectively. Anti-double stranded DNA antibody was in the normal range. Brain magnetic resonance imaging did not show any parenchymal lesion or perfusion abnormalities. Peripheral blood smear indicated anisocytosis, poikilocytosis, and the presence of schistocytes, and ADAMTS13 activity was low at 16%.In view of TTP with low SLE activity, hydroxychloroquine, methylprednisolone 1 g pulse, IVIG 400 mg/kg for 5 days and plasmapheresis for 5 days were prescribed. The patient's platelet count increased to 231,000/uL. Four cycles of rituximab were administered weekly with high dose glucocorticoids. After 4 cycles of rituximab therapy, her platelet count had increased to 305,000/uL. Therefore, the glucocorticoids dose was reduced gradually with the addition of tacrolimus 2 mg. The platelet count increased to 337,000/uL 2 months later. After 1 year and 2 months, the patient developed headache with severe thrombocytopenia at 10,000/uL. ADAMTS activity was 0.5%. Four cycles of rituximab were administered weekly with plasmapheresis for 5 days. Thrombocytopenia recovered to 298,000/uL. Presently, she takes low dose prednisolone (daily 5 mg) and hydroxychloroquine (daily 300 mg) and her platelet count is maintained at more than 200,000/uL. Table 3This patient was admitted for SLE-related immune thrombocytopenia twice before her first TTP episode. At the third event, her platelet counts were similar, but her serum total bilirubin was elevated, and neurologic symptoms developed, which was different with the previous episodes. With no abnormal findings on brain imaging, confirmation of microangiopathic hemolytic anemia on a peripheral blood smear, and plasma exchange immediately conducted, this patient achieved rapid improvement in her mental status. In addition, rituximab therapy followed, resulting in overall improvement without any sequelae or recurrence.A review of 105 cases of SLE and TTP showed that the mortality was 12.4%, with infection and renal damage contributing to the poor outcomes in TTP-associated SLE.In a comparison of outcomes of immune-mediated TTP between patients with SLE (n = 8) and those without TTP (n = 10), mortality rates were higher, with a longer duration of treatment before remission, in patients with SLE compared in patients without SLE. Inhibition of B cell proliferation and suppression of autoantibody production by anti-CD20 antibodies was revealed to be effective in controlling acute flare-ups of SLE, including lupus nephritis and severe thrombocytopenia.[20–] Moreover, rituximab therapy that blocks anti-ADAMTS13 antibody formation has been found to be effective in acquired TTP.Rituximab is a chimeric monoclonal antibody against the CD20 antigen present on B cells, leading to B cell depletion.,19 Inhibpenia.20– MoreoverOne case report showed that a 34–year-old female presented with syncope and skin rash with hemolytic anemia and thrombocytopenia was diagnosed TTP and SLE simultaneously.In summary, we describe a patient with SLE and immune thrombocytopenia in whom TTP developed, and was treated with plasmapheresis and rituximab therapy successfully, though the patient experienced a disease relapsed after 18 months, which was controlled by the same management.Conceptualization: Ju-Yang Jung, Hyoun-Ah Kim.Data curation: Ju-Yang Jung, Ji-Won Kim, Hyoun-Ah Kim.Formal analysis: Hyoun-Ah Kim.Investigation: Ju-Yang Jung.Methodology: Ju-Yang Jung.Supervision: Chang-Hee Suh, Hyoun-Ah Kim.Visualization: Ju-Yang Jung, Ji-Won Kim, Hyoun-Ah Kim.Writing – original draft: Ju-Yang Jung, Hyoun-Ah Kim.Writing – review & editing: Ji-Won Kim, Chang-Hee Suh, Hyoun-Ah Kim.