The emergence of multidrug-resistant pathogens and the lack of new antimicrobial drugs is a major public health concern that needs urgent and innovative solutions. Endophytic fungi living in unique niches such as in endosymbiosis with plants are increasingly drawing attention as alternative sources of novel and chemically diverse compounds with unique mechanisms of action.Sclerocarya birrea were screened for bioactivity against a panel of indicator bacteria. Three bioactive endophytic fungi  were selected and identified through ITS-rDNA sequencing. The whole broth extracts of the three selected isolates were further screened against contemporary drug-resistant bacterial pathogens. This was followed by partial purification by solid phase extraction and GC–MS analysis of bioactive fractions.In the present study, ten endophytic fungi isolated from the medicinal plant, Alternaria alternata species (strains P02PL2 and P02MS1) and Nigrospora oryzae (strain P02MS2A). The whole broth extracts from N. oryzae P02MS2A exhibited a MIC of one μg/mL and 16 μg/mL against gram-negative, MDR Pseudomonas 5625574 and gram-positive MRSA 25775 clinical isolates, respectively. After partial purification and GC–MS analysis of whole broth extract from A. alternaria PO2MS1, 2-fluorobenzoic acid heptadecyl was putatively identified as the active compound in fraction C of this extract. This compound was also putatively identified in fraction E of A. alternata P02PL2, fraction B of A. alternata P02MS1 and fraction B of N. oryzae P02MS2A, and interestingly, all these fractions retained activity against the two MDR clinical isolates.The bioactive endophytic fungi were identified as The putative identification of 2-fluorobenzoic acid heptadecyl compound showing a broad-spectrum of activity, more especially against gram-negative MDR contemporary pathogens is highly encouraging in the initiative at developing novel drugs to combat multi-drug resistance.The online version contains supplementary material available at 10.1186/s12906-023-04157-8. The increasing problem of antimicrobial resistance (AMR) coupled with the emergence of new pathogens has been identified by the World Health Organisation (WHO) as one of the top ten global priorities that need urgent attention . Over thTo ensure a steady flow of new antimicrobial drug candidates into the clinical pipeline, there are increasing research efforts on hit identification and hit-to-lead optimization programs . To do tEndophytic fungi are microorganisms that colonize and proliferate in the inner plant tissues without causing any apparent harm . InsteadStaphylococcus aureus ATCC 25923, S. aureus CS , methicillin resistant S. aureus (MRSA) 25775, and multidrug resistant (MDR) Pseudomonas 5625574).More recently, an increasing interest in exploring endophytic fungi has been sparked by the discovery that they can produce the same or similar compounds to their plant hosts. In this way, the ethnobotanical knowledge of medicinal plants provides a promising lead to isolate fungi with interesting properties . This alSclerocarya birrea. This medicinal plant was collected from the South Coast region of the Durban Municipality, KwaZulu-Natal . The plant was sent to the University of KwaZulu-Natal (UKZN) School of Life Science Herbarium for taxonomic identification and registration (voucher number 18234). Isolation of endophytic fungi was done following a modified protocol described by Shara et al. pyrazine-1,4-dione, hexahydro-3-  , that haic acid) , anti-inic acid) , antibacic acid) , antifunic acid) , and antic acid) .A. alternata P02MS1  and gram-negative (MDR Pseudomonas) pathogens listed in the WHO priority list of pathogens of interest with the potential to escape existing currently available antimicrobial regimen. It would be interesting to understand the mechanism of action of this compound, its toxicity in human cell lines, and to link the biosynthetic pathway responsible for its synthesis in the fungal genome.In this study, all Fractions from  N. oryzae P02MS2A against gram-negative multidrug-resistant (MDR) pseudomonas 5625574 and gram-positive MRSA 5627679 indicate that the active compound in the crude extract is highly effective with broad-spectrum antimicrobial properties. The activity observed against contemporary gram-negative multidrug-resistant clinical isolate is encouraging since there has been slow progress for production of antibiotics active against gram-negative pathogens has been launched in the past decades [In this study, three endophytic fungal isolates showed a diverse chemical profile with diverse compounds some of which have no previously reported biological activities and might represent some novel activities and mechanisms that were not captured by the employed bioactivity assays in this study. The lower MICs exhibited by whole broth extract of decades , 71. SinAll procedures were conducted in accordance to the guidelines and the study was approved by the UKZN Biomedical research ethics committee, reference number: BREC/00002127/2020. The ethics committee of the University of KwaZulu Natal permitted the use of the plant specimen for research purposes.Additional file 1: Figure S1. Optimization of antibacterial activity production from whole broth crude extract of endophytic fungi isolated from S. birrea. (A) antimicrobial activity against S. aureus(ATCC25923) and (B) antimicrobial activity against S. aureus(C.S) for 20 days. The controls included control 1 (fungi-free MEB crude extract) and control 2 (0.2% DMSO). Figure S2. GC-MS chromatogram illustration of compounds detected contained on Fraction B of A. alternata P02PL2 whole broth extract. Figure S3. GC-MS chromatogram illustration of compounds detected contained on Fraction E of A. alternata P02PL2 whole broth extract. Figure S4. GC-MS chromatogram illustration of compounds detected contained on Fraction B of A. alternata P02MS1 whole broth extract. Figure S5. GC-MS chromatogram illustration of compounds detected contained on Fraction B of N. oryzae P02MS2A whole broth extract.Additional file 2: Table S1. Antibacterial activity of whole broth crude extracts from selected endophytic fungi. Table S2. Putative compounds detected by GC-MS on bioactive Fraction B of A. alternata P02PL2 whole broth extract. Table S3. Putative compounds detected by GC-MS on bioactive Fraction E of A. alternata P02PL2 whole broth extract. Table S4. Putative compounds detected by GC-MS on bioactive Fraction B of A. alternata P02MS1 whole broth extract. Table S5. Putative compounds detected by GC-MS on bioactive Fraction C of A. alternata P02MS1 whole broth extract. Table S6. Putative compounds detected by GC-MS on bioactive Fraction B of N. oryzae P02MS2A whole broth extract.


O-β-glucoside (C3G) is the most important component and is widely distributed in various colored edible plant materials, especially berries, cherries, black rice, purple corn, etc. In recent decades, several studies have reported the therapeutical properties of C3G. C3G has various biological properties and health benefits, such as antioxidant, antimicrobial, anti-inflammatory, antidiabetic, anti-obesity, neuroprotective, anticancer, etc. In this review, we summarized the in vitro and in vivo studies in relation to the role of C3G in obesity-related complications. Several mechanistic studies demonstrated that C3G maintains the metabolism of glucose, fatty acids, and lipids by regulating different genes and signaling pathways. It could be concluded that the consumption of C3G protects healthy individuals from obesity-related issues by maintaining body weight and regulating their metabolism and energy balance. This review provides some important signaling pathways/targets of C3G to facilitate the prevention and treatment of obesity, leading to the development of important food supplements.Obesity has become a major health issue worldwide and obese individuals possess higher levels of adipose tissue when compared with healthy individuals. Obesity is highly associated with the development of different chronic diseases, such as diabetes, cardiovascular diseases, hypertension, cancers, etc. Previous studies established that anthocyanin compounds play an important role in attenuating obesity-related consequences. Among various anthocyanin compounds, cyanidin-3- Obesity is one of the most important health problems worldwide with considerable illness and mortality. According to the WHO, more than 2.8 million deaths occur every year due to obesity-related consequences. In 2016, approx. 