Escherichia coli (ExPEC) and epidemic clones of E. coli in association with New Delhi metallo-β-lactamase (blaNDM) in septicaemic neonates are rare. This study captured the diversity of 80 E. coli isolates collected from septicaemic neonates in terms of antibiotic susceptibility, resistome, phylogroups, sequence types (ST), virulome, plasmids, and integron types over a decade (2009 to 2019). Most of the isolates were multidrug-resistant and, 44% of them were carbapenem-resistant, primarily due to blaNDM. NDM-1 was the sole NDM-variant present in conjugative IncFIA/FIB/FII replicons until 2013, and it was subsequently replaced by other variants, such as NDM-5/-7 found in IncX3/FII. A core genome analysis for blaNDM+ve isolates showed the heterogeneity of the isolates. Fifty percent of the infections were caused by isolates of phylogroups B2 (34%), D (11.25%), and F (4%), whereas the other half were caused by phylogroups A (25%), B1 (11.25%), and C (14%). The isolates were further distributed in approximately 20 clonal complexes (STC), including five epidemic clones . ST167 and ST131 (subclade H30Rx) were dominant, with most of the ST167 being blaNDM+ve and blaCTX-M-15+ve. In contrast, the majority of ST131 isolates were blaNDM-ve but blaCTX-M-15+ve, and they possessed more virulence determinants than did ST167. A single nucleotide polymorphism (SNP)-based comparative genome analysis of epidemic clones ST167 and ST131 in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of antibiotic-resistant epidemic clones causing sepsis calls for a modification of the recommended antibiotics with which to treat neonatal sepsis.Longitudinal studies of extraintestinal pathogenic IMPORTANCE Multidrug-resistant and virulent ExPEC causing sepsis in neonates is a challenge to neonatal health. The presence of enzymes, such as carbapenemases (blaNDM) that hydrolyze most β-lactam antibiotic compounds, result in difficulties when treating neonates. The characterization of ExPECs collected over 10 years showed that 44% of ExPECs were carbapenem-resistant, possessing transmissible blaNDM genes. The isolates belonged to different phylogroups that are considered to be either commensals or virulent. The isolates were distributed in around 20 clonal complexes (STC), including two predominant epidemic clones (ST131 and ST167). ST167 possessed few virulence determinants but was blaNDM+ve. In contrast, ST131 harbored several virulence determinants but was blaNDM-ve. A comparison of the genomes of these epidemic clones in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of epidemic clones in a vulnerable population with contrasting characteristics and the presence of resistance genes call for strict vigilance. Escherichia coli remains an important cause of neonatal sepsis in both developing and developed nations , is not always distinct, particularly in the neonate, as breaches in the gut barrier may allow commensal E. coli to cause disease  and as a successful pathogen that can cause sepsis, meningitis, urinary tract infection, osteomyelitis, wound-site infection, pyelonephritis, etc. testifies to its versatility , of which B2 and, to a lesser extent, D, are considered to be ExPECs, whereas A and B1 are considered to be commensals , has spread across the globe within a span of about 10 years . Previously, it was believed that ARGs impose a “fitness cost” and that wer ARGs . The virwer ARGs . The epiwer ARGs . Althoug10 years –15. Seve E. coli . In deve E. coli . The pre E. coli .blaNDM in an effort to understand any epidemiological pattern that is emerging across the years.ExPECs have been well-studied, but there are considerably fewer studies that explore the ExPECs that cause neonatal infections/sepsis , 20. We E. coli were identified from the blood of septicaemic neonates (2009 to 2019), and, furthermore, 10 E. coli isolates were identified from sources other than blood, such as endotracheal aspirate (n = 2), stool (n = 2), pus (n = 2), and peritoneal fluid (n = 4), not necessarily from the same neonates from whom blood was collected.70 50, 16 mg/L, MIC90, 64 mg/L) except one   and third-generation cephalosporins , amikacin , gentamicin , ciprofloxacin , trimethoprim-sulfamethoxazole , aztreonam , and meropenem . The isolates were susceptible to only colistin and tigecycline. Thirty-five out of 80 isolates screened positive for the combination disk test, which indicated the production of MBL. Isolates that were positive in the combination disk test had higher minimum inhibitory concentration (MIC) values (MIC 2 mg/L) . The othblaNDM. The blaNDM-ve isolates (n = 45) were comparatively more susceptible to other antibiotics, whereas the blaNDM+ve isolates were highly resistant. There was a significant difference between the two groups in their nonsusceptibility to certain antibiotics, such as amikacin , gentamicin , and the second and third-generation cephalosporins   .blaNDM or not, possessed several β-lactamases, such as blaCTX-M, blaTEM, blaOXA-1, and blaSHV in percentages of 73%, 44%, 41%, and 28%, respectively. The most common metallo-β-lactamases found in the isolates was blaNDM (44%). Serine-β-lactamases such as blaOXA-48-like carbapenemase were present along with blaNDM in three isolates  (rmtB (34%), armA (13%), and rmtC (4%) were present. The efflux pump modulator gene qepA was present in a single isolate (EN5101) (blaNDM. The fluoroquinolone resistance genes aac-(6’)-Ib-cr (51%), qnrB (21%), qnrS (16%), oqxA (8%), and oqxB (11%) were also present. The carriage of multiple resistance determinants, such as β-lactamases, 16s rRNA methylases, plasmid-mediated quinolone resistance (PMQR), and AmpC, were higher in the blaNDM+ve isolates  . Aminogl(EN5101)  that cohisolates , compareiderably .n = 35) were found to possess blaNDM as the major carbapenemase. A sequence analysis of the blaNDM revealed the presence of different NDM-variants, such as NDM-1 (n = 12), NDM-5 (n = 19), NDM-7 (n = 3), and NDM-15 (n = 1). The common mutation present in NDM-5, NDM-7, and NDM-15 was M154L, compared to NDM-1. Additional mutations  were also present  isolates (n = 3). Two of these isolates (EN5378 and EN5379) were indistinguishable by PFGE  were analyzed. Additional resistance determinants, such as blaCTX-M, blaTEM,blaCMY,rmtB, and aac(6′)-Ib-cr were also found to cotransfer in different combinations with blaNDM. A detailed characterization of the blaNDM+veE. coli isolates and their transconjugants (TCs) and TFs is provided in Out of 35 blaNDM was probably via the IncF group  as most TCs harbored these plasmids. The carriage of blaNDM was mostly via the IncF group  and, to a lesser extent, via IncX3, X4, R, A/C, Y, as reflected by the conjugation results. In addition, the typing of the F-plasmid (pMLST) using the WGS data suggested that F2:A-:B and F36:A4:B (FAB formula) were the common plasmid types associated with blaNDM-1 and blaNDM-5, respectively  and whole genome sequencing (WGS). Different incompatibility groups, such as A/C, L/M, FII, FIIK, FIIS, FIA, FIB, FIB-M, HIB-M, HI1α, I1α, I1γ, X1, X2, X3, U, Y, N, L, P, and R were found along with different types of Col plasmids, including Col(pHAD28), Col(BS512), Col(MG828), Col440I, ColVc, Col156, and ColRNAI. The carriage of ectively .blaNDM+ve isolates. At the upstream, blaNDM was always associated with ISAba125, either in full-length (15/35) or truncated (18/35). Two isolates (EN5095 and EN5169) possessed IS5 with a remnant of ISAba125 within close proximity of blaNDM. In another isolate (NBE0004), the insertion of ISKpn26 between ISAba125 and blaNDM was found. bleMBL was always found to be present downstream of all of the blaNDM-producing isolates. Four different downstream combinations were found in the individual isolates in this region (Both the PCR and WGS data were considered for the study of the genetic environment of the s region .n = 21), and it contained different genes  in their variable regions and conferred resistance against trimethoprim and streptomycin. In addition, In54 and In191 were identified in 8 and 7 isolates, respectively. A new class 1 integron, namely, In2101  was detected in a single isolate (NBE0004), according to the INTEGRALL database . 12 isolates were found to be carrying more than one class 1 integron , and it revealed that isolates were clonally diverse with four clonal clusters that were found across four different years: cluster 1 , cluster 2 , cluster 3 , and cluster 4  . Based on the PFGE result, WGS was performed for isolates (n = 30) that included all of the distinct isolates and one representative from each different cluster  were distributed in the blaNDM-possessing isolates , the phylogroup distribution was as follows: A (50%), C (30%), and B2 (20%). Phylogroup B2 isolates were identified each year, and they persisted throughout the 10 years. There was an increasing prevalence of phylogroup A over time (23% to 35% from 2011 to 2019). No such trend was noticed for the other phylogroups (All of the isolates (blood and non-blood) belonged to six different phylogenetic groups, including A, B1, B2, C, D, and F. The majority of the strains belonged to phylogroup B2 (34%), and this was followed by A (25%), C (14%), D (11.25%), B1 (11.25%), and F (4%). For a single isolate, the phylogroup was undetected. An analysis of the blood isolates showed a distribution of B2 (38%), A (21%), D (13%), B1 (13%), C (11%). and F (4%). For isolates that were collected from different body sites, other than blood , and their respective clonal complexes were detected using the goEBURST algorithm. The STIPs were also converted to the corresponding clonal complexes in the Warwick scheme (STW), as this scheme is easy to compare with global strains (Table S1). In addition, the blaNDM+ve isolates were subjected to WGS, and the STWs for these isolates were determined from the WGS data.The sequence types of all isolates (blood and non-blood) were analyzed by the Institut Pasteur scheme (STblaNDM) were classified into 29 different STIPs: STIP2, 3, 4, 6, 8, 21, 33, 36, 43, 53, 58, 66, 129, 146, 294, 369, 471, 477, 479, 506, 635, 664, 678, 686, 816, and 922 as well as the three novel STIP974, 1134, and 1135. STIP2 (20%) was predominant, and it was followed by STIP43 (16%), STIP471 (7.5%), STIP58 (5%), STIP3 (3.75%); STIP53 (3.75%), and other lone STIPs. The predominant clone STIP2 was associated with phylogroup A. Other STIPs, such as STIP43, STIP58, and STIP471, were members of phylogroups B2, B1, and C, respectively. The isolates of phylogroup B2 were further divided into five subgroups: subgroups I (STWc131), III (STWc127), IV (STWc141), VI (STWc12), and VII (STWc14) (Table S1). STIP2, which was the most predominant STIP, corresponds to the epidemic clones STW167 and STW617. Similarly, STIP43, STIP471, and STIP477 correspond to STW131, STW410, and STW405, respectively, which are also globally disseminated high-risk clones. All STIPs with their respective clonal complexes (in STIP and STW) are mentioned in (Table S1).All 80 isolates (with or without IP974 was found in a single isolate (EN5101)   belonginblaNDM-1 . The blaW167 was the major epidemic clone that was significantly associated with blaNDM-5  . STW410 persisted throughout the years with a low prevalence, and diversity in serotypes were observed. Two of them were blaNDM-1+ve with two different serotypes (O78:H18 and O8:H9), whereas the other two isolates were blaNDM-5+ve with somatic antigen O-negative.During the last few years (2016 to 2019) of the study period, ST<0.0001) , whereaslaNDM-ve  isolatesn = 19), the most predominant VFs were traT (86.25%) and fyuA (71.25%), and these were followed by fimH (56.25%), iucC (51.25%), papC (42.5%), usp (36.25%), papA (35%), MTII (33.75%), PAI (32.5%), papG (25%), iha (25%), hlyA (16.25%), E.ciron (15%), sfa/foc (9%), and afa (6.25%), with ibeA being found only in a single isolate and with cdtB and cvaC being completely absent , and two subclades . The isolates were of two serotypes, namely, O16:H5 and O25:H4, for clades A and C, respectively. All of the isolates harbored blaCTX-M-15 with chromosomal mutations in gyrA , parC , and parE  that conferred elevated fluoroquinolone resistance. Only one isolate (EN5115) possessed blaCTX-M-27 belonging to the C1-M27 subclade. A specific group of resistance genes, including aadA5, dfrA12, mdf(A), mph(A), sul1, tetA, aac-6’-Ib-cr, and blaCTX-M-15, was found in almost all of the isolates. None of the isolates possessed blaNDM, except for four  that were found to be positive for blaNDM-1 from the H30Rx subclade and to be carrying some additional resistance determinants . Virulence determinants, such as heme uptake (chuA), curli fibers (csgBDFG), type 1 fimbriae (fimABCDEFGHI), yersiniabactin siderophore , outer membrane protein A (ompA), pap operon (papBCDFGH), and others , were present in STW131. papGII was present in all of the H30Rx isolates. The pathogenicity-associated island (PAi) mostly encoded the papGII that was found in 50% of the isolates. The plasmid types, as revealed by pMLST, were as follows: F2:A1:B-, F29:A-:B10, and F36:A1:B20 and F1:A1:B16 for the H30Rx and F1:A2:B20 for the C1-M27 subclade.The STW131 contextual SNP analysis, 48 genomes from NCBI were included. The NCBI genomes were largely blood isolates, although some (n = 11) were urine isolates. In total, STW131 genomes from 10 countries were included, and the most distant isolate  was a single isolate from the USA, as indicated by it having the longest branch length. The SNP phylogenetic tree is formed by two main branches with several subbranches. Isolate EN5382 (acting as the reference) was the only isolate from this study in the top branch , as the two other strains were clonal with EN5132 within this study. The isolates EN5132 and NBE0008 were within 20 SNPs and were collected during 2013, within a period of 3 months, whereas the isolate EN5179 sits on a separate branch and is 39 SNPs distant from the pair but is blaNDM-ve , especially CTX-M. The top branch was largely -M-15+ve . GenomeslaNDM-ve . AdditiolaNDM-ve .W167 (n = 12) isolates revealed some common patterns of resistance and virulence determinants. For the resistance determinants, blaNDM-5/-7/-15, aadA2, dfrA12, mph(A), sul1, and chromosomal mutations in gyrA , parC (S80I), and parE (S48A) were common in all of the strains. In addition to PCR data, WGS helped to explore more VF determinants, such as common pilus (ecpABCDE), lamin binding fimbriae (elfACDH), type I fimbriae (fimDFG), type IV pilli (pilw), invasins (ibeBC), yersiniabactin siderophore , Klebsiella sp. specific lipopolysaccharide (rfb locus), heat resistance (hra), increased serum survival (iss), and serum resistance-associated outer membrane protein (traT), which were present in all of the isolates. A serotype-based characterization showed that all of the isolates shared the same O -antigen biosynthesis gene cluster , such as STW617 and STW1702, did not possess any fimH types.The high-risk clone ST cluster . In addiblaNDM+ve isolates belonged to STW167; however, a SNP analysis on WGS data further revealed genetic divergence within the STW167 isolates  were single-locus variants of STW167, and this is denoted on the tree (W167 isolates within this study were >50 pairwise SNPs distant from each other (12 out of 35 isolates . A contethe tree . The SNPch other .blaNDM+ve isolate or due to an epidemic clone was tested (Tables S3A and B). No statistically significant association was noted between mortality and the presence of NDM (Table S3A). A neonate was twice as likely (odds ratio = 2.009615) to have expired when sepsis was due to an epidemic clone than when sepsis was due to a nonepidemic clone (Table S3B). However, the association between mortality and the presence of an epidemic clone was not statistically significant . This is possibly due to the small sample size, and, thus, it cannot be interpreted whether the results from Klebsiella pneumoniae and Acinetobacter baumanii have lately been noted as the foremost causes of neonatal sepsis in low-income and middle-income countries (LMICS) (E. coli still remains relevant (Tables S2A and B).The vulnerability of the neonates and the increasing antimicrobial resistance in causative pathogens lead to morbidity and mortality in this population . Althoug (LMICS) , E. coliE. coli causing sepsis over a decade. 74 out of 80 (92.5%) E. coli isolates across the years were multidrug-resistant (MDR), and 44% were carbapenem-resistant , indicating that the modification of the WHO guidelines for the treatment of sepsis is long overdue  or blaNDM-5/-7 (thereafter). The growing predominance of other variants of NDM-1, particularly NDM-5 and NDM-7, has been noted in several studies in India as well as across South East Asia  structure that consisted of ISAba125 (either full-length or truncated) at the 5′ end and ISCR1 (IS91-like element insertion sequence common region 1 family transposase) at the downstream regions. This has also been observed in other blaNDM+ve isolates , and others that were narrow . The IncX3 plasmids that were identified here were found in 5 out of 22 isolates carrying blaNDM-5/-7 and in other previous studies in humans, animals, and the environment , and insertion sequences (IS), are the main vehicles for the dissemination of antibiotic resistance genes. The isolates . ISCR1 ice genes , 29. As f blaNDM . blaNDM ironment . The ass studies , 32.IPs. This indicates that the majority of infections or acquisitions were independent and that cross-transmission in the unit was limited to specific cases. This unit caters to high-risk deliveries and out-born  admissions, and this probably accounts for the diversity of the strains that was noted.The ExPECs in the study were distributed among 6 phylogroups  and 27 STblaNDM was predominantly present in these isolates, notably within the epidemic clone STW167 isolates -Ib and aac-(6’)-Ib-cr, and, importantly, blaCTX-M-15. This indicates that although both blaNDM-1 and blaCTX-M-15 are plasmid mediated, their acquisition remains different. blaNDM is primarily associated with STW167, whereas blaCTX-M-15 is associated with both STW167 and STW131.A large proportion of the septicaemic isolates belonged to phylogroup A 25%), and isolates . Studies%, and isth NDM-5 , 33, 34.E. coli are the members of phylogroups A, B1, and C  with serotypes O25:H4 and subclade H30Rx. Other B2 subgroups, including III (STWc127), IV (STWc141), VI (STWc12), and VII (STWc14) were also isolated here and are known to be prevalent among ExPEC strains , and this was also noted in this study (B2 + D = 45%). Most of the B2 isolates that were studied belonged to ST strains .W167 and STW131 were further analyzed. The genomic relatedness of the STW167 and STW131 study isolates were compared, both internally and with a selection of global isolates available in NCBI. As most of the STW167 strains in this study possessed blaNDM, these genomes were compared to other blaNDM+ve global STW167 strains. Most of the study STW167s (except for a few that were within 15 pairwise SNPs) were distinct and possessed few VFs. The serotypes of the STW167 study strains were O101:H9 with an rfb locus and fimH was absent. In contrast to the results of our study, a recent study focusing on the genomics of STW167 reported serotype O89b:H4 possessing Klebsiella sp. specific K48 capsular type (mucoid colony morphology) . Considering this, the two predominant epidemic clones STphology) . O89b:H4phology) , 33. In W131, the basis of selection of global strains for comparison with the study strains was irrespective of the presence of NDM, as blaNDM was not frequent in these strains. Most previous studies have noted that the STW131 isolates were homogenous , mph(A), sul1, tetA, aac-6’-Ib-cr, and blaCTX-M-15) were associated with this lineage, as observed here  and STW131 (4/15), the prevalence of VFs also differed in STW167 and STW131. Tourret et al. indicated that extraintestinal virulence resulted from the additive effects of VFs, meaning that the higher numbers of VFs in the B2 strains attest to their disease-causing potential  and susceptible isolates. The comparison of genomes, including the study and global strains, reveals the diversity of ExPECs. The accessory genome plays an important role in antibiotic resistance and also in the diversity of the strain causing sepsis. It is difficult to demarcate commensal and pathogenic E. coli, as an equal proportion of strains from both groups caused sepsis. The increasing numbers of carbapenem-resistant high-risk clones is worrisome. As the pool of whole-genomes continues to increase, our understanding of ExPECs will be enriched, and both classical and molecular studies remain important tools in sepsis and antimicrobial resistance research.This longitudinal study focusing on neonatal bloodstream infection shows clear differences in the sequence types and serotypes between carbapenem-resistant , peritoneal fluid (PF), pus, and stool, from 2011 to 2014 were also analyzed.Bacterial isolates were primarily isolated from the blood of septicaemic neonates (newborns within 28 days of birth) over a period of approximately 10 years (2009 to 2019) from a tertiary care center, namely, the IPGME&R and SSKM hospital of Kolkata in India. Isolates were identified as E. coli isolates were identified via biochemical tests (2009 to 2017) and by using a Vitek 2 compact system (2018 to 2019), using a card Vitek 2 GN .The susceptibility to different antibiotics, such as piperacillin (100 μg), cefoxitin (30 μg), cefotaxime (30 μg), ciprofloxacin (30 μg), trimethoprim-sulfamethoxazole (1.25 mg/23.75 μg), aztreonam (30 μg), amikacin (30 μg), gentamicin (10 μg), meropenem (30 μg), colistin (10 μg), and tigecycline (15 μg)  was quantified via a conventional disk diffusion assay for the isolates that were collected from 2009 to 2017  and by ublaVIM, IMP, SPM-1, GIM-1, SIM-1, KPC, SME, SPM, NDM, GES, OXA-48 like)    -Ib-cr, qepA, oqxA, oqxB) (PCR was carried out for the detection of carbapenemases (48 like) , 43, β-l, CTX-M) , 