1.9 billion adults were obese . GeneticAnthocyanins are widely found in colored fruits and vegetables. . They arO-β-glucoside (C3G) is an important biologically active component . They are formed by a flavylium cation backbone that is hydroxylated in different positions to produce diverse anthocyanidins . Cyanidia proton . In plana proton . The maia proton ,34.The excessive consumption of energy-rich foods and an inactive lifestyle are believed to be the main causes of obesity-related insulin resistance and abnormal glucose metabolism. Scientific studies confirmed that anthocyanins may lower the metabolic risks linked with obesity due to their potent antioxidant and anti-inflammatory properties. The dysfunction of adipocytes is highly correlated with the onset of obesity and insulin resistance. It is believed that obesity and the improvement of insulin sensitivity can be prevented by regulating the secretion of adipocytokine and the expression of adipocyte-specific genes. An excessive amount of circulating free fatty acids has been proven to be potentially associated with obesity, insulin resistance, and diabetes. In this context, lipolysis inhibition is the main target for reducing free fatty acids and improving insulin sensitivity. A study reported that C3G effectively suppressed the release of free fatty acids and glycerol from 3T3-L1 adipocytes during hyperglycemia. C3G treatment downregulated the hexosamine biosynthetic pathway by increasing the AMP-activated protein kinase activity, decreasing the glutamine:fructose 6-phosphate aminotransferase activity (GFAT), and reducing the production of cellular UDP-N-acetylglucosamine. Furthermore, C3G downregulated the adipose triglyceride lipase expression by attenuating the forkhead box O1 (FoxO1) transcription factor . C3G effC3G markedly inhibited basal adipocyte lipolysis in differentiated human fat cells. A mixture of docosahexaenoic acid and C3G reduced the tumor necrosis factor-α (TNF-α) secretion and enhanced insulin-induced lipogenesis . MatsukaOne of the promising approaches for treating obesity is to convert white adipocytes into brown-like adipocytes. In 3T3-L1 adipocytes, C3G stimulated phenotypic modifications into white adipocytes, such as higher levels of multilocular lipid droplets and mitochondrial content. In addition, C3G treatment increased the expression of the mitochondrial genes in 3T3-L1. Moreover, C3G improved the differentiation of preadipocytes by activating the CCAAT/enhancer-binding protein β (C/EBPβ) via increasing the level of intracellular cyclic adenosine 3′5′-monophosphate (cAMP) . RecentlA study indicated that C3G treatment significantly regulated protein kinase B (Akt) phosphorylation, the sensitivity of adipocytes to palmitic acid, GLUT-1 and GLUT-4 glucose transporters, and hexokinase-II in palmitic acid-induced human SGBS adipocytes. In addition, the cells pretreated with C3G exhibited a significant reduction in the mRNA levels of pro-inflammatory cytokines -6, IL-8, and monocyte chemoattractant protein-1 (MCP-1)) in palmitic acid-induced human SGBS adipocytes . C3G mar2O2- or TNF-α via the downregulation of the c-Jun N-terminal kinases (JNK) signaling pathway [In 3T3-L1 adipocytes, a study found that C3G treatment effectively reverted the insulin resistance induced by H pathway . C3G and pathway . In macr pathway . Further pathway .2+ was increased in the cyanidin-stimulated cells [Further, AMPK plays a key role in preventing hepatic lipid metabolism by regulating the downstream acetyl CoA carboxylase and carnitine palmitoyl transferase 1 pathways. A study indicated that C3G regulated hepatic lipid homeostasis through an AMPK-dependent signaling pathway . In 3T3-ed cells . Cyanidied cells . In meseed cells . Zhang eed cells  investiged cells .Saulite et al.  investig50 values of 188.28 µM [50 at 59.4 μM). Further, C3R showed a considerable reduction in the cholesterol uptake in free cholesterol as well as mixed micelles in addition to the suppression of the NPC1L1 expression in Caco-2 cells [Aronia melanocarpa exhibited the strongest inhibitory activity against α-amylase and lipase. However, cyanidin 3-galactoside, cyanidin-3-arabinoside, and cyanidin 3-xyloside did not show any effect on the α-amylase and lipase enzymes. Further, Xie et al. [Pancreatic lipase is one of the most essential digestive enzymes for metabolism as well as for the absorption of triglycerides to monoglycerides and free fatty acids. In the body, the level of total cholesterol is decreased when inhibiting the lipase enzyme activity . In this88.28 µM  and 0.2688.28 µM . A recen88.28 µM . In anot-2 cells . Chen et-2 cells  found the et al.  suggesteSimilar to the in vitro studies, numerous in vivo studies have been carried out to determine the effect of C3G on obesity-associated mechanisms . The C3GIt is well known that brown adipose tissue burns energy to generate heat. The previous studies found that C3G can improve the thermogenic ability of brown adipose tissue. Han et al.  evaluateThe combination of peptides and C3G markedly improved the glucose uptake with or without insulin. In addition, this combination suppressed the expression of the angiotensin II receptor, type 1 (AGTR-1), and activated the insulin receptor substrate 1 and GLUT4 expressions . In dietAspergillus oryzae inhibited lipid and glucose metabolism via the activation of hepatic insulin signaling and the AMPK pathways in OVX rats [Aronia melanocarpa extract inhibited adipogenesis by downregulating the expressions of the CCAAT/enhancer-binding protein, PPAR, sterol regulatory element-binding protein-1c, PPARγ coactivator-1, acetyl-CoA carboxylase 1, ATP-citrate lyase, fatty acid synthase, and adipocyte protein 2 [Aronia melanocarpa treatment expressively decreased the levels of TNF-α, IL-6, and IL-1β in the serum. Further, C3G improved HFD-induced obesity by activating AMPK and suppressed HFD-induced inflammation by activating the phosphorylation of STAT3 and suppressing NF-κB p65 in the nucleus [Titta et al.  demonstrOVX rats . A studyOVX rats . BlackbeOVX rats . A C3G-erotein 2 . The antrotein 2 . In HF-d nucleus .Anthocyanins, such as C3G, C3R, and pelargonidin-3-glucoside, isolated from Chinese mulberry significantly inhibited body weight gain, reduced insulin resistance, lowered the size of adipocytes, and attenuated the accumulation of lipids and the secretion of leptin . The supO-glucoside, interacted with planar lipid membranes and formed conductive units.The improvement in the thermogenesis of brown adipose tissue is an efficient strategy for alleviating obesity. A study exhibited that C3G improved the energy expenditure and thermogenesis of brown adipose tissue in mice induced with HF diet obesity. Additionally, C3G upregulated the UCP1 expression as well as other thermogenic genes in inguinal white adipose tissue and brown adipose tissue . In db/dC3G is an important anthocyanin compound and is widely found in various fruits and vegetables. Previous studies established that C3G supplementation offers numerous health benefits to the consumer. In this review, we discussed the effect of C3G treatment on obesity-associated mechanisms under different in vitro and in vivo models. The mechanisms preventing obesity highly depend on their capacity to control the consumption of food and energy metabolism and improve inflammatory response, insulin resistance, and glucose metabolism. In addition, C3G can effectively inhibit the pancreatic lipase enzyme. A variety of pathways and gene expressions play a crucial role in preventing obesity-related consequences. In particular, AMPK is an important pathway, attributed to the prevention of hepatic lipid metabolism, and PPARs play a key role in energy metabolism. The IRS-1/PI3K/Akt pathway and GLUT4 gene expression are mainly responsible for insulin sensitivity and the NF-κB signaling pathway is for inflammatory response. The published papers clearly showed that C3G could be a potential agent for facilitating the prevention and treatment of obesity-associated complications owing to its multi-targeted activities. Although C3G can effectively attenuate obesity-associated complications, further clinical studies leading to the development of C3G-enriched functional foods are required.


Chimeric antigen receptor (CAR)-T cell therapy has been used to treat pediatric refractory or relapsed mature B-cell non-Hodgkin lymphoma (r/r MB-NHL) with significantly improved outcomes, but a proportion of patients display no response or experience relapse after treatment. To investigate whether tumor-intrinsic somatic genetic alterations have an impact on CAR-T cell treatment, the genetic features and treatment outcomes of 89 children with MB-NHL were analyzed.89 pediatric patients treated at multiple clinical centers of the China Net Childhood Lymphoma (CNCL) were included in this study. Targeted next-generation sequencing for a panel of lymphoma-related genes was performed on tumor samples. Survival rates and relapse by genetic features and clinical factors were analyzed. Survival curves were calculated using a log-rank (Mantel-Cox) test. The Wilcox sum-rank test and Fisher’s exact test were applied to test for group differences.TP53 (66%), ID3 (55%), and ARID1A (31%). The incidence of ARID1A mutation and co-mutation of TP53 and ARID1A was high in patients with r/r MB-NHL . CAR-T cell treatment significantly improved survival in r/r MB-NHL patients (P = 0.00081), but patients with ARID1A or ARID1A and TP53 co-mutation had poor survival compared to those without such mutations.A total of 89 driver genes with somatic mutations were identified. The most frequently mutated genes were ARID1A or TP53 and ARID1A co-mutation are insensitive to initial conventional chemotherapy and subsequent CAR-T cell treatment. Examination of ARID1A and TP53 mutation status at baseline might have prognostic value, and risk-adapted or more effective therapies should be considered for patients with these high-risk genetic alterations.These results indicate that children with MB-NHL harboring The online version contains supplementary material available at 10.1186/s12935-023-03122-2. Mature B-cell non-Hodgkin lymphoma (MB-NHL) accounts for 50–60% of non-Hodgkin lymphomas (NHLs) in children and adolescents –3 and isChimeric antigen receptor (CAR)-T cell therapy is an emerging therapy that has been used to treat patients with r/r lymphoma, with significant improvement in clinical outcomes and an overall response rate of 52–82% –11. In oTherefore, we selected a cohort of 89 Chinese pediatric MB-NHL patients to analyze the relationship between mutation status and genetic characteristics and CAR-T cell therapy. We sought to understand the intrinsic molecular characteristics of r/r MB-NHL patients who are not sensitive to CAR-T cell therapy in an attempt to identify molecular predictors associated with this treatment. Identifying patients at high risk of relapse who may not benefit from immunochemotherapy and/or CAR-T cell therapy can provide a clinical basis for improving clinical management and treatment strategies for these lymphoma patients.A total of 89 patients treated at multiple clinical centers of the China Net Childhood Lymphoma (CNCL) from February 2019 to September 2021 were included in this retrospective study. Targeted next-generation sequencing (t-NGS) with a panel of