44, AmpCT, FOX) , 16S rRNnd armA) , and plaA, oqxB) , 47.blaNDM and blaOXA-48 were subjected to Sanger sequencing in an automated DNA sequencer  and were aligned with GenBank reference gene sequences that are available in NCBI (http://www.ncbi.nlm.nih.gov/genbank) (Amplicons of genbank) . The resblaNDM-harboring plasmid (donor) to sodium azide-resistant E. coli J53 (recipient) was performed in a solid-state mating assay using a medium supplemented with MEM (2 mg/L) and sodium azide (100 mg/L). For some isolates, conjugation was not successful. Hence, purified plasmid DNA was electrotransformed using electrocompetent E. coli DH10B cells  in a Gene Pulser II . Transformants (TFs) were screened in the presence of MEM (2 mg/L). The presence of blaNDM was confirmed, along with other β-lactamase genes, via PCR in transconjugants (TCs) and TFs (The transfer of the  and TFs . The MIC and TFs .blaNDM+ve wild-type isolates (WT), along with their TCs and TFs, was determined via PCR-based replicon typing (PBRT) assay using a PBRT Kit  (https://cge.food.dtu.dk/services/pMLST/).The plasmid replicon typing of the , Italy) , 49. In blaNDM was determined via PCR using two sets of primers  for nomenclature . The similarity between the isolates was generated based on the Dice correlation coefficient. The unweighted pair group method with arithmetic mean (UPGMA) was selected for a cluster analysis with 1.5% tolerance and optimization. Isolates showing at least 95% similarity were considered to be identical  (blaNDM+ve and blaNDM-ve) were also prepared for WGS. Genomic DNA was extracted by using a Wizard Genomic DNA Purification Kit (Promega) for the short read paired-end sequencing (2 × 150 cycles). The library preparation was carried out using a Nextera XT Kit . The WGS was executed on an Illumina platform , using an Illumina NovaSeq 6000 sequencer. The sequences were assembled and processed for further analysis (Supplementary Text).Based on the results of the PFGE, all of the distinct http://clermontyping.iame-research.center/); (ii) MLST for sequence type identification (https://cge.food.dtu.dk/services/MLST/); (iii) SeroTypeFinder (https://cge.food.dtu.dk/services/SerotypeFinder/) for serotype identification; (iv) VirulenceFinder and VFDB for virulence gene identification (https://cge.food.dtu.dk/services/VirulenceFinder/); (v) FimTyper for fimH allele determination (https://cge.food.dtu.dk/services/FimTyper/); (vi) CHTyper (https://cge.food.dtu.dk/services/CHTyper/) for C-H type determination; (vii) ResFinder (https://cge.food.dtu.dk/services/ResFinder/) and Resistance Gene Identifier  for resistance gene identification; (viii) PlasmidFinder for the detection of plasmid replicon types (https://cge.food.dtu.dk/services/PlasmidFinder/); (ix) the integral site for the nomenclature of the integron sequences ; and (x) ISfinder for the identification of IS elements (https://isfinder.biotoul.fr/). Where applicable, all of the PCR data were corroborated with the genome data.Using the short read data, the following online-based services were achieved: (i) phylogroup by Clermont typing (W131 and STW167 (blaNDM+ve) study isolates with relevant E. coli genomes were collected from NCBI, following a literature search in PubMed (Supplementary Text). IQtree (v2.0) was used to generate a SNP phylogeny, and iTOL (v6.0) was used to annotate and visualize the phylogenetic tree. Additionally, a core genome phylogenetic tree was generated for all of the study blaNDM+ve isolates using Panaroo (v.1.2.8) and IQtree (v2.0).Two phylogenies were performed, based on single nucleotide polymorphisms (SNP). STE. coli phylogenetic groups . The discrimination of phylogroups A or C and D or E were further carried out by using the C and E specific primers in a singlex PCR assay  (C) (IP) were converted to the equivalent STCs of the Warwick scheme (STW)  was performed using the Institut Pasteur MLST scheme  (_seqdef) . The clodef) (C) . The STsme (STW)  ; (ii) toxins ; (iii) siderophores ; (iv) invasins (invasion of brain endothelium factor [ibeA]); (v) protectins and serum resistance ; and (vi) miscellaneous   , 56.Neonatal mortality and related clinical and bacterial factors from a sample of 70 observations (fewer observations for some factors due to missing data) were studied. This was done by comparing the proportions of patients in different groups. For some bacterial factors, a bivariate analysis was carried out to test associations with neonatal mortality. The Chi-square test of association was conducted where appropriate. All of the data analyses were executed using Stata version 17.0.The samples were collected as a part of the routine diagnosis of sepsis. Hence, a waiver of informed consent was granted. The study protocol was reviewed and approved by the Institutional Ethics Committee of ICMR-National Institute of Cholera and Enteric Diseases . Patient records and information were anonymized and deidentified prior to analysis.PRJNA548120 (EN5134), PRJEB33565 (EN5356 and EN5358), and PRJNA801430 (the rest of the isolates).All of the genome data were submitted to the NCBI database under the BioProjects


Among the differentially abundant genes were ten regulatory genes, including IRF1, IRF6, HERC5, HERC6, IFIH1, IFIT2, IFIT5, IFI35, RSAD2, and UBQLNL. Gene ontology (GO) and KEGG pathway analysis was conducted, and we found that processes and pathways associated with the blood testes barrier (BTB), immunity, inflammation, and DNA methylation in gametes were preferentially enriched. We also found abnormal expression of genes related to infertility  and down-regulation of testosterone in monkeys with dietetic obesity. This work not only provides an important reference for research and treatment on male infertility caused by obesity, but also valuable insights into the effects of diet on gene expression in testes.Obesity is a metabolic disorder resulting from behavioral, environmental and heritable causes, and can have a negative impact on male reproduction. There have been few experiments in mice, rats, and rabbits on the effects of obesity on reproduction, which has inhibited the development of better treatments for male subfertility caused by obesity. Nonhuman primates are most similar to human beings in anatomy, physiology, metabolism, and biochemistry and are appropriate subjects for obesity studies. In this investigation, we conducted a transcriptome analysis of the testes of cynomolgus monkeys on high-fat, high-fructose, and cholesterol-rich diets to determine the effect of obesity on gene expression in testes. The results showed that the testes of obese monkeys had abnormal morphology, and their testes transcriptome was significantly different from that of non-obese animals. We identified 507 differentially abundant genes (adjusted  Accordise BMI ≥ 0 kg/m2, re obese .Obese men are more likely to have decreased sperm concentration, and to have sperm with abnormal morphology, damaged chromatin, and reduced motility ,6. BMI iAt present, a consensus has been reached on the adverse effects of obesity from a high-fat or high-sugar diet on male reproduction. Non-human primates, as the animals closest to humans, can be used as important disease models to study human metabolic diseases . Up to nMacaca fascicularis) were provided by Spring Biological Technology Development Co., Ltd. . Ten male monkeys (age ≥ 9 years) were used in this study, five in the control group, which were fed with normal chow diet, and five in the obesity group, which were fed a diet high in fat, fructose, and cholesterol. The normal chow diet was made according to the Nutrient Requirements of Nonhuman Primates . The high-fat diet was purchased from Beijing Keao Xieli Feed Co., Ltd.  with 24% fat, 15% fructose, and 1% cholesterol. Both groups of monkeys were fed for 15 months. Afterward, monkeys with BMI > 42 were assessed as overweight. The temperature of the animal room was 22–25 °C, the humidity was 48–65%, and the light time was 12 h a day. All monkeys were housed in individual cages with access to water and food ad libitum. In the end, the monkeys were anesthetized and then treated with bloodletting. The testes were quickly collected, and both sides were placed in liquid nitrogen or 4% paraformaldehyde.All experimental protocols involving nonhuman primates were approved by the Laboratory Animal Care Committee of Xi’an Jiaotong University. Cynomolgus monkeys  water for 3 min. They were stained with H & E then immersed in 90%, 95%, and 100% alcohol for dehydration, and lastly in two changes of xylene, and were mounted using neutral resin.g for 10 min to collect the supernatant. The monkey testosterone enzyme-linked immunosorbent assay kit  was used to assay testosterone. The coated plate was removed after equilibrating at room temperature for 1 h. Standard wells were added with 50 μL different concentrations of standards, and blank and sample wells were added with 50 μL sample diluents and samples, respectively. Then, 100 μL horseradish peroxidase (HRP)-labeled antibody was added to each well, and the plate was covered and incubated in the 37 °C incubator for 1 h. After that, the plate cover was removed, and the contents of the microtiter wells were thoroughly aspirated after washing each well five times with 350 μL washing buffer. Subsequently, the plate was incubated at 37 °C for 15 min (without direct exposure to intense light) after the addition of 50 μL substrates A and B to each well. Finally, the absorbance (OD) of each well was measured at 450 nm after adding 50 μL stop solution to each well.About 200 mg of testicular tissue, the middle portion of the left testis, was weighed and rinsed with pre-chilled phosphate-buffered saline (PBS) to remove residual blood. PBS was added at a weight-to-volume ratio of 1:9; the tissue samples were treated on ice for 5 min with TissueMaster™ Handheld Homogenizer , and the homogenates were centrifuged at 5000× g for 5 min at 4 °C. The supernatants were transferred to another 2 mL centrifuge tube and mixed with 0.3 mL of chloroform/isoamyl alcohol. After shaking for a few seconds, the extracts were centrifuged at 12,000× g for 10 min at 4 °C, and the supernatants were transferred to clean 1.5 mL centrifuge tubes. Equal volumes of isopropanol were then added to the transferred liquid, mixed, and centrifuged at 12,000× g for 20 min at 4 °C. The supernatants were removed and discarded, and the precipitated RNA was rinsed with 1 mL of 75% ethanol. Samples were placed in a biosafety cabinet for air-drying, and then dissolved in 25 µL to 100 µL of diethyl pyrocarbonate (DEPC)-treated water. RNA quality was checked by A260/280 using a NanoDrop spectrometer and concentration was determined with an Agilent 2100 bioanalyzer .Total RNA was extracted from tissues using TRIzol  following the company’s instructions. After weighing out 60 mg of tissue, it was flash-frozen in liquid nitrogen and ground to powder. The powder was transferred to a 2 mL centrifuge tube, dissolved in 1.5 mL TRIzol, and centrifuged at 12,000× Ribosomal