lymphoma-related genes was performed on tumor samples  after first-line chemotherapy. Relapsed or refractory (r/r) was defined as disease that was refractory (never obtaining a CR) or relapsed after first-line chemotherapy and had a partial response (PR) or no response (NR) as the best response to at least 2 cycles of salvage chemotherapy. Poor prognostic outcome was defined as meeting at least one of the following criteria: deceased, unresponsive to treatment and/or disease relapse.t-NGS was used to detect the mutation status of 262 driver genes related to lymphoma to identify somatic mutations, including single-nucleotide variants (SNVs) and short insertions and deletions (InDels) in the patients. First, DNA was extracted from tumor tissue samples. The genomic DNA was fragmented to approximately 200 bp by enzyme digestion, and then end repair, adaptor ligation and PCR (polymerase chain reaction) were carried out to complete prelibrary construction. A complete set of probes provided by Agilent was used to capture the sequences of 262 genes , and the target fragments were enriched to generate the library. A NextSeq550 sequencer (Illumina) was used for 2*150 bp sequencing analysis. The human genome hg19 was used as a reference for sequence alignment and base identification. Mutation information was annotated by the information screening function based on public databases . The variant allele frequency (VAF) was calculated by the number of mutants reads over the number of total reads (Supplementary 1.6).p values of 0.05 or below were considered statistically significant.Statistical analyses were carried out with SPSS statistics v.25 and R software v3.6.2. Actuarial survival analysis was estimated by the Kaplan–Meier method, and survival curves were generated using a log-rank (Mantel‒Cox) test. The Wilcox sum-rank test and Fisher’s exact test were applied to test for group differences. Overall survival (OS) was calculated from the date of the last CAR-T cell infusion to the date of last follow-up or death, and disease-free survival (DFS) was defined only for patients who achieved CR and was calculated from the date of CR after the last CAR-T cell treatment to the date of relapse or death regardless of cause. Follow-up visits for MB-NHL cases are scheduled every 3 to 6 months for the first 2 years and once every 6 months after 5 years. All patients were followed up until 30 September 2021.The clinical data of the 89 patients are summarized in Table The median follow-up was 11.4 months (95% CI: 9.9–13.5 months). The one-year OS of r/r MB-NHL was 43.5% .The mutational profiles of 89 MB-NHL patients are presented in Supplemental Figure TP53 , followed by ID3 , ARID1A , CCND3 , DDX3X , and GNA13    and those with r/r MB-NHL (n = 48), it was found that the age of the latter was significantly higher than that of the former  Fig. . There aTP53 , ID3 , CCND3 , DDX3X , MYC , and GNA13 . In 48 r/r patients, the most commonly mutated gene was TP53 , followed by ID3 , ARID1A , CCND3 , DDX3X , and GNA13 . Compared with MB-NHL, TP53 (75% vs. 54%) and ARID1A (35% vs. 7%) showed a higher incidence of mutation than IR MB-NHL  were the main type for TP53. The TP53 R248 W/V/Q (4 vs. 2) mutation was more common in IR MB-NHL, but the TP53 R273H/C (8 vs. 3) mutation was more common in r/r MB-NHL patients. Patients with r/r MB-NHL showed a higher prevalence of frameshift and truncating mutations than IR MB-NHL, and these mutation patterns were the major pathogenic mutations found for ARID1A, leading to a loss of its function .Further analysis of the VAF showed a significantly higher percentage of VAF for Among the r/r MB-NHL patients, 40 were treated with CAR-T cell therapy. Twenty-two patients (55%) achieved CR and 12 patients (30%) achieved PR, with an Overall response rate (ORR) of 85% (34/40), according to the outcome assessment at the end of each CAR-T cell treatment.P = 0.00081). The results showed that CAR-T cell treatment significantly improved the outcome of r/r MB-NHL.The difference in r/r MB-NHL survival probability between patients treated with and without CAR-T cells was compared . It is worth noting that the median survival in patients treated with CAR-T cell therapy was more than one year , with a one-year survival rate of 52.6% and a two-year survival rate of more than 47.3% . In comparison, patients who did not receive CAR-T cell treatment had poor prognosis, with a median survival of less than one month , 25) and those who did not respond (CAR-T nonresponse patients (NR or PD), 15) to CAR-T cell therapy.ARID1A mutation (65% vs. 24%) and the VAF of the gene mutation were significantly higher in patients who did not respond to CAR-T cell therapy than in patients who did respond  than those who did respond . Survival analysis also showed that patients with TP53 mutations had a shorter median survival of 4.08 months (95% CI: 2.37-NA month) than those without TP53 mutations  with co-mutations of TP53 and ARID1A had a lower response rate to CAR-T cell treatment than the other groups. Only 5 of 14 patients showed a response,the overall response rate was only 36%  .Kaplan-Meier survival curves of the effects of ARID1A and TP53 co-mutation had poor outcome after treatment when compared to those without such mutations.Therefore, although CAR-T cell treatment significantly improved survival in r/r MB-NHL patients, patients with ARID1A mutations are associated with poor outcome after CAR-T cell therapy in children with r/r MB-NHL. At the same time, a significant finding was that co-mutation of ARID1A and TP53 in Chinese children with r/r MB-NHL is associated with insensitivity to chemotherapy and CAR-T cell treatment.The outcome of r/r MB-NHL in children is very poor , 24, 25.ARID1A is one of the most commonly mutated genes in cancers [ARID1A impacts transcription initiation and elongation [EZH2 methyltransferase activity, steroid receptor modulation and regulation of p53 targets [ARID1A has a crucial role in regulating gene expression that drives oncogenesis or tumor suppression and that deletion of ARID1A promotes tumor progression. In this study, the incidence of ARID1A was higher in r/r MB-NHL patients than in IR patients (35% vs. 7%), indicating that ARID1A mutations are enriched in resistant disease and that these patients might be insensitive to initial chemotherapy. A study in ovarian cancer showed that ARID1A alterations may also mediate resistance to platinum chemotherapy and estrogen receptor degraders/modulators [ARID1A deficiency may render patients insensitive to initial chemotherapy and be associated with relapse. Moreover, consistent with other studies, most of the ARID1A mutations we detected are classified as loss-of-function mutations, which were dispersed throughout the coding sequence [ARID1A gene function [ cancers –29. Exprongation , 31, par targets –34. Studpatients % vs. 7%,sequence . Most offunction , 38 and ARID1A mutations have lower rates of response and survival after CAR-T cell therapy. CAR-T cell exhaustion is partly attributed to prolonged exposure to the immunosuppressive microenvironment, upregulation of inhibitory receptors, and persistent CAR stimulation by antigen [ARID1A can modulate the tumor immune microenvironment, which underlies its correlation with sensitivity to immunotherapy [ARID1A improves T-cell persistence and that anti-ARID1A deficiency promotes mutability and potentiates therapeutic antitumor immunity, unleashing tumor immunity in vivo [ARID1A gene function and decreased T-cell persistence may be barriers to immune checkpoint blockade and the effectiveness of CAR-T cell immunotherapy [ARID1A mutation is associated with increased expression of PD-L1, which reduces the level of antitumor immune response and may promote immune escape of tumor cells [ARID1A is also reportedly closely related to DNA mismatch repair and microsatellite instability [ARID1A mutation. These conclusions support our view that ARID1A mutations are associated with poorer clinical benefit in patients treated with CAR-T cells. Furthermore, ARID1A alterations may be a risk factor for insensitivity to chemotherapy or CAR-T cell therapy, and patients should be monitored and regularly followed up.CAR-T cell therapy is a good option for pediatric patients with r/r MB-NHL ; however antigen , 40. Theotherapy . Julia A in vivo ; howeverotherapy . In addior cells , 46. ARItability , which iTP53 mutation and its association with drug resistance is well known in many malignancies [TP53 aberrations are valuable prognostic markers in CD19-CAR-T cell recipients [TP53 mutations showed a trend of adverse outcomes after CAR-T cell therapy, but without reaching a significant level; hence, further research is needed. Multiple studies in solid tumors have demonstrated an inverse relationship between TP53 and ARID1A mutations and even found ARID1A and TP53 mutual exclusivity in ovarian clear cell and uterine endometrioid carcinomas [ARID1A and TP53 in r/r MB-NHL that was associated with CAR-T cell insensitivity. ARID1A has been shown to interact with ligand-bound nuclear hormone receptors and TP53 through its C-terminal domain and to stimulate the transcriptional activity of these transcription factors [ARID1A loss probably affects multiple aspects of TP53-regulated chromatin and promotes squamous differentiation and acquisition of invasive properties [ARID1A and TP53, and their molecular mechanisms need to be further studied. In recent years, it has been shown that a high VAF load is associated with poor outcome in patients with hematologic tumors [The negative prognostic impact of gnancies , and somcipients . Nonethercinomas , 48. Rem factors , 49. A noperties . These mc tumors  and we aTP53 or ARID1A have been studied in the clinic and laboratory, including combined epigenetic inhibitors to form combined lethal targeted therapies and immune checkpoint inhibitor treatment [We have identified molecular abnormalities that may affect the