RNA (rRNA) was isolated using target-specific oligos. After solid-phase reversible immobilization (SPRI) selection, the RNA was separated into multiple small fragments under the treatment of divalent cations. The separated RNA pieces were copied into first-strand cDNA with reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase and Ribonuclease H (RNase H). The RNA template was digested by this method; a fresh substitute strand was synthesized, and double-stranded cDNA was generated by replacing dTTP with dUTP. These cDNA pieces possess a sole ‘A’ base and afterwards, sequencing adapters were ligated to the ends. After treatment with uracil DNA glycosylase (UDG), the merger of dUTP quenches the second strand during amplification. Products were enriched by PCR to establish the final cDNA library. Fragment size distribution was measured with an Agilent 2100 Bioanalyzer, and libraries were quantified and evaluated for quality with real-time quantitative PCR (RT-qPCR) using TaqMan probes. Valid libraries were sequenced end-to-end on the BGISEQ-500/MGISEQ-2000 system .https://biosys.bgi.com, accessed on day 1 January 2022). The expression level of the gene was calculated by RSEM (v1.3.1) [http://www.geneontology.org/, accessed on 1 May 2022) and KEGG  enrichment analysis was performed by Phyper  based on a hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold . Results of the testosterone assay were compared with Student’s t test using SPSS ver20 statistics software . A p < 0.05 was considered statistically significant. Moreover, the inter-assay is less than 15%; the intra-assay is less than 10%; and the minimum detection concentration is less than 0.1 ng/mL.Analysis of RNA-sequencing data was conducted using the ‘Dr. Tom’ system ((v1.3.1) . The hea(v1.3.1) . GO , interferon regulatory factor 6 (IRF6), homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain (RLD) containing E3 ubiquitin protein ligase family member 5 (HERC5), HECT and RLD containing E3 ubiquitin protein ligase family member 6 (HERC6), interferon induced with helicase C domain 1 (IFIH1), interferon induced protein with tetratricopeptide repeats 2 (IFIT2), interferon induced protein with tetratricopeptide repeats 5 (IFIT5), interferon induced protein 35 (IFI35), radical S-adenosyl methionine domain containing 2 (RSAD2), Ubiquilin like (UBQLNL), six initial genes and fifty-two associated genes were found  included anchored components of membranes, extracellular regions, extracellular spaces, anchored components of the plasma membrane, eukaryotic 80 S initiation complex, integral component of the membrane, membranes, Coding Region Determinant (CRD)-mediated mRNA stability complex, cell surface, and pi-body A. Among Among the 163 up-regulated genes, the top ten biological processes (BPs) were enriched in basement membrane organization, DNA methylation involved in gamete generation, cation transport, inhibition of neuroepithelial cell differentiation, methionine catabolic process, deoxyribonucleoside monophosphate catabolic process, polarity specification of proximal/distal axes, regulation of mRNA stability involved in response to stress, 4-nitrophenol metabolic process, pallium cell proliferation in forebrain, and P granule organization A. Among CXCR3) chemokine receptor binding, actin binding, phospholipid scramblase activity, and actin filament binding  were enriched in small molecule binding, copper ion binding, retinal isomerase activity, endochitinase activity, 4-nitrophenol 2-monooxygenase activity, voltage-gated proton channel activity, all-trans-retinyl-palmitate hydrolase, 11-cis retinol forming activity, all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity, deoxyribonucleoside 5′-monophosphate N-glycosidase activity, and type I activin receptor binding A. Among  binding B.Among the 163 up-regulated genes, the top 20 KEGG pathways were enriched in glycosylphosphatidylinositol (GPI)-anchor biosynthesis, various types of N-glycan biosynthesis, Th1 and Th2 cell differentiation, viral protein interaction with cytokine and cytokine receptor, cysteine and methionine metabolism, graft-versus-host disease, steroid hormone biosynthesis, type I diabetes mellitus, chemical carcinogenesis-DNA adducts, signaling pathways regulating pluripotency of stem cells, arachidonic acid metabolism, and others A. Among TDRD5), chloride voltage-gated channel 2 (CLCN2), mcrorchidia1 (MORC1), regulatory factor X8 (RFX8), spermatogenesis and oogenesis-specific basic helix-loop-helix 1 (SOHLH1), interleukin 2 receptor subunit beta (IL2RB), multi-ciliate differentiation and DNA synthesis-associated cell cycle protein (MCIDAS), and zona pellucida binding protein (ZPBP), were abnormally highly expressed in obese monkeys (NFIA), protein-tyrosine phosphatase non-receptor type 11 (PTPN11), TSC22 domain family member 3 (TSC22D3), mitogen-activated protein kinase 6 (MAPK6), phospholipase C beta 1 (PLCB1), defective in cullin neddylation 1 domain containing 1 (DCUN1D1), lipin 1 (LPIN1), and glycine amidinotransferase (GATM), were abnormally under-expressed  database A. In the monkeys B, while xpressed C. Testosxpressed D.Many important findings about reproduction, fertility, and development were based on experiments with mice, rats, and rabbits. However, human physiology and metabolism differ in many ways from that in these animals, which has limited the development of better treatments for sterility, infertility, and other problems caused by obesity . NonhumaThe results of transcriptome analysis showed a significant change in gene expression in the testes of obese monkeys. Key driver analysis showed that ten genes were closely related to immunity or inflammatory processes, which suggested that dietetic obesity could lead to chronic inflammation of the testes. This condition could be one pathogenic mechanism leading to infertility in obese people. Using relatively strict threshold screening, 163 up-regulated genes and 344 down-regulated genes were identified in testes from obese monkeys. The biological process with the highest score among the 163 up-regulated genes is basement membrane organization, which is closely related to the BTB, suggesting that obesity may cause damage to the BTB and the dysfunction of spermatogenesis by negatively affecting the expression of regulatory genes related to basement membrane organization. Enterogenic endotoxin production by intestinal microbiota, which can be induced by a high-fat or high-sugar diet, can activate toll-like receptors (TLRs) related to inflammation on testicular microvascular endothelial cells and release inflammatory factors . InflammThe DNA methylation level of spermatozoa showed a dynamic change different from that of somatic cells during spermatogenesis . The metTDRD5 protein is essential for pachytene piRNA biogenesis and may play an important role in male infertility. Previous studies found that TDRD5 was abnormally expressed in the testes of individuals with azoospermia [SOHLH1 is a testis-specific basic helix-loop-helix transcription factor which is essential for spermatogonial differentiation. Mutations in the SOHLH1 protein resulted in non-obstructive azoospermia [MCIDAS is necessary for the generation of functional multi-ciliated cells in the efferent ducts that are required for spermatozoa to enter the epididymis [MCIDAS reduced the generation of multiple motile cilia and caused male infertility [ZPBP is localized to the acrosome in human spermatozoa, and males lacking ZPBP1 were sterile, with abnormal round-headed sperm lacking forward motility [TSC22D3 is a widely expressed dexamethasone-induced transcript that has been proposed to be important in immunity and adipogenesis. TSC22D3-deficient males were infertile and exhibited severe testicular dysplasia and a high number of apoptotic cells within the seminiferous tubules [MORC1 is expressed in germ cells (spermatogonia and spermatocytes) of the testis [MORC1 is also essential for the facilitation of DNA methylation and transposon repression in the male embryonic germ cells. The finding showed that mutations in MORC1 caused the loss of male-specific germ cells and infertility in mice [NFIX, one of the members, is expressed in spermatocytes and plays an important role in spermatogenesis. The deficiency of NFIX resulted in multinucleation in spermatocytes, structural defects in the synaptonemal complex, etc., which ultimately led to the inhibition of spermatogenesis [PTPN11, also known as SHP2 (SH2-containing tyrosine phosphatase 2), is essential for the self-renewal of spermatogonial stem cells and the production of other male germ cells [PTPN11 resulted in Noonan and LEOPARD syndromes, both of which manifested reproductive defects in the reproductive system such as male infertility [PLCB1 is a vital regulator of the acrosome reaction in spermatozoa. Mutations in PLCB1 reduced the rate of acrosome reaction, fertilization rate, and the probability of embryo development to mulberry or blastocyst [DCUN1D1, also known as SCCRO (squamous cell carcinoma-related oncogene), is an important regulator of neddylation in mammals. An animal study found various abnormal morphologies that appeared in the spermatozoa of DCUN1D1-/- mice, such as macrocephaly and multiple flagella. In addition, these aberrant spermatozoa indicated the presence of abnormal intercellular bridges, which were probably responsible for specific infertility and inability to release mature spermatozoa of DCUN1D1-/- mice [TDRD5, SOHLH1, MCIDAS, and ZPBP were abnormally expressed in the testes of obese monkeys compared with normal-weight controls, suggesting that the dysfunction of those genes and their related biological functions should be given prominence in studies of human obesity-induced male infertility.ospermia ,38. SOHLospermia ,40. MCIDididymis . Mutatioertility . ZPBP ismotility . TSC22D3 tubules . MORC1 ie testis . Besides in mice . The NFIogenesis . PTPN11,rm cells . It is arm cells . Mutatioertility ,50. PLCBastocyst , resulti-/- mice . Here, wDespite the rapid development of multiomics, research on obesity or the effects of a high-calorie diet on the testicular transcriptome have mainly been confined to rats and mice with few studies focused on humans or non-human primates. In our research, we investigated the influence of a typical modern diet, high in fat and fructose, and cholesterol-rich, on gene expression in the testes of cynomolgus monkeys, which afforded valuable insights into the effects of diet and obesity on male infertility.Dietetic obese monkeys showed abnormal testicular morphology, and their testes transcriptome was significantly altered. The differentially abundant genes were predominantly associated with blood–testes barrier function, immunity, inflammation, DNA methylation involved in gametogenesis, decreased testosterone level, and abnormal expression of infertility-related genes. This work will provide an important basis for future research and the development of treatments for male infertility caused by obesity.