outcome of CAR-T cell therapy in r/r MB-NHL patients, and there is an urgent need to find new solutions. In recent years, with the development of drugs and clinical research, target drugs related to reatment , 53. CARreatment . In addiARID1A in children with r/r MB-NHL treated with CAR-T cells, the sample size was limited. Validation using larger cohorts and prospective trials are warranted to guide CAR-T cell product selection.This study has several limitations. First, sequencing of tumor samples was driven by clinical decision-making, potentially leading to selection bias. However, we note that population features and outcomes were similar between patients who underwent sequencing and those who did not. Finally, to our knowledge, although this is the first analysis evaluating the role of ARID1A mutations are common in pediatric MB-NHL. We demonstrated that children with MB-NHL harboring ARID1A or TP53 and ARID1A co-mutation were insensitive to initial conventional chemotherapy and subsequent CAR-T treatment and were associated with disease progression or relapse. Our data suggest that ARID1A and TP53 mutation status should be considered when prognostic factors are evaluated before CAR-T treatment, and this finding would help clinicians customize tailored treatments for their patients.In summary, our study analyzed the genetic features and treatment outcomes of 89 children with MB-NHL, and Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons’ stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity. The directions of visual motion are encoded in the retina by direction-selective ganglion cells. This study shows that the direction-selective J-RGCs in the mouse retina receive spatially asymmetric inputs from starburst amacrine cells even though their dendritic arbors show little overlap. Most of the synaptic connections in the vertebrate retina are located in the inner plexiform layer (IPL). The IPL is organized into functionally distinct sub-laminas and the neural processes of retinal ganglion cells (RGCs), amacrine cells and bipolar cells often stratify in restricted sub-laminas . It is bMotion detection is a fundamental task of the visual system. This process starts in the retina ,3. DSGCsHow synaptic inputs contribute to the direction selectivity of DSGCs is a fundamental and important issue ,3,5. TheJ-RGCs are OFF DSGCs in the mouse retina . They haWe set out to uncover this source by functionally dissecting J-RGCs’ circuit but arrived at a surprising conclusion that the direction selectivity of J-RGCs also depends heavily on the inhibitory inputs from SACs despite their minimal co-stratification in the IPL. Asymmetric connections between J-RGCs and SACs were also observed. Our results identify the synaptic inputs and presynaptic partners of J-RGCs and highlight the critical role of SACs in the emergence of direction selectivity in the mouse retina. The noncanonical connections between J-RGCs and SACs, 2 narrowly stratified neurons in different sub-laminas, also underscore that care should be given when looking for connections between retinal neurons by matching their stratification patterns.Most J-RGCs have highly asymmetric dendritic arbors. Their PD is the dorsal-to-ventral direction on the retina, which is the same as the soma-to-dendrite direction for J-RGCs . Since JThe excitatory inputs to J-RGCs are from the OFF pathway, and the inhibitory inputs include both ON and OFF components . We usedStrategies for the generation of direction selectivity often involve spatial and temporal offsets in different synaptic inputs ,20. We tWe also recorded EPSCs during a white noise stimulus composed of randomly flickering bars and obtained the excitatory receptive field (RF) by reverse correlation. A similar space-time slant was observed , left. UWe next examined whether specific types of synaptic transmissions were involved in the generation of this space-time slant in the excitatory RF. Pharmacological blockade of inhibitory inputs with picrotoxin  and strychnine , N-Methyl-D-aspartic acid (NMDA) inputs with DL-2-Amino-5-phosphonopentanoic acid (APV) or cholinergic inputs with hexamethonium (HEX) in the retina had no effects Figs  and S2A.The spatiotemporal offset revealed in the asymmetric structure of J-RGCs’ RFs was reported to be associated with their direction selectivity . Notablym) was when only the neuron’s excitatory or inhibitory inputs were direction-selective (start) of the cell was between the equilibrium potentials for the inhibitory and excitatory inputs (Einh and Eexc), direction selectivity of ΔVm could be observed -2-glycine (DCG-IV) first. PTX blocks the GABAergic inhibitory pathways including those of SACs’. DCG-IV is a type 2 metabotropic glutamate receptor agonist, which specifically blocks all the outputs of SACs in the retina [We tested this hypothesis pharmacologically using PTX and  in SACs ,34–37. TWe then activated ChR2 in JamB-CreER/ChAT-Cre/Ai32 retinas under the same drug cocktail. Significant IPSCs could be reliably recorded from J-RGCs when ChR2 was activated in SACs. No IPSCs were observed in control retinas where ChR2 was not expressed in SACs . Moreoven = 4 cells) corresponds well with the latency of the IPSCs recorded in J-RGCs  and their difference is within the range of monosynaptic transmission [The drug cocktail blocked all the non-GABAergic transmissions, therefore, the connections between J-RGCs and SACs are either direct (monosynaptic) or via serial GABAergic connections. Under our experimental setup, the latency of the optogenetically evoked photocurrents recorded in SACs  and were performed according to the Guideline for Ethical Review of Animal Welfare (GB/T 35892–2018) and the General Requirements for Animal Experiments (GB/T 35823–2018) of the People’s Republic of China.f/+ and Ai9/Cx36f/+ were crossed together (Cx36f/f from EMMA # 02510). Tamoxifen  was injected intraperitoneally 3 times at P1/2/4 for the induction of CreER. Both male and female adult mice (1 to 6 months old) were used in our study.Mice were housed in the 12/12 h light/dark specific-pathogen-free environment. JamB-CreER mice were kindly provided by Joshua Sanes. They were crossed with the reporter line Ai9 (Jackson Lab # 007909) for targeted recording. In the optogenetic experiments, triple positive mice of JamB-CreER, ChAT-Cre (Jackson Lab # 006410) and Ai32 (Jackson Lab # 024109) mice were used. To remove Cx36 selectively in J-RGCs, JamB-CreER/Cx362, 2.5 mM KCl, 1.6 mM MgCl2, 22 mM NaHCO3, 10 mM glucose) at room temperature. The dorsal halves of the retinas were used for recordings. For cell-attached recordings, both the extracellular and pipette solutions were the Ringer’s solution. For whole-cell recordings, pipettes were filled with an intracellular solution  and the retinas were superfused with artificial cerebrospinal fluid . To isolate the excitatory and inhibitory synaptic inputs, cells were held at the reversal potential of inhibition (−67 mV) and excitation (0 mV). Series resistance was not compensated and only cells with series resistances less than 20 MΩ were used. Signals were acquired with MultiClamp 700B amplifier (Molecular Devices), digitized at 10 kHz and low-pass filtered at 1 kHz.Mice were dark-adapted for over 2 h before sacrificed under dim red light. The retinas were isolated under infrared light and then put into oxygenated Ringer’s solution . The JamB-CreER/ChAT-Cre/Ai32 retinas were carefully characterized first. Cre-mediated expression could be induced occasionally in some bipolar and non-SAC amacrine cells, possibly due to weak expression of JamB-CreER in these cells. Whole-cell recordings were performed only from the retinas without non-SAC amacrine cells labeled. All the EYFP positive amacrine cells in the recorded retinas were confirmed to be ChAT-positive SACs by postrecording immunostaining.2 within a spot of about 108 μm diameter, the location of which was positioned by the computer-controlled stage; 12.5 μm PTX (Tocris), 0.25 μm STR, 10 μm DCG-IV (Tocris), 300 μm HEX, 50 μm APV, 10 μm CNQX, and 1 μm TTX  were used for the pharmacological experiments. All drugs were purchased from Sigma except where noted.ChR2 was activated using a high-power LED  through the objective lens with the mean light intensity of 2.4 mW/mm4 photons/s/μm2 at 500 nm; M cone, 6.7 × 104 photons/s/μm2 at 511 nm; S cone, 3.9 × 102 photons/s/μm2 at 370 nm. Illuminance levels were modulated in Figs Visual stimuli, generated with a custom program and delivered from a computer-driven Acer K130 projector, were focused onto the photoreceptors through the custom-made microscope condenser at a scale of 8 μm/pixel and a frame rate of 60 Hz. White light was used, and the average intensity for all the stimuli under the neutral density filter (Thorlabs) with optical density at 3.0 was equivalent to the following photon flux values for the 3 mouse photoreceptors: rod, 5.9 × 10The RF centers of the recorded J-RGCs were determined by a manually controlled small flashing spot near the somas and were used to center all the subsequent stimuli. To measure the direction selectivity, a square black dot (160 × 160 μm) moving at 800 μm/s  and the resting conductance Grest (3.60 nS) were the average values from 8 recorded J-RGCs. Ge and Gi from the same 8 J-RGCs were calculated as describe previously [e, PD Gi, ND Ge, and ND Gi. Gaussian noise and temporal offset matching the actual level of the noise in the recorded conductance were then added and the results were taken as the input Ge and Gi to the equation. Different resting membrane potential (Vstart) values (from −65 mV to −40 mV at a step of 5 mV) were tested.To simulate the membrane potential responses to motion under different conditions, a conductance-based integration model  was usedeviously , alignedThe vector sum magnitude, the spiking and excitatory RF, and the simulation of the excitatory responses with a linear model were computed and performed as described previously ,30. SpacPD and PeakND denote the peak responses  to PD and ND motion, respectively.DSI was computed as below:To quantify the contributions of individual component flashes to the apparent motion stimulus, multiple linear regression was performed to fit the response to apparent motion using the responses to each flash.t test (two-sided). All the statistical details can be found in the figure legends, including the exact meaning and value of n. Statistical significance was defined at p < 0.05.No statistical method was used to predetermine sample size. All data are reported as the mean ± standard error of mean (SEM). Data distribution was in general assumed to be normal and not formally tested; in the figures, data points are shown. Data sets were compared using 1 sample, paired or unpaired S1 Fig(A) Schematic diagram of the flash bar stimulus and representative IPSC traces recorded from the same J-RGC in (B) The spatiotemporal profile for the ON inhibitory conductance (Gi) responses. Similar to (PDF)Click here for additional data file.S2 Fig(A) Representative excitatory RFs after bath application of PTX + STR, APV, HEX, and TTX. Scale bar, 200 μm. (B) Representative EPSCs during motion stimulus from a J-RGC in JamB-CreER/Ai9/Cx36f/f mice (right) and a J-RGC in the control JamB-CreER/Ai9/Cx36f/+ mice (left). Traces are aligned to the estimated time when the leading edge of the moving spot entered the RF center (dotted line). Shaded area around the traces, mean ± SEM, n = 16 trials. (C and D) Comparison of EPSC amplitudes (C) and DSI values (D) after removing Cx36 selectively in J-RGCs. Control group includes the data from both Cx36+/+ and Cx36+/− mice. Error bars, SEM. In C, control group PD vs. ND, paired t test, ***, p < 0.001, n = 24 cells; Cx36−/− group PD vs. ND, paired t test, *, p < 0.05, n = 6 cells; PD from control vs. Cx36−/− group, unpaired t test, ***, p < 0.001; ND from control vs. Cx36−/− group, unpaired t test, **, p < 0.01. In D, unpaired t test; NS, not significant; n = 24/6 cells for control/Cx36−/− group. (E) Representative EPSCs recorded during motion stimulus before (left) and after (right) bath application of PTX and STR. Shaded area around the traces, mean ± SEM, n = 8 trials. (F and G) Comparison of EPSC amplitudes (F) and total charges (G) between PD and ND motion under control and inhibition blocked condition. n = 5 cells. Data for this figure are in (PDF)Click here for additional data file.S3 Fig(A) Simulated J-RGCs’ responses with actual (left) and modified (middle and right) synaptic inputs. Similar to start = Einh. Shaded area around the traces, mean ± SEM. Ge, excitatory conductance. Gi, inhibitory conductance. (B and C) Peak depolarization of simulated responses to PD and ND motion under the DSe (B) and DSi (C) conditions across different Vstart. Responses under the control condition are included for comparison. (D) DSI values of simulated responses under control, DSe and DSi conditions across different Vstart. Error bars, SEM. n = 100 trials. Data for this figure are in (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(A) Representative OFF IPSC responses evoked by apparent motion stimulus and smooth motion stimulus. (B) Comparison of OFF IPSCs evoked by apparent motion stimulus and smooth motion stimulus. Each point on the graph compares the mean IPSC amplitudes of a J-RGC’s response in a 20 ms time bin between smooth motion  and apparent motion . Six J-RGCs’ responses are included. Dashed: line of identity. Data for this figure are in (PDF)Click here for additional data file.S6 Fig(A) Polar plots of a J-RGC’s average spiking responses to motion in 8 directions under control condition and after bath application of PTX and PTX + STR. n = 10 trials. (B) Polar plots of a J-RGC’s average spiking responses to motion in 8 directions before and after bath application of DCG-IV. n = 10 trials. (C–E) Comparison of J-RGCs’ direction selectivity measured by the vector sum magnitudes of the motion responses before and after bath application of PTX , PTX+STR , and DCG-IV . Paired t test; **, p < 0.01; ***, p < 0.001. (F) Vector sum magnitudes of J-RGCs’ responses to the moving spot stimulus under different luminance levels. Data from 23 randomly chosen OFF RGCs and 23 genetically defined ON-OFF DSGCs [n = 6/11/3 J-RGCs for optical density = 4.0/3.0/2.0 group. Dotted lines in C–F: vector sum magnitude = 0.12, mean + 2 × SD of the OFF RGC group. Error bars, SEM. Data for this figure are in FF DSGCs  are incl(PDF)Click here for additional data file.S7 Fig(A) Inputs for the deconvolution. Cyan, distribution of light intensity within the LED spot. Magenta, normalized IPSC peak amplitudes of a J-RGC to the activation of SACs by LED spots centered at different distances from the soma of the J-RGC. (B) Hotspots of SAC inputs revealed by deconvolution using the inputs in A. (C) Comparison of convolved result (convolution of the cyan curve in A and connection strength in B) and recorded result (the magenta curve in A). (D–F) The same as A–C, except using the data from another example J-RGC for deconvolution. (G) A summary for the hotspots of SAC inputs from 6 J-RGCs. Error bars, SEM. (H) Comparison of convolved result (convolution of the cyan curve in (PDF)Click here for additional data file.S8 Fig(A and D) Representative EPSCs (A) and IPSCs (D) recorded from a J-RGC during PD and ND motion at 800 μm/s (left) and 2,000 μm/s (right). Traces are aligned to the estimated time when the leading edge of the moving spot entered the RF center (dotted line). Shaded area around the traces, mean ± SEM, n = 10 trials.  Summary of the peak amplitudes and DSI values of motion evoked OFF EPSCs (B and C) and IPSCs (E and F). In B and C, n = 13 cells; in E and F, n = 9 cells. (G) The spiking responses of a J-RGC to PD and ND motion at 800 μm/s (left) and 2,000 μm/s (right). (H) Comparison of DSI values at 800 μm/s and 2,000 μm/s. n = 12 cells. Dotted line: DSI = 0.3. (I) Polar plots for the average spiking responses of a J-RGC to motion in 8 directions at 800 μm/s and 2,000 μm/s. n = 11 trials. (J) Comparison of direction selectivity measured by vector sum magnitudes of J-RGCs’ responses to 8 directions of motion at 800 μm/s and 2,000 μm/s. Dotted line: vector sum magnitude = 0.12. n = 6 cells. Error bars, SEM. In B, C, E, F, H, and J, paired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. Data for this figure are in (PDF)Click here for additional data file.S9 Fig(A) Representative average synaptic currents recorded from a J-RGC under different holding potentials. Traces are the average of 6 repeats, with different colors indicating different holding potentials. Arrow, onset of a dark spot stimulus. (B) I-V curve from A at the time of peak conductance (magenta dashed line in A). This is the I-V relationship for a mixture of all synaptic inputs. (C and D) The same as A and B but with the bath application of PTX + STR to block all the inhibitory inputs. The reversal potential for remaining currents was calculated to be 9.7 ± 3.3 mV, n = 3 cells. Data for this figure are in (PDF)Click here for additional data file.S10 Fig(A) An example where both the orientation-selective and direction-selective components of the J-RGC’s response could be observed using the drifting grating stimulus. Dashed green line, orientation of the J-RGC dendrites. (B and C) A J-RGC’s responses with different degrees of orientation and direction selectivity under 2 drifting grating stimuli: 320 μm period, 640 μm/s in B; 160 μm period, 320 μm/s in C. The J-RGC dendrite orientations are illustrated in green. Blue arrow, vector sum magnitude.(PDF)Click here for additional data file.S11 Fig(A) Representative IPSCs recorded during PD (left) and ND (right) motion before (blue) and after (red) bath application of 300 μm HEX to block the cholinergic transmission. Shaded area around the traces, mean ± SEM, n = 16 trials. (B) Summary of the effect of HEX on the peak amplitudes of motion evoked IPSCs. Paired t-test; *, p < 0.05; NS, not significant; n = 4 cells. Data for this figure are in (PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5’D2-RS – 3’D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response. T-cell receptor beta (TRB) chains are one of the essential components of the antigen recognition complex in T cells. The power of T-cell receptors (TR) to recognize a wide variety of possible pathogen peptides presented by the major histocompatibility (MH) proteins is provided by the enormous diversity of the variable domains of both alpha (TRA) and beta (TRB) chains , 2. ThisThere are two canonical types of RS; each type of RS contains a conserved 7-mer and 9-mer separated by a less conservative 12- or 23-nucleotide spacer, forming 12-RS and 23-RS, respectively. Recombination is initiated by binding RAG-1 and RAG-2 recombinase to RS sites, leading to specific DNA cleavage and hairpin generation at the edges of the coding regions . OpeningPrevious studies , 14 demoThese D-D rearrangements were detected in all DNA samples from thymic cells and peripheral blood mononuclear cells (PBMC), from all three analyzed species  chains, which contain three D genes and produce the only conventional partial D-D rearrangements, TRDD2-TRDD3. The distribution of the D-J junction length has a single peak  and then with V genes using 5’D1-RS (12-RS). To investigate this possibility, we identified TRB D-D junctions in partial TRB D-J rearrangements  T cells to understand whether V-D-D-J rearrangement-derived TRB chains are genuinely productive and are thus capable of recognizing epitopes similar to conventional TCRs. We successfully identified D1-D2 regions in the structures of complete productive TCR V-D-J rearrangements in memory T cells of 15 individuals -J diversity.Deleting J(1-6)-C1 cluster during D-D recombination mechanically increases the frequency of J2 genes in TRB generation. This new knowledge is the basis for the further