Grain shape is an important agronomic trait directly associated with yield in rice. In order to explore new genes related to rice grain shape, a high-density genetic map containing 2193 Bin markers (526957 SNP) was constructed by whole-genome resequencing of 208 recombinant inbred (RILs) derived from a cross between ZP37 and R8605, with a total genetic distance of 1542.27 cM. The average genetic distance between markers was 0.76 cM, and the physical distance was 201.29 kb. Quantitative trait locus (QTL) mapping was performed for six agronomic traits related to rice grain length, grain width, length-to-width ratio, thousand-grain weight, grain cross-sectional area, and grain perimeter under three different environments. A total of 39 QTLs were identified, with mapping intervals ranging from 8.1 kb to 1781.6 kb and an average physical distance of 517.5 kb. Among them, 15 QTLs were repeatedly detected in multiple environments. Analysis of the genetic effects of the identified QTLs revealed 14 stable genetic loci, including three loci that overlapped with previously reported gene positions, and the remaining 11 loci were newly identified loci associated with two or more environments or traits. Locus 1, Locus 3, Locus 10, and Locus 14 were novel loci exhibiting pleiotropic effects on at least three traits and were detected in multiple environments. Locus 14, with a contribution rate greater than 10%, influenced grain width, length-to-width ratio, and grain cross-sectional area. Furthermore, pyramiding effects analysis of three stable genetic loci showed that increasing the number of QTL could effectively improve the phenotypic value of grain shape. Collectively, our findings provided a theoretical basis and genetic resources for the cloning, functional analysis, and molecular breeding of genes related to rice grain shape. Rice is one of the world’s most important staple crops, providing sustenance for over half of the global population. However, the current level of rice production is still insufficient to meet the demands of population growth. It is projected that the overall demand for rice will increase in the coming decades, particularly in Africa and Asia ,2,3. TheGS3 [GL3.1 [GL4 [GLW7 [GW2 [GW5 [GW8 [GS5 [TGW6 [TGW2 [qTGW3 [Grain shape traits in rice primarily include grain length (GL), grain width (GW), grain length-to-width ratio (GLWR), and so on. These traits are typically quantitative in nature, influenced by multiple genes, although a few are controlled by single or double genes ,8. StudiGS3 , GL3.1 [3 [GL3.1 , GL4 [13L4 [GLW7 . Genes mLW7 [GW2 , GW5 [16GW5 [GW8 , and GS5GW8 [GS5 . AdditioS5 [TGW6 , TGW2 [2W6 [TGW2 , and qTG2 [qTGW3 , which pGL7/GW7 gene located on chromosome 7 influences both GL and GW. Upregulation of GL7 leads to decreased transverse cell division and increased longitudinal cell division, resulting in thinner and longer grains with improved appearance [GL2/GS2 gene located on chromosome 2, which is a major QTL that simultaneously influences GL, GW, and grain weight in rice [GW6a [GS9 [Some QTLs that control grain traits exhibit pleiotropy, simultaneously affecting multiple traits related to rice grains. For example, the pearance ,23. Anot in rice ,24. Addice [GW6a  and GS9 W6a [GS9  also exhWith the rapid development of high-throughput sequencing technology in recent years, the cost of sequencing has continuously decreased. Single nucleotide polymorphism (SNP) has emerged as a third-generation molecular marker technology, which allows for the rapid acquisition of a large number of polymorphic markers in a short period of time. It has become a preferred choice for constructing high-density genetic maps due to its advantages of high throughput, high marker density, and time and labor efficiency. This technology provides an effective means for exploring and identifying important agronomic trait QTLs, facilitating subsequent fine mapping and breeding applications .In recent years, a number of researchers have localized rice grain shape QTLs by using a high-density genetic map constructed by whole genome resequencing of recombinant inbred lines in rice ,29,30. IIn this study, we aimed to construct a high-density bin genetic map by performing whole-genome resequencing of two superior parents, R8605 and ZP37, along with their hybrid-derived population consisting of 208 RILs. Subsequently, we evaluated the grain-related traits of the RIL population in three distinct environments. Leveraging the high-density genetic map, we successfully identified novel and consistent QTLs associated with rice grain-related traits. The ultimate goal of this research was to establish a foundation for the cloning, functional analysis, and molecular breeding of genes involved in rice grain-related traits.This study examined and statistically analyzed six grain-related traits of the parents ZP37, R8605, and the 208 RILs in three different environments . The resUsing the GBS method, we conducted whole-genome resequencing on ZP37/R8605 parents and their derived 208 RIL progenies. ZP37 and R8605 parents yielded 12.75 Gbp and 12.21 Gbp of clean data, respectively. The total data volume for the 208 RILs was 972.76 Gbp, with an average of 4.68 Gbp per family. The Q30 score reached over 80% for all samples. A total of 804,031 polymorphic SNPs were detected between the parents, which were filtered and refined to obtain 526,957 high-quality, biallelic, and homozygous SNPs. These SNP markers were converted into 2193 bin markers, which were assigned to 12 linkage groups corresponding to the 12 chromosomes of rice. The total genetic map distance of the 12 chromosomes was 1542.27 cM, with marker numbers ranging from 71 to 308, averaging 182.75 per chromosome. The genetic distances ranged from 68.68 to 193.02 cM, with an average genetic distance of 128.52 cM. The average genetic and physical distances between adjacent bin markers were 0.76 cM and 201.29 kb, respectively . Among tQTL analysis was performed using the constructed high-density genetic map and phenotypic data of six grain-related traits in 208 RIL progenies and their parents across three environments. QTLs with overlapping confidence intervals for the same trait in different environments were grouped together as the same QTL. A total of 39 QTLs associated with grain morphology were identified , distribMost of the detected QTLs were located within the same interval or in close proximity. The specific results for each grain-related trait QTL localization were as follows:qGL-7-1, and qGL-7-3 were detected in a single environment, while qGL-2, qGL-7-2, and qGL-8 were stable QTLs detected in multiple environments. The other QTLs showed positive additive effects except for qGL-2, which exhibited a negative additive effect. This finding indicated that the beneficial allele for qGL-2 originated from R8605, while the beneficial alleles for the other five QTLs were derived from ZP37.GL: In the three environments, five major-effect QTLs were identified on chromosomes 2, 7, and 8. The PVE ranged from 1.74% to 6.96%. Among them, two QTLs, qGW-3-1 and qGW-4-1, were detected in a single environment, while qGW-3-2, qGW-4-2, and qGW-8 were stable QTLs detected in multiple environments. The two QTLs on chromosome 4 exhibited negative additive effects, while the three QTLs on chromosomes 3 and 8 showed positive additive effects. It is worth noting that qGW-8 on chromosome 8 was not only detected in all three environments but also had a consistently high PVE (PVE ≥ 9.21), and its beneficial allele originated from ZP37.GW: A total of five GW QTLs were detected across chromosomes 3, 4, and 8 in the three environments. The PVE of individual QTLs ranged from 3.23% to 14.10%. Among them, two QTLs, qGLWR-2-1, qGLWR-2-2, qGLWR-4, qGLWR-5, and qGLWR-7) were detected in a single environment, while the QTL qGLWR-8 was a stable QTL repeatedly detected in all three environments, with contribution percentages of 14.20%, 13.83%, and 12.46%, respectively. It was a high-contributing QTL, and its beneficial allele originated from ZP37.GLWR: A total of six GLWR QTLs were detected across chromosomes 2, 3, 4, 7, and 8 in the three environments. The PVE of individual QTLs ranged from 1.45% to 14.20%. Among them, five QTLs  were detected in a single environment, while two QTLs (qTGW-3 and qTGW-8-1) were stable QTLs detected in multiple environments. Except for qTGW-2 and qTGW-3, which exhibited positive additive effects, the other seven QTLs showed negative additive effects. This finding indicated that the beneficial alleles for increasing TGW mainly originated from R8605.TGW: A total of eight QTLs for TGW were detected across chromosomes 2, 4, 5, 7, and 8 in the three environments. The PVE of individual QTLs ranged from 2.09% to 5.75%. Seven QTLs  was a pleiotropic locus that affected GL, TGW, and PL. It was mapped to the interval of Block5989-Block6012 on chromosome 8, with a physical distance of 22,065,235-23,447,755 bp. Within this position, a G protein gamma subunit GGC2 [Through genetic effect analysis of all identified 39 grain-related QTLs, a total of 14 loci with stable genetic effects were obtained, involving 30 QTLs . Among tPGL2 , is asso [OsPUP7  that simnit GGC2  regulateThe other 11 newly identified loci in this study were detected in different environments, indicating their stable genetic effects and highlighting the value and reliability of high-density genetic maps for QTL analysis. Locus 4 and Locus 12 were repeatedly detected as QTLs for a single trait in two environments. The remaining nine loci were associated with multiple traits, among which Locus 1, Locus 3, Locus 10, and Locus 14 exhibited genetic pleiotropy, controlling three or more traits and detected across multiple environments. Locus 1, located within the interval of Block1305–Block1318 on chromosome 2, corresponded to the physical position of 29,677,627–30,291,539 bp. It simultaneously influenced traits such as GL, TGW, and PL. Locus 3, located within the interval of Block1968–Block1974 on chromosome 3, corresponded to the physical position of 16,148,731–16,617,377 bp. It simultaneously affected traits such as GW, TGW, and AS. Locus 10, located within the interval of Block5433–Block5490 on chromosome 7, corresponded to the physical position of 20,606,977–22,680,384 bp. It simultaneously influenced traits such as GL, TWG, AS, and PL.Notably, Locus 14 had the most prominent genetic effect, with a contribution rate exceeding 10%. It was located within the interval of Block6167–Block6169 on chromosome 8, corresponding to the physical position of 27,555,403–27,911,017 bp. It simultaneously affected traits such as GW, GLWR, and AS.This study selected three QTLs with stable genetic effects, namely Locus 1, Locus 10, and Locus 13, for further analysis of their pleiotropic effects on multiple traits, such as GL, PL, and TGW, across multiple environments. First, the RIL population was divided into two genotypic groups, aa, and bb, based on the bin-marked genotypes within different QTL intervals, excluding the heterozygous type. The differences in corresponding traits between the two genotypic groups were then evaluated.qTGW-2 controlled by Locus 1, which did not reach significance in the 2019 environment for TGW, the remaining traits showed significant or highly significant differences across all three environments  signaling, plant hormone perception and homeostasis, transcriptional regulatory factors, and epigenetic modifications. It has been reported that  of rice . In thisand size . On chrotein APG .The utilization of the same mapping population across multiple environments for QTL mapping is highly significant for aggregation breeding. It enables the detection of stable QTLs with minimal environmental influence, which can be consistently identified. Additionally, the identification of pleiotropic QTLs is valuable in breeding, as multiple traits can be simultaneously selected using a single genomic region ,43. In tGS3, GL2/OsGRF/GS24, GW8, GL3.1/ospplkl1, and GLW7/OsSPL13 has shown that introducing multiple favorable alleles can effectively increase GL and improve rice yield [Pyramide breeding is a viable approach for increasing yield. In previous studies, the aggregation analysis of multiple QTLs related to GL and GW, such as ce yield ,10,13. T1 hybrid was obtained, and subsequent generations were generated through multiple generations of self-pollination using the single seed descent method. This resulted in the construction of a set of RIL populations (F8:9 generation). Both the parental lines and RILs were planted in Nanning City, Guangxi, China  for early-season cultivation in 2019 and late-season cultivation in 2020. Additionally, in 2022, they were also planted in Guiping City, Guangxi, China . These planting locations were labeled as 2019, 2020, and 2022, respectively. The sowing and transplanting of the early-season planting took place on 1 March and 2 April each year, respectively, while those for the late-season planting occurred on 18 July and 4 August, respectively. Each RIL or parental line was arranged in a randomized complete block design, with six rows per line and six plants per row, spaced at 20 cm × 20 cm intervals. Single seedling transplanting was employed, and field management followed standard local agricultural practices.In the present study, ZP37, a local variety collected by our research group, was characterized by strong and stout stems, numerous secondary branches, and strong disease resistance. R8605, on the other hand, was a hybrid rice restorer line developed by the Rice Research Institute of Guangxi Academy of Agricultural Sciences. It exhibited characteristics such as exceptionally long panicles, cold tolerance, and high-quality and high-yield traits. Using R8605 as the paternal parent and ZP37 as the maternal parent, an FDuring the rice maturation stage, we collected three plants at random from each plot for harvesting. The harvested grains were naturally dried, and any empty grains were removed from the samples. To analyze the grain-related traits, we utilized the SC-G automatic seed phenotyping system . The system was operated following the provided instructions, with precise settings and appropriate threshing thresholds to ensure accurate measurements. Six specific traits were analyzed in this study, including GW, GL, GLWR, area size (AS) of grain, perimeter length (PL) of grain, and TGW. The grains were then weighed using an electronic balance, and the average values from the three plants were recorded as the final phenotypic values for each trait.The data were recorded, analyzed, and processed using WPS Office Excel. Histograms depicting the different traits of the RIL population across multiple environments were created. Statistical analysis was performed using DPS v9.01, and significant differences were detected using the LSD method. Correlation analysis was conducted using SPSS Statistics 26.8:9 generation) were collected. The DNA extraction and sequencing analysis were entrusted to Biomarker Technologies Co., Ltd. . The specific steps were as follows. First, DNA extraction was performed using the CTAB method. After sample validation, the DNA was randomly fragmented using the Covaris ultrasonic disruptor. The fragmented DNA was subjected to end repair, 3′ end adenylation, sequencing adapter ligation, purification, and PCR amplification to construct sequencing libraries. The Illumina sequencing platform was used to generate raw sequencing data, known as sequencing reads. The data underwent filtering steps to remove adapter contamination, reads with an N content exceeding 10%, and low-quality reads. This resulted in obtaining high-quality clean reads for subsequent analysis. The sequencing reads were aligned to the reference genome of the rice variety ‘Nipponbare’ (IRGSP-1.0) using the BWA software . Only uniquely aligned paired-end reads were retained. Subsequently, the GATK software (version 3.6-0-g89b7209) [During the tillering stage of rice, tender leaves of both the parental lines and the RIL population (F89b7209)  was empl89b7209)  was emplThe high-density genetic map constructed based on whole-genome resequencing was used for trait mapping using the composite interval mapping (CIM) method. A significance threshold (PIN) of 0.001 was set, and the genome-wide scan was performed with a step size of 1 cM. The LOD threshold was determined through 1000 permutations using the PT (permutation test) method. Initially, a confidence level of 99% was set, and if no mapping interval was identified, it was reduced to 95% or 90%. If still no result was obtained, the PT test results were not considered, and the LOD threshold was manually lowered to 3.0. If no interval was identified at 3.0, it was further reduced to 2.5 or 2. The LOD value at the peak was considered the LOD value of the corresponding QTL. The effect of the QTL was estimated based on the bin marker at the peak position, and the additive effect and the contribution rate of each QTL to the trait were calculated. QTLs were named following the principles previously described . A positThe genotyping results of all bin markers within each mapping interval were analyzed. The genotypes of the 208 offspring in this interval were classified as “aa” and “bb” types. If different genotypes of bin markers were observed within the interval, it indicated the occurrence of recombination events, and such intervals were classified as heterozygous intervals. Stable QTL refers to the loci that can be repeatedly identified across multiple environments. The stable QTLs with higher contribution rates were selected. The distribution of enhancing alleles in the RIL population was analyzed. The RIL population was grouped based on the pyramiding of different favorable genes. The performance of traits in each group was evaluated to analyze the aggregation effect of different numbers of favorable genes.In the present study, we constructed a high-density genetic map of a RIL population derived from the cross between R8605 and ZP37. This map consisted of 2193 bin markers with an average physical distance of 201.29 kb between markers. A total of 39 QTLs related to rice grain traits were identified in three different environments, with the mapping intervals ranging from 8.1 kb to 1781.6 kb and an average physical distance of 517.5 kb. By analyzing the genetic effects of the identified QTLs, 14 stable genetic loci (involving 30 QTLs) were obtained. Among them, four loci were novel loci exhibiting pleiotropic effects, controlling at least three traits and detected in multiple environments. Furthermore, pyramiding effects analysis of three loci showed that as the number of QTL increased, the phenotypic values of grain traits could be effectively improved. Collectively, our newly identified QTLs could be used in future breeding programs to enhance rice yield.


IDH) is the most mutated metabolic gene in human cancer. Mutations in this gene have been identified in a high percentage of gliomas, in acute myeloid leukemia (AML) and in many other malignancies. Mutant IDH causes tumour initiation, possibly through accumulation of the oncometabolite D2-hydroxyglutarate, a product of normal metabolism with limited known function in mammals. Many studies have tried to elucidate the biological consequences of mutant IDH and to find effective inhibitors targeting this enzyme. This review delves into the cellular, biochemical and molecular consequences of mutant IDH and the therapeutic strategies for treating IDH mutant cancers.Isocitrate dehydrogenase (IDH) is mutated in many gliomas and other cancers. Physiologically, IDH converts isocitrate to α-ketoglutarate (α-KG), but when mutated, IDH reduces α-KG to D2-hydroxyglutarate (D2-HG). D2-HG accumulates at elevated levels in IDH mutant tumours, and in the last decade, a massive effort has been made to develop small inhibitors targeting mutant IDH. In this review, we summarise the current knowledge about the cellular and molecular consequences of IDH mutations and the therapeutic approaches developed to target IDH mutant tumours, focusing on gliomas.Altered metabolism is a common feature of many cancers and, in some cases, is a consequence of mutation in metabolic genes, such as the ones involved in the TCA cycle. Isocitrate dehydrogenase ( Moreover, unlike IDH1 and 2, the chemical reaction catalysed by IDH3 is tightly regulated by substrate availability and allosteric effectors , nicotinamide adenine dinucleotide (NAD+), Mg2+/Mn2+and Ca2+), product inhibition (NADH and α-KG) and competitive feedback inhibition (adenosine 5′-triphosphate (ATP)) [The isocitrate dehydrogenase (IDH) family includes three enzymes, IDH1, IDH2 and IDH3, ubiquitously expressed in human cells. Although all these enzymes catalyse the same chemical reaction, i.e., the oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (α-KG), each enzyme has unique biological properties. IDH1 is localised to the cytoplasm and peroxisomes of the cell, where it has a role in glucose and lipid metabolisms and protects against reactive oxygen species (ROS) . IDH2 anto NADPH , while te (ATP))  to avoidIDH1 and IDH2 have been identified in many cancers, such as low-grade gliomas (LGG) (>70%), secondary glioblastoma  (55–88%), primary glioblastoma (5–14%) [In recent decades, recurrent somatic mutations in astoma 5–% [14,15, (5–14%) ,17, cart (5–14%) ,23,24, i (5–14%) ,31,32,33 (5–14%) ,40,41,42 (5–14%) ,44,45,46delines) –88%, pri (5–14%) ,57,58,59IDH1 and IDH2, no tumour-associated mutations for any IDH3 genes  have been reported [In contrast to reported .IDH changes are missense mutations, which predominantly affect the aminoacidic residues R132 and R100 in IDH1, and the equivalent residues R172 and R140 in IDH2 [ in IDH2 ,12,34,47 in IDH2 ,61. Spec in IDH2 ,61, resu in IDH2 ,61. Sinc in IDH2 ,38,61,62 in IDH2 , mainly  in IDH2 ,64. Cons in IDH2 ,69,70,71 in IDH2 .IDH1 and IDH2 mutations appear mutually exclusive [IDH1 gene, which is mainly converted to the R132H variant. IDH1 is most frequently mutated in chondrosarcoma and cholangiocarcinoma, specifically in the