improvement of the well-established TRB generation models , 19, whiThe “short” D-D group cannot be traced directly into complete TRB since they do not have recognizable parts of D genes. At this point, we can only hypothesize that they may contribute to shorter TRB fractions which are reported to participate in the immune response to common infections and are abundant and public , 23. TheThe traces of the “long” group can be partially detected in partial and complete TRB rearrangements. Meanwhile, the “long” fraction is clearly contributing to antigen recognition being directly detected in memory T-cell subsets in this study.In summary, this study analyzed an almost unnoticed component of the TCR diversity generation process. While we do not completely understand the extent to which it affects T-cell immunity in health and disease, we have clearly established it’s existence. We believe this newly identified rearrangement profile is significant for both clinical and basic scientific knowledge. Partial TRB D-D rearrangements can be applied as a novel marker for monitoring minimal residual disease , 25, cloth donor, dissection of the thymus was a standard part of the heart surgery), DNA from thymi of the same mouse, and the same rat  from 15 healthy human individuals with the age range of 30-50 years, one wild-type mouse  and one wild-type rat ; DNA from the thymus of one human donor  density gradient centrifugation according to manufacturer’s protocol. Bulk CD4+ and CD8+ T cells and CD19+ B cells were isolated from PBMC suspension using a magnetic separation approach with Dynabeads Positive Isolation Kits . DNA from all collected samples was isolated by FlexiGene DNA kit .The library for high-throughput sequencing was obtained in two sequential PCR reactions . The firObtained amplicons were purified using 1X AmPure XP beads (Beckman Coulter) and used as a template for the second PCR. The second (indexing) 25 µl PCR contained 1X Turbo buffer, 2.5 units of HS Taq polymerase, 200 µM of each dNTP, and 1 µl of Unique dual indexes primers . The amplification profile was as follows: 94°C for 3 min, followed by 15 cycles of 94°C for 20 sec, 57°C for 20 sec, and 72°C for 40 sec. Obtained amplicons were purified using 0.8X AmPure XP beads , pooled, and sequenced on Illumina NextSeq or MiSeq machine with coverage ~100,000 reads per sample  was used as a template in a 25 µl PCR reaction containing 1X Turbo buffer, 2.5 units of HS Taq polymerase, 200 µM of each dNTP and 0.2 µM of each primer specific to signal joint flanks Table 1.The amplification profile was 94°C for 3 min, followed by 45 cycles of 20 sec of 94°C, 20 sec of 58°C and 40 sec of 72°C. Signal joints for each analyzed species were amplified in separate PCR. Capillary sequences of the target PCR products were obtained as a service .TRB D-D, D-J, and TRD D-D rearrangements were extracted from fastq files using MiXCR 3.0 software  with addTRB V-D-J rearrangements were extracted from previously published deep TCR-seq dataset  for bulkThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PRJNA952099 (SRA).The studies involving humans were approved by the Pirogov Russian National Research Medical University local ethics committee. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were gifted by another research group. Written informed consent for participation was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and institutional requirements. The use of animals was approved by local committee for the control of the keeping and use of laboratory animals.AK: the idea, study design, funding, data analysis, manuscript drafting. AS, AM, LB and IK: performed the experiments and data analysis. YL, IM, DC: resources, samples, mentorship, manuscript drafting. All authors contributed to the article and approved the submitted version.


K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants, K-Ras4A and K-Ras4B, arise originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A and K-Ras4B and their role in breast ductal carcinoma. Total RNA was extracted from breast tumors, and the NATs were obtained via mastectomy. Patients were selected from new cases of breast cancer with no prior history of chemotherapy. Relative mRNA expression was calculated based on a pairwise comparison between the tumors and the NATs following normalization to the internal control gene. Predictive values of the transcript variants were examined by ROC curve analysis. p = 0.01) and 2.47 (p = 0.001), respectively. The K-Ras4A/K-Ras4B ratio was lower in the tumors than that of the normal tissues. ROC curve analysis revealed the potential of K-Ras4A (AUC: 0.769) and K-Ras4B (AUC: 0.688) in breast cancer prediction. There was also a significant association between K-Ras4B expression and HER2 statues (p = 0.04). Furthermore, a significant link was detected between K-Ras4A expression and pathological prognostic stages (p = 0.04). A statistically significant increase was found in K-Ras4A and K-Ras4B expression with the mean fold changes of 7.58 (Our findings reveal that the expression levels of K-Ras4A and K-Ras4B is higher in the tumor compared to the normal breast tissues. Increase in K-Ras4A expression was more significant than that of K-Ras4B.  Histologically, breast cancer is divided into two main overarching groups: carcinomas and sarcomas[2]. Breast carcinomas start in the epithelial cells, and consist of either ductal or lobular types, depending on their origin[3]. About one-third of human cancers with epithelial origin have dysregulated Ras/MAP/ERK signaling pathway[4]. Following activation of the cell surface receptors by extracellular ligands, Ras GTPase triggers signal transducing, and subsequently cell growth, differentiation, and survival. The gain-of-function mutations of the Ras proto-oncogenes deregulates cancer signaling pathways, leading to tumorigenesis and metastasis[5]. K-Ras is the most clinically significant members of the Ras subfamily and frequently mutated gene contributing to tumorigenesis in human. Numerous mutations of K-Ras are detected in a variety of cancers, including colorectal, lung, and pancreatic, but rarely in breast cancer.Despite recent developments in the diagnosis and treatment of human diseases, cancer is still the most prominent cause of mortality, worldwide. Iranian National Population-Based Cancer Registry has reported the age-standardized incidence rate of breast cancer as 34.53 per 100,000 and considered breast malignancies the most common cancer among Iranian women[8]. Functionally, the key difference between K-Ras4A and K-Ras4B exists in their post-translation modifications. C-terminal hyper-variable regions are typically presented on all Ras proteins. It consists of a CAAX motif, which undergoes cysteine farnesylation and permits an amplified Ras localization to the plasma membrane. K-Ras4B has a charge-mediated polybasic sequence that is located in the upstream of the CAAX motif and contains multiple lysine residues[9] by which K-Ras4B interacts with calmodulin. Since ductal tissues contain a high level of calcium and calmodulin, K-Ras4B could contribute to tumorigenesis by activating calmodulin/PI3Kα/Akt pathway[10]. K-Ras4A comprises polybasic region, as well as cysteine residue for palmitoylation modification, which also occurs in H-Ras and N-Ras[11]. K-Ras4A exists in both farnesylated/ nonpalmitoylated and farnesylated/ palmitoylated forms, contributing to two distinct signaling pathways as K-Ras4B and N-Ras, respectively. This characteristic of K-Ras4A explains its involvement in a broader range of cancers[12]. K-Ras gene has two main transcript variants, known as K-Ras4A and K-Ras4B. Dissimilarity between these two isoform roots in the alternative splicing of exon 4, resulting in K-Ras4A with 189 amino acids vs. 188 residues in K-Ras4BHerein, we aimed to study the expression of K-Ras4A and K-Ras4B and determination of their ratio in a series of ductal carcinoma of breast samples. Given the fact that K-Ras4A and K-Ras4B act differently in cancers, we intended to reveal which variant is the main transcript involving in breast tumor pathogenesis.PatientsBreast cancer cases were selected from the patients referred to the Department of Surgery, Sina Hospital, Tehran, Iran. The volunteered individuals participated in the study based on the following inclusion criteria, i.e. new cases of breast ductal carcinoma confirmed by biopsy. Exclusion criteria were patients who received no chemotherapy, radiotherapy, and hormone therapy before surgery and sampling. The patients were examined by an expert surgeon and evaluated according to the standard imaging procedures. Sample collection and verificationth edition). Seventy tumors and the matched NATs were obtained via mastectomy and then sectioned into two replicates. One replicate was instantly immersed into liquid nitrogen containers for long-term storage, and the other one was examined by an expert pathologist. Presence of ER, PR, and HER2 was assessed by IHC assay. Envision method was applied as a two-step technique, based on the dextran polymer technology. The antibody against Ki67 nuclear antigen was used to determine the cell proportion of luminal tumor subtypes. The cut-off value of 14% was considered to define the Ki67 index. Since our study was conducted on the patients who performed surgery as the initial treatment, we assigned them into the pathologic prognostic stage  were used as the template for complementary DNA synthesis carried out by using BioFact™ RT Series kit . Quantitative real-time RT-PCR assay was set up by selecting PUM1 gene as an internal control. K-Ras4A, K-Ras4B, and PUM1 specific primers indicated in . Amplification was performed by Biofact SYBR-Green High-ROX master mix  as follows: 95 °C for 15 min; 40 cycles at 95 °C for 20 s and 60 °C for 40 s. Real-time RT-PCR was performed in duplicates for each sample, as well as no template control. The reaction mixtures were run