variant R132C [R132HIDH1 mutated tumours have lower levels of genome-wide DNA methylation, an increased gene expression and the worst prognosis compared to patients affected by tumours with other IDH1/2 mutations [xclusive ,40, withxclusive ,36,42. Int R132C . Interesutations .R132H has a relatively low ability to produce D2-HG compared to other mutations, such as IDH1R132C, which is highly more efficient [The reason for the tissue specificity of the various IDH mutations in different cancers is unclear. However, specific IDH mutations differ in their capacity to produce D2-HG ,74. For fficient ,74. Potefficient .It is well recognised that IDH mutations have a biological impact both intracellularly and as part of the tumour microenvironment, favouring tumour formation and recurrence. In recent decades, many functional studies have focused on understanding the molecular mechanisms responsible for the formation of IDH mutant tumour. These studies mainly focus on the functional significance of D2-HG accumulation, but despite many efforts, the biological mechanism driving tumour formation is not completely clear. Considering that current therapies fail to demonstrate improved outcomes, IDH-induced biochemical alterations should be adequately understood and assessed as potential targets. Here, we summarise the current knowledge of IDH-mutated human malignancies, focusing on glioma, discussing the oncogenic role of mutant IDH in tumour formation and the therapeutic opportunities.IDH mutations result in significant alterations and reprogramming of the cellular metabolic pathways. Indeed, the production of D2-HG leads to significant reduction and drainage of TCA cycle substrates, and thus, carbohydrate sources ,65. ConsIn IDH mutant cells, a considerable reduction in α-KG has been reported by most, but not all studies ,70,71,76LDH gene was the main reason for the lack of LDH expression [Compared to other tumours, IDH mutant gliomas show a distinctive metabolic behaviour. In most cancers, the need for an adequate energy supply is mediated by the increased expression of lactate dehydrogenase (LDH) . LDH catpression ,84. Moreover, IDH mutant gliomas rely less on the glycolytic pathway and more on oxidative phosphorylation to produce energy than wild-type (WT) IDH1 gliomas . The relIDH determine an increased affinity of the enzyme for NADPH and α-KG [+ ratio. This disrupts the reducing equivalents of biochemical reactions in the cell, accumulating reactive oxygen species (ROS) [IDH1 and IDH2 are essential sources of NADPH in the cytoplasm and mitochondria, respectively . Mutatioand α-KG ,61, leades (ROS) ,88. Excees (ROS) .Consequently, ROS accumulation is fundamental and a hallmark of cancer biology, especially for IDH-mutated gliomas. Cells derived from IDH1 mutated gliomas exhibit strong oxidative stress, evident by the increased expression of manganese superoxide dismutase . The eleEpigenetic alterations affect gene expression without causing permanent changes to the DNA sequence as mutations would. Epigenetic changes range from DNA methylation, non-coding RNA molecules  and covalent modifications to histones, such as acetylation, methylation and phosphorylation ,95. IDH The Cancer Genome Atlas (TCGA) database found a strict correlation between IDH mutation and the development of a unique DNA methylation signature [Analysis of DNA methylation changes of glioblastoma (GBM) samples from ignature ,98. Thisignature . Howeverignature . A studyignature .Methyltransferases and demethylases control the methylation pattern of the DNA. During the DNA demethylation process, the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) is catalysed by the methylcytosine dioxygenases TET  in an α-KG-, oxygen- and iron-dependent manner. In addition, TETs concomitantly catalyse cytosine demethylation steps by converting 5-hmC to 5-formyl cytosine (5-fC) and then to 5-carboxyl cytosine (5-caC) . 5ca-C oAdditionally, D2-HG aids histone methylation by inhibiting histone demethylases . One notIt is now recognised that histone and CpG island hypermethylation patterns are predominantly found in IDH mutant glioma stem cells (GSCs) . StudiesThe D2-HG produced by mutant IDH accumulates tomM ranges intracellularly ,62 and eThe microenvironment surrounding glioma is rife with a variety of immune cells, including tumour infiltrating lymphocytes (TILs), natural killer (NK) cells, tumour-associated macrophages (TAMs), microglia, myeloid-derived suppressor cells (MDSCs), neutrophils, dendritic cells and fibroblasts .Cxcl-2, Ccl-2 and C5 [+T lymphocytes compared to their WT counterpart; this was also confirmed using a syngenic mouse model of glioma, where expression of mutant IDH or treatment with 2-HG reduced the infiltration of cytotoxic CD8+T lymphocytes and levels of T-cell-associated chemokines within tumours. Interestingly, these effects were reversible using a specific inhibitor of mutant IDH1 [Increasing evidence denotes that mutant IDH can decrease the levels of tumour immune surveillance, enabling cancer cells to escape it. Various glioma studies have shown that mutant IDH affect tumour immune-cell infiltration and function by modulating chemokine secretion. For instance, IDH1 mutation has been shown to reduce leukocyte chemotaxis into gliomas in a syngenic mouse model by repressing the expression of key chemoattractant cytokine genes, including 2 and C5 . Other sant IDH1 . Accordiant IDH1 . The inh1 with a pro-inflammatory signature and M2 with an anti-inflammatory signature [Many recent studies have also indicated that IDH mutation modifies TAM phenotypes to influence glioma growth. TAMs are the primary innate immune effector cells in malignant gliomas and can be in vivo separated into Mignature . Mutant ignature . Anotherignature . Conversignature .Mutation in IDH1 plays an essential role in TAM activation. D2-HG can be taken up by TAMs using membrane transporters, favouring the activation of the M1 state of TAMs . An in v+ and CD8+ T cells was inhibited by treatment with D2-HG, while T-cell proliferation and IFNγ production were both inhibited in a dose-dependent manner [Other recent studies have shown that migration of both CD4t manner . This prThe role of D2-HG in immune suppression was further elucidated using genetically engineered CAR-T cells. CAR-T cells were generated, which were either a knock-out (KO) or overexpression (OE) of the D2-HGDH enzyme—D2-HG dehydrogenase—the enzyme responsible for the conversion of D2-HG to α-ketoglutarate. The D2-HGDH-KO and -OE CAR-T cells were tested using the cell line NALM6 overexpressing mutant IDH1. The results show that D2-HGDH-OE CAR-T cells had a profound rate of tumour cell death compared to D2-HGDH-KO CAR-T cells. Mice bearing mutant IDH1 tumours showed a significant increase in their survival following treatment with D2-HGDH-OE CAR-T cells. It was later revealed that the mechanism behind this was the reduction in D2-HG, and therefore, the removal of immune suppression, which increased memory cell differentiation and cytokine production ,116.+ T-cell inhibition [+ T cells to perform glycolysis by direct inhibition of lactate dehydrogenase (LDH). In vitro studies using purified, recombinant LDH protein and exogenous D2-HG showed a non-competitive inhibition of the enzyme, leading to altered glycolytic flux in the CD8+ T cells. Application of D2-HG to CD8+ T cells led to a reduction in the production of IFN-γ and therefore also in its autocrine effects on gene expression. Encouragingly, the results in vitro are also found in vivo when analysing samples from IDH mutant tumours. These glioma samples demonstrate reduced IFN-γ-related gene expression and reduced lactate levels due to the inhibition by D2-HG [A recent study provides new evidence for mechanistic insight into the effects of D2-HG on CD8hibition . Notaranby D2-HG .Together, these data show that mutant IDH enzyme, via the aberrant production of D2-HG, leads to suppression of the immune system in terms of activation and infiltration. This leads to an immunologically “cold” tumour microenvironment, which improves overall patient survival but limits treatment options, as immune-targeted therapies are rendered ineffective. Further understanding of the difference in the immune system’s role in IDH WT and mutant gliomas will be essential to finding ways to target these tumours effectively.IDH1, in cells treated with exogenous 2-HG, in IDH mutant tumours and in brains of mouse embryos expressing idh1 R132H [idh1 was expressed [Contrasting data exist in the literature regarding the ability of mutant IDH to increase the levels of hypoxia and the formation of new blood vessels within the tumour. It was initially hypothesised that reduced levels of α-KG  and the h1 R132H ,70,118. h1 R132H , and folh1 R132H ,120 or ixpressed . Furtherxpressed ,122. Recxpressed .Similarly, a gene set variation analysis of a cohort of TCGA patients has shown that IDH1/2 mutations were associated with inhibiting angiogenesis signalling pathways. The same study also found that IDH mutant gliomas tend to have a lower relative cerebral blood volume compared to the IDH WT counterpart, indicative of a decreased angiogenesis of IDH mutant samples compared to the WT . HoweverPatients affected by IDH mutant gliomas present more frequently with epileptic activity than patients with IDH wild-type gliomas ,127. UnfGlioma cell invasion is one of the main reasons for tumour recurrence, and thus, poor prognosis. The exact mechanisms underlying invasion are yet to be elucidated. Mathematical modelling using data obtained from MRI scans has demonstrated that IDH mutant tumours are less aggressive but more diffuse and invasive than their WT counterparts . To explIn colorectal cancer cells, D2-HG was shown to promote motility and invasion of cancer cells. Exogenous administration of D2-HG directly induced epithelial–mesenchymal transition (EMT) by increasing the genetic expression of ZEB1, a master regulator of EMT. Interestingly, D2-HG was applied at 250 µM, while endogenous production of D2-HG by IDH mutation has been known to reach the mM range . This woInterestingly, in vivo studies in which IDH1 R132H was selectively induced in the brain subventricular zone (SVZ) of the adult mouse demonstrated that SVZ cells gained the ability to proliferate ectopically and spread outside of the SVZ into the surrounding brain regions. This process does not occur in healthy mice . This in+ to NADH [IDH3 is the third member of the IDH family and a vital enzyme of the TCA cycle. As mentioned previously, IDH3 catalyses the irreversible conversion of isocitrate to α-KG, meanwhile reducing NAD to NADH . NADH, p to NADH .Increasing experimental evidence indicates that IDH3 is involved in the epigenetic regulation of the genome. The α-KG is an obligatory co-factor of the α-KG-dependent dioxygenases, a superfamily of enzymes involved in many biological processes, including epigenetic regulation of gene transcription . IDH3, bIDH3A and IDH3B have been linked to inherited retinal diseases (IRDs), which are a leading cause of blindness in children and adults [IDH3A has been associated with a severe form of encephalopathy, which causes neurological defects from birth and retinal degeneration [Loss-of-function and missense mutations in d adults ,148,149.d adults ,157,158,neration .IDH3 has not been found mutated in any tumours. In a screen of 47 glioblastoma samples, no IDH3 mutations were found [Currently, re found . Howeverre found ,163,164.IDH3α is overexpressed in various human cancer cell lines  and cancMTA1), an oncogene regulating cancer progression and metastasis [Recently, IDH3α has been found to promote the progression of HCC by inducing expression of metastasis-associated 1 , AG-881 (vorasidenib) and AG-221 (enasidenib) are the second generation of selective, reversible drug inhibitors produced, which the F.D.A. approves for the treatment of acute myeloid leukaemia ,170. AG-R140Q ,171. TheR140Q . Howeverhibition . Subsequhibition  with subR132H mutant cell lines selectively [Celgene/Agios developed AG-120 (Ivosidenib) to optimise the AGI-5198 for human therapeutic applications . Broad sectively . A signiectively . More anR132H mutant glioma xenograft mouse model [Regarding mouse models, a dose-dependent 2-HG depletion has been observed across cell types, including an IDH1se model ,192,193.se model . Afterwase model . In the se model . The latse model . Howeverse model . Clinicase model . On the R132H mutation [R132C, G, H, L, S [R140Q and IDH2R172K mutant enzymes, 2-HG inhibition was also shown, and cellular differentiation was increased [AG-881, also named Vorasidenib, is the only oral pan-inhibitor for IDH1 and IDH2 mutations developed by Celgene and Agios Pharmaceuticals ,177,196. H, L, S . In TF-1ncreased . Accordincreased . The medncreased . Doses >ncreased . ApproxiR132 mutants in AML human-derived cell lines and genetically engineered lines of solid tumours with reduced proliferation and induction of differentiation. In astrocytoma xenograft IDH1R132H mouse models, the drug effectively penetrated the BBB with prolonged survival. Two ongoing AML and solid tumour trials are reviewed here [R132X mutant advanced solid tumours, including gliomas [BAY-1436032 is a relatively new species, non-competitive IDH1m inhibitor with adequate pre-clinical results in AML and glioma models . This cowed here . The gli gliomas   is linked to hypermethylation at sites for cohesion and CCCTC-binding factor (CTCF), leading to the reduced affinity of this protein . The CTFDNA is susceptible to various damaging agents from within the body, such as replication errors and toxins, and from outside the body, such as UV and ionising radiation . BecauseAs mentioned, the tumourigenic effects of D2-HG are proposed to derive from modulating the α-KG-dependent dioxygenases, many of which are involved in histone and DNA methylation, but also enzymes playing critical roles in the DNA damage response, such as alkB homologue (ALKBH) enzyme ,220. WanD2-HG can also compromise the HR of DNA damage response . Sulkows+, which is needed for PARP during TMZ-induced BER, using GMX1778 and inhibiting Nicotinamide phosphoribosyltransferase (NAMPT) using FK866, eliminates the remaining repair activity of PARP [Conversely, inhibiting the neomorphic activity of mutant IDH1 with specific small molecule inhibitors reversed the deficiency in HR, with the number of double-strand breaks almost returning to that seen in IDH1 WT cells. Interestingly, the PARP inhibitor sensitivity induced by mutant IDH1 is present and functional in both patient-derived AML and glioma cells . Taken t of PARP ,222. Thihttps://clinicaltrials.gov/ct2/show/study/NCT02152982, accessed on 20 March 2023).Additionally, a currently active phase II/III trial—NCT02152982—investigates whether the combination of TMZ and PARP inhibitor veliparib is more effective at treating newly diagnosed glioblastoma than TMZ alone. The recruited patients must have MGMT promoter methylation, as this indicates that tumours are sensitive to TMZ and that the added inhibition of PARP DNA repair would further exacerbate DNA damage, leading to a higher rate of cell death . At the TERT gene, which encodes for the telomerase, the enzyme responsible for maintaining the structure of telomeres Consequently, cells would be stunted in their growth, as they would not be able to maintain the length of their telomeres. However, TERT loss is usually accompanied by the deletion of ATRX and IDH1 mutation, which, when combined in cells, is associated with alternative lengthening of telomeres (ALT) [The telomere lengthening is a mechanism cancer cells employ to maintain their growth over time . Low-graes (ALT) . ALT is es (ALT) . In low-es (ALT) . Howeveres (ALT) . In concBy producing D2-HG, IDH mutant enzyme drains some of TCA cycle intermediates. Therefore, anaplerotic pathways are recruited to compensate for the loss of TCA cycle metabolites, such as α-KG. Understanding and delineating the new metabolic alterations will allow us to evaluate potential druggable targets as novel therapeutic options.+ ions is important for the electron transport and metabolism of redox reactions, including glycolysis, TCA cycle and serine biosynthesis. Furthermore, by affecting the activity of several enzymes, NAD functions as an essential element for various signalling pathways. In the cell, NAD is produced from two different pathways, the de novo pathway or the ubiquitous salvage pathway  [NAD is an indispensable co-factor in the cell, as carrying Hrnative) . Even thrnative) .NAPRT1 gene [In IDH mutant glioma cells, the alternative salvage pathway of NAD is reduced due to the epigenetic silencing of the RT1 gene . Thus, iRT1 gene . Thus, iMoreover, in IDH mutant cells, glutaminolysis is the major pathway of metabolic compensation due to a lack of isocitrate . Thus, tROS are predominantly elevated in IDH-mutated tumours . It was One main consequence of IDH mutation is its ability to block normal cellular differentiation. Studies performed in various IDH mutant malignancies have demonstrated that mutant IDH can halt normal cell differentiation, causing an expansion of progenitor cells, which is an important step in cancer development ,237,238.In the glioma context, Rohle et al. demonstrated that inhibition of mutant IDH by using a small IDH mutant selective inhibitor, AGI-5198, hampered the in vitro and in vivo growth of an oligodendroglioma cell line (TS603), harbouring an endogenous IDH1-R132H mutation. Treatment of mice xenografts with AGI-5198 caused an upregulation of several genes involved in glial differentiation, while the in vitro treatment of TS603 cells with AGI-5198 showed a loss of repressive histone marks at the promoters of astrocytic marker genes. This study demonstrated that, at least in this model, targeting mutant IDH1 can impair glioma development in vivo, which is related to changes in cellular differentiation .More recently, an undifferentiated glioma cell line, BT142, which expresses endogenous IDH mutation and has an undifferentiated cell state was treated with a mutant IDH inhibitor (MRK-A). MRK-A caused decreased expression of some key stem cell markers in vitro indicating an onset of differentiation. Treatment of mice bearing orthotopic BT142 tumours with MRK-A showed a reduction in the 5mC DNA signature, indicating a release of the hypermethylation phenotype seen in IDH mutant tumours. The treatment with MRK-A in vivo showed a significant upregulation of 245 genes, which is in line with removing the hypermethylated phenotype. Mice that received MRK-A treatment showed an extended survival and reduced Ki67 staining compared to their vehicle-treated control group . This stIDH inhibitors as differentiation therapy have also been shown in haematological malignancies, such as acute myeloid leukaemia (AML). Wang et al. developed a specific inhibitor for the most commonly occurring IDH mutation in AML, IDH2-R140Q, AGI-6780. Treatment with this inhibitor of an erythroleukemia cell line, which ectopically expressed IDH2-R140Q, decreased the levels of 2-HG and released these cells from the block to differentiation. Similar studies on IDH2-mutated primary human AML cells showed a reduction in 2-HG levels and an increased differentiation of mutated AML cells . These dIDH1-mutated tumours [More recent studies have shown that AG-120 (ivosidenib) treatment decreased intracellular levels of 2-HG and induced differentiation in models of  tumours ,239. Rec tumours . AG-120  tumours .The simultaneous use of an IDH1 inhibitor, BAY1436032, and a DNA methyltransferase inhibitor, azacitidine, has proven to be a novel, clinically relevant drug combination in treating AML. The frequency of leukaemia stem cells (LSCs) in PDX models was analysed, and it was found that they were decreased 4.1-fold with azacytidine alone and 117-fold with BAY1436032 alone. However, the frequency of LSCs was reduced by 470-fold when the drugs were given sequentially and by 33,150-fold when given simultaneously. These data show that it is important to continue evaluating the synergistic effects of different drugs. Similarly, azacitidine has also been shown to reverse the differentiation block caused by mutations in IDH2 in chondrosarcoma cell lines . This raThe use of ivosedenib or enasidenib in treating mutant IDH AML presents a risk of the patient developing differentiation syndrome (DS) . While tIDH1 and IDH2, and a few other metabolic genes, such as succinate dehydrogenase (SDH) and fumarate hydratase (FH), have provided compelling evidence for metabolic alteration in human cancer development and progression. However, the mechanism from altered metabolism to tumourigenicity is still unclear.In last decades, mutations identified in Increasing genetic evidence has indicated that mutant IDH1 is a driver of gliomagenesis and a rational therapeutic target, leading to a massive effort to design and test specific IDH mutant inhibitors. Although targeted inhibitors of mutant IDH have shown promise in IDH mutant acute leukaemia patients, no benefits have been obtained for patients affected by gliomas, despite the reduction in D2-HG after treatment with inhibitors. Here, we outline key metabolic alterations, epigenetic changes and redox imbalance, which might not be uniform across IDH mutant gliomas and ongoing targeted therapeutic options, with several clinical trials specific to IDH inhibition recently published. However, more light needs to be shed on the possible oncogenic role of D2-HG in IDH mutant malignancies, and more research needs to be focused on the metabolic, cellular and molecular consequences associated with IDH mutations in addition to the accumulation of D2-HG. New IDH1 mutant glioma models are needed to examine the spectrum of responses to treatment, which may be observed clinically following the administration of IDH1 mutant inhibitors. Considering that current therapies fail to demonstrate improvement in glioma treatments, IDH-induced biochemical alterations should be adequately understood and assessed as potential targets.