on StepOne Plus® . The Ct values were obtained from the data output of the instrument software. The melting curves were created for each amplified fragment to determine the PCR specificity. Raw amplification data were transferred to LinReg software, and linear standard curves were generated.Total RNA was extracted from the tumors and NATs using GeneAll®RNA extraction kit  according to the manufacturer's instructions. RNA samples with high purity . Gene expression variations with more than two-fold changes were considered significant. Fold change significance was shown by ggplot2 and ggsignif packages in R language environment. The pheatmap package was applied to visualize log2Fc against the selected pathological parameters. ROC curve assessment Relative distribution of K-Ras4A and K-Ras4B expression was analyzed by ROC curve. pROC package in the R was used for plotting ΔCt of the transcripts. A logistic regression model was recruited to calculate the AUC and 95% CIs. Sensitivity and specificity were calculated to compare the predictive values of the transcripts by caret and lattice packages. Statistical analysis The sample size was calculated by EpI Info 7.0.9.34 software. To reject the null hypothesis of no K-Ras4A and K-Ras4B effects, the significance level was chosen at 5% to ensure a power of 80%. The collected clinical and pathological data were statistically analyzed using SPSS version 24. Correlation studies were performed by Chi-square tests.Patients' clinicopathological characteristicsThe mean age of patients was 56.52 ± 13.37 years old, ranging from 31 to 82. Thirty-five percentage of the patients had a positive family history of cancer among their first- or second-degree relatives . DespiteK-Ras4B and K-Ras4A gene expression © software identified a significant increase in K-Ras4A and also K-Ras4B mRNA expression level in the breast tumors compared with NATs. The fold change of K-Ras4B was 2.47, with 95% CI = 0.37-24.25 and p = 0.001. Of note, the increase in the expression level was even more remarkable for K-Ras4A compared to K-Ras4B, displaying a fold change of 7.58, 95% CI = 0.57-119.42, and p = 0.01 .The significance of the K-Ras4A and K-Ras4B expression was determined based on the clinicopathological features of the patients. The correlations were studied using the Chi-square test, and the values lower than 0.05 were considered significant. Samples were classified into two groups: high expression (fold change >2) and low expression (fold change ≤2) levels. K-Ras4B expression was significantly associated with HER2 status . Each patient signed a written informed consent. All the authors have read and approved the contents of the final manuscript and agreed to publicize this manuscriptThe raw data supporting the conclusions of this article are available from the authors upon reasonable request. MMM: project development, data collection, and manuscript writing/editing. ZMH: data collection and manuscript editing; LG: data collection and management, and manuscript editing; RM: project development, data analysis, and manuscript editing. SS: project development, data collection and management, data analysis, and manuscript writing/ editing. The authors declare no competing interests.This study has received no financial support from any agency in the public, commercial, or nonprofit sectors.


IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms. IL-6 and CRP levels on the severity of periodontitis in CAD in Indonesia were also evaluated. This case-control study was conducted with mild and moderate–severe chronic periodontitis groups. A path analysis test was conducted with Smart PLS with a 95% confidence interval to determine the significant variable for chronic periodontitis. Our study revealed that the effects of IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms on IL-6 levels and CRP levels were not significant. IL-6 and CRP levels were not significantly different between the two groups. We found that IL-6 levels had a significant effect on CRP levels in periodontitis patients with CAD . IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms had no effect on the severity of chronic periodontitis in CAD patients in the Indonesian population. We also observed no apparent effects of the influence of gene polymorphisms in IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C genes. Although the IL-6 and CRP levels were not significantly different between the two groups, IL-6 levels affected CRP levels in periodontitis patients with CAD.Interleukin 6 (IL-6) and C-Reactive Protein (CRP) play an important role in chronic periodontitis with coronary artery disease (CAD). Genetic factors can affect a person’s risk of CAD, which affects one-third of the population. This study investigated the role of  Coronary artery disease (CAD) is a cardiovascular disorder due to atherosclerosis or atherosclerotic occlusion of the coronary arteries . It is ePeriodontitis is defined as a chronic infectious disease of supporting tissues of teeth. It is associated with gene variations and environmental interaction ,7. This The relationship between periodontitis and cardiovascular disease can develop through direct and indirect pathways. The direct pathways include bacterial invasion and infection, and the indirect pathways include increases in C-Reactive protein (CRP) and interleukin-6 (IL-6) ,14. Seve572 C/G and 174 G/C genes. The two SNPs influencing IL-6 expression and serum levels are associated with periodontitis susceptibility; however, their geographic and ethnic distribution is distinct [IL-6 -572 may be a genetic risk factor for periodontitis patients in Asian populations, especially the Chinese population [IL-6 -174 polymorphism is rare in the Chinese population [IL-6 -572 C/G [IL-6 -572 G/C is correlated with plasma concentrations of IL-6 [Polymorphisms in the promoter region of the IL-6 gene trigger variations in the transcription . The mosdistinct ,23. The pulation . The IL-pulation . Several-572 C/G ,27,28. F of IL-6 .-717 A/G, and -757 T/C genes [CRP -757 T/C and chronic periodontitis [CRP -717 polymorphisms have been shown to be associated with CAD [IL-6-572C/G and CRP+1444 C/T [CRP gene polymorphisms affect CRP levels in serum. Moreover, about 35–40% of variations in CRP levels between individuals are inherited . Polymor/C genes . A signidontitis . They pldontitis . Meanwhiwith CAD  and periwith CAD . Studies1444 C/T ,35.IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms; IL-6 levels; and CRP levels in chronic periodontitis related to CAD patients in Indonesia. Therefore, we investigated the roles of IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms; IL-6 levels; and CRP levels on the occurrence of chronic periodontitis related to CAD patients.To our knowledge, there are no studies on This study was an analytic observational study with a case-control study. The. inclusion criteria were CAD patients with at least 20 teeth who underwent coronary angiography from December 2021 to October 2022 at dr. Doris Sylvanus Palangka Raya, Indonesia, and did not undergo six months of periodontal treatment. CAD patients with moderate–severe periodontitis were used as a case group, and patients with mild chronic periodontitis were used as a control group. The exclusion criteria were not using antibiotics and anti-inflammatories in the last three months, acute myocardial infarction, pregnancy, fever, pneumonia including COVID-19, and kidney failure. The sample size of each group was 40, and the samples were obtained by consecutive sampling.Chronic periodontitis was diagnosed according to the assessment of the probing depth in 6 index teeth  by measuring the depth of the buccal, mesial, lingual, and distal areas [Stenosis was considered significant if at least 70% of the main coronary arteries were in one angiographic projection, at least 50% of two projections, and 50% of the main coronary arteries  using QuBlood samples were collected through the cubital vein. Samples were put into each vacutainer containing EDTA anticoagulant. Peripheral blood mononuclear cell (PBMC) isolation with the addition of Ficoll was performed using the Genomic DNA Mini Kit (Blood/Cultured Cell) , according to the manufacturer’s recommended procedures. The results of DNA extraction were stored at −20 °C until further analysis. DNA quantification was measured using the NanoDrop TM One spectrophotometer .® GTXpress Master Mix 2X , 1.25 μL TaqMan® SNP Genotyping Assay 20X, 1-20 ng DNA sample, and nuclease-free water to a final volume of 25 μL. Amplification was performed using a CFX 96 Touch™ Real-Time PCR  with a setting of 20 s of enzyme activation at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C and annealing extension for 1 min at 60 °C. Sample genotype was measured based on each allele’s Relative Fluorescence Unit (RFU). Materials for real-time PCR were TaqMan® Pre-Designed SNP Genotyping Assay Reagent, Thermo Fisher Scientific Baltics UAB V. Graiciuno 8 LT-02241 Vilnius Lithuania, cat No. 4351379, C32343415_10 (size S) (rs3093059), C318207_10 (size S) (rs2794521), and C__11326893_10 (size S) (rs1800796). QuantStudio 5  was used as a thermocycler.The PCR mix consisted of 12.5 μL TaqManIL-6 -572 C/G gene polymorphism (rs1800796): 5′-GTAAAACGACGGCCAGTAGTGGGCTGAAGCAGGTGA-3′ (36-mer). Reverse Primer (with M13 tail): 5′-GCGGATAACAATTTCACACAGGCTTTGTTGGAGGGTGAGGG-3′ (41 -mer).Forward Primer (with M13 tail) CRP -757 A/G gene polymorphism (rs3093059): 5′-GTAAAACGACGGCCAGTCCTTTGGAAAAGATGTATTCGG-3′ (39-mer). Reverse Primer (with M13 tail): 5′-GCGGATAACAATTTCACACAGGGACTCTACTACAAAGGATACGG-3′ (44-mer).Forward Primer (with M13 tail) CRP -717 T/C gene polymorphism (rs2794521): 5′-GTAAAACGACGGCCAGTCCTTTGGAAAAGATGTATTCGG-3’ (39-mer. Reverse Primer (with M13 tail): 5′-GCGGATAACAATTTCACACAGGGACTCTACTACAAAGGATACGG-3′ (44-mer). DNA sequencing confirmation was conducted by 1st BASE DNA Sequencing Division, Apical Scientific Laboratory, Selangor, Malaysia.Forward Primer (with M13 tail) IL-6 levels in blood serum were measured by electrochemiluminescence immunoassay (ECLIA) analysis. The result was expressed with pg/mL units. Additionally, the result was determined by a calibration curve with a two-point calibration and a master curve provided through a reagent barcode, with normal values ≤7.00 pg/mL .CRP levels were determined based on quantitative HsCRP in serum with the latex immunoassay principle. Agglutination was detected as a change in absorbance at a wavelength of 572 nm with the turbidimetric or immunoturbidimetric method and normal values ≤10 ng/mL .IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms; IL-6 levels; and CRP levels was determined. Genotype and allele frequencies were compared by Hardy–Weinberg equilibrium. t-test was used if data distribution was normal. The Mann–Whitney test was used if data were not normally distributed. The path analysis test was conducted with Smart PLS 3.04 GmbH, Gewerbering, Oststeibek, Germany, to determine the dominant variable for chronic periodontitis. A 95% confidence interval was used and was considered a significant difference if p ≤ 0.05.Data were analyzed using SPSS statistics software for Windows, version 26, IBM Corp, Armonk, NY, USA. A chi-square test was used to determine the relationship between polymorphisms and chronic periodontitis. Continuous data were presented as mean ± standard deviation (SD) while dichotomous with frequency and percentage. Continuous data were tested for normality distribution with the Shapiro–Wilk test. An ANOVA test was performed if the distribution was normal, while Kruskal–Wallis test was used if data were not normally distributed. The relationship between IL-6 -572 C/G homozygote minor GG 5 gene polymorphism (6.3%), the CRP -717 T/C homozygote CC 5 gene (6.3%), and the CRP -757 A/G minor homozygote GG 2 gene (2.5%), as shown in https://www.ncbi.nlm.nih.gov/snp/rs1800796, accessed on 23 February 2023) . The frequency of the C allele was higher than the T allele compared to Asian populations .Our subjects were predominantly male (72.5%) patients who were over <60 years (83.8%) and had hypertension (78.8%). Our study showed that they did not suffer from diabetes mellitus (DM) (70%), and the statuses of dyslipidemia, smoking or non-smoking, and obesity were almost the same in both groups. We found the IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms were 0.540, 0.764; 0.099, 0.144; 0.196, 0.907 , according to the Hardy–Weinberg equilibrium. We observed no significant difference between the mild and moderate–severe chronic periodontitis groups. Therefore, the two groups were considered homogeneous (p > 0.05).The frequency distributions of IL-6 -572 C/G gene polymorphism in patients with moderate–severe periodontitis and mild periodontitis with CAD. The genotypes and alleles of the two study groups were not statistically different (p = 0.233).IL-6 -572 C/G gene polymorphism, the percentage of the GG genotype was higher in the moderate–severe periodontitis group (10%) than in the mild periodontitis group (2.5%). Our study demonstrated that 5% of the GG genotype was found in the moderate–severe periodontitis group, while none of the GG genotype was found in the mild periodontitis group based on the CRP -757 A/G gene. We also found that the percentage of the CC genotype was lower in the moderate–severe periodontitis group (5%) than in the mild periodontitis group (7.5%) based on the CRP -717 T/C gene. Genotypes CC, CG, and GG and alleles C and G in the IL-6 -572 C/G gene; genotypes AA, AG, and GG and alleles A and G in the CRP -757 A/G gene; and genotypes TT, TC, and CC and alleles T and C in the CRP -717 T/C gene were not significantly different between the two groups (p > 0.05).Regarding the IL-6 -572 C/G gene. We found that the average IL-6 level was higher in the GG genotype than in the CC and CG genotypes; however, it was not statistically significant (p = 0.579). The CRP -757 A/G gene polymorphism showed a lower average HsCRP level in the GG genotype compared to the AA and AG genotypes. However, it was not significantly different (p = 0.842). The average HsCRP level was lower in the CC genotype (4.580 mg/L) compared to the TT and TC genotypes ; however, there was no apparent significant difference (p = 0.490).IL-6 and HsCRP levels were higher in the moderate–severe chronic periodontitis group than in the mild periodontitis group. Our results showed that the IL-6 levels in the mild and moderate–severe periodontitis groups were 11.245 ± SD 8.646 and 12.982 ± 11.752, respectively. Moreover, the CRP levels in the mild periodontitis and moderate–severe periodontitis groups were 7.675 ± SD 9.143 and 9.420 ± 15.548, respectively .IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms; IL-6 levels; and CRP levels had no apparent effects on the severity of chronic periodontitis in CAD based on the path analysis . Interestingly, IL-6 levels affected CRP levels in periodontitis patients with CAD .analysis . Our resIL-6-572 C⁄G gene polymorphism and severe chronic periodontitis-related CAD in Indonesia. The mild and. moderate–severe periodontitis groups showed no significant differences in the. proportions of genotypes CC, CG, and GG or alleles C and G. We also found that IL-6 levels were the highest in the GG genotype compared to AA and AG; however, this was not statistically significant.The present study found no association between IL-6 -572 C/G gene polymorphisms on chronic periodontitis in CAD, which remain unclear. Based on the previous study, the IL-6 -572 C ⁄G gene polymorphism did not correlate with chronic periodontitis susceptibility, but it was significantly different between the CAD and non-CAD groups [The etiology and pathogenesis of periodontal disease are complex . The indD groups .IL-6 -572 is not associated with periodontitis in the Iranian population [IL-6 -572 C/G polymorphism and CAD. The CG and GG genotypes increase the risk of periodontitis-related CAD [IL-6 -572 gene polymorphism [Polymorphisms have been reported to be related with periodontitis. These genetic risk factors play an important role in Asian populations, especially the Chinese population , and in pulation . Polymorpulation . Additiopulation . A recenated CAD . Gene poated CAD ,47. The morphism , idiopatmorphism , DVT [49morphism .CRP -757 A/G and CRP -717 T/C gene polymorphisms for chronic periodontitis in CAD. Our study showed no significant difference in the. proportions of the AA, AG, and GG genotypes or the A and G alleles (-757 A/G). We did not observe significant differences in the proportions of the genotypes TT, TC, CC, T, and C (-717 T/C) and the CRP levels in both groups. The highest mean CRP levels were in the AA genotype in the CRP -757 gene and the TC genotype in the CRP -717 gene, but they were not statistically significant. Similar studies also reported a significant association of CRP -757 T/C with chronic periodontitis in South India and high-risk TT genotypes [No studies have focused on the enotypes . The C aenotypes ,50. Howeenotypes . This coenotypes .CRP-717 T/C gene is not related to periodontitis [CRP -717 polymorphism predisposes to CAD allele A [The dontitis ,53 includontitis . It provdontitis . Anotherallele A . A relatallele A . Our stuallele A , the difallele A ,56.IL-6 -572 was lower than in the Asian population. In contrast, its frequency in the European population reached 95% [CRP -757 showed a frequency of 14.4%, which is lower than the frequencies in Asia (16%) and the European population (6%) [CRP -717 is 26.9% higher than in the Asian population (16%) and in Europe (28%) [-717 C/T dominant C allele with homozygote recessive in Iranian population with chronic periodontitis [In the present study, the frequency of the G allele at ion (6%) . The frepe (28%) . A studydontitis . Periododontitis . The IL-IL-6 gene. It is involved in immunological, infection, inflammatory mechanisms, and polymorphisms in the IL-6 gene region that affect physiological function and other cytokine imbalances [Our study emphasizes the previous study concerning the significant effect of IL-6 on increasing CRP levels in chronic periodontitis with CAD. IL-6 is an inflammatory mediator encoded by the balances ,62.Periodontitis is associated with increased IL-6  and CRP Increased CRP levels in periodontitis have been reported ,69,70. SIL-6 and CRP play important roles in forming CAD plaques ,78,79,80The results of the current study showed that the CAD with moderate–severe periodontitis group presented a higher level of IL-6 and CRP in comparison with the mild periodontitis group; however, our results were not statistically significant. This difference could be due to differences in examination methods, especially the probing depth limit , the invIL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C gene polymorphisms; IL-6 levels; and CRP levels on the severity of periodontitis in CAD, especially in the Indonesian population. However, there are several limitations. First, the sample size was only obtained from one center. Second, the type of polymorphisms studied were limited to the IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C genes. Third, our study focused on the severity of chronic periodontitis in CAD and did not involve a normal group (without periodontitis). Further studies are needed to involve healthy controls and more centers.This study is the first study to analyze the effects of IL-6 -572 C/G, CRP -757 A/G, and CRP -717 T/C C/G gene polymorphisms. Polymorphisms were not affected by the severity of chronic periodontitis, IL-6 levels, and CRP levels. In addition, IL-6 levels affected CRP levels in moderate–severe periodontitis patients with CAD. Further studies are warranted to involve IL-6 and CRP polymorphisms at other loci and with larger samples.The chronic periodontitis population with CAD had the majority of CC, AA, and TT genotypes in