Bone contributes to supporting the body, protecting the central nervous system and other organs, hematopoiesis, the regulation of mineral metabolism , and assists in respiration. Bone has many functions in the body. Recently, it was revealed that bone also works as an endocrine organ and secretes several systemic humoral factors, including fibroblast growth factor 23 (FGF23), osteocalcin (OC), sclerostin, and lipocalin 2. Bone can communicate with other organs via these hormones. In particular, it has been reported that these bone-derived hormones are involved in glucose metabolism and diabetic complications. Some functions of these bone-derived hormones can become useful biomarkers that predict the incidence of diabetes and the progression of diabetic complications. Furthermore, other functions are considered to be targets for the prevention or treatment of diabetes and its complications. As is well known, diabetes is now a worldwide health problem, and many efforts have been made to treat diabetes. Thus, further investigations of the endocrine system through bone-derived hormones may provide us with new perspectives on the prediction, prevention, and treatment of diabetes. In this review, we summarize the role of bone-derived hormones in glucose metabolism, diabetic kidney disease, and cardiovascular disorders. Diabetes has become a major social health problem throughout the world. Diabetes is caused by both insufficiency of insulin secretion from pancreatic β cells (mainly type 1 diabetes) and increased insulin resistance in the liver and peripheral tissues due to the presence of lifestyle factors including obesity, lack of exercise, and an unbalanced diet (mainly type 2 diabetes). In addition, diabetic complications have been characterized by micro- or macrovascular dysfunction. However, the concept of diabetic complications has become extensive and complicated ,6,7,8,9.It has been revealed that bone secretes several hormones and systemically regulates the functions of other organs . CurrentSLC34A1 and SLC34A3, respectively [2D] level by suppressing the expression of CYP27B1 and enhancing the expression of CYP24A1 [CYP27B1 and CYP24A1 encode 25-hydroxyvitamin D-1α-hydroxylase and 25-hydroxyvitamin D-24-hydroxylase, respectively. Because 1,25(OH)2D enhances the expression of NaPi-Ⅱb, which is encoded by SLC34A2 in the intestine, FGF23 reduces intestinal phosphate absorption by suppressing the 1,25(OH)2D level and the expression of NaPi-Ⅱb [FGF23 was the first bone-derived hormone to be reported. FGF23 was identified as a gene or a humoral factor responsible for hypophosphatemic rickets/osteomalacia ,13. FGF2ectively . At the  CYP24A1 . CYP27B1 NaPi-Ⅱb . Finally NaPi-Ⅱb ,21. α-Kl NaPi-Ⅱb ,27. Rece NaPi-Ⅱb . In addi NaPi-Ⅱb ,30, iron NaPi-Ⅱb ,32, hypo NaPi-Ⅱb , erythro NaPi-Ⅱb  were rep NaPi-Ⅱb .Fgf23 gene results in hypoglycemia and increased peripheral insulin sensitivity [Hypophosphatemia was reported to be the cause of poor glycemic control by decreased insulin secretion due to the fact of impaired adenosine triphosphate (ATP) production in pancreatic islets ,38. Furtsitivity ,46. Howesitivity ,48,49,50sitivity . WhetherThe impact of FGF23 on diabetes is quite complex. In some studies, diabetes was associated with higher blood FGF23 levels ,50,52, w2 persisted after adjustment for eGFR, linking a higher FGF23 level with adverse outcomes, even in patients without impaired kidney function [Recent reports showed that a higher blood FGF23 level is a novel predictor of all-cause and cardiovascular mortality in patients with type 2 diabetes ,64,65,66function . Furtherfunction ,68. It wfunction . Patientfunction . Furtherfunction ,26,28,71function , vasculafunction ,74, impafunction , inductifunction , and stifunction . While tfunction . Becausefunction ,23,79, tOsteocalcin (OC) is a bone-derived hormone directly associated with glucose metabolism . OC is pSubsequently, it was reported that OC increases the secretion of insulin from pancreatic β cells and insulin sensitivity in peripheral tissues through experimental models using OC knockout mice . A new cIt was revealed that ucOC has multiple functions , includiSince the discovery of the metabolic functions of OC in rodents , evidencOn the other hand, several issues remain to be clarified. First, large-scale prospective clinical research concerning ucOC is currently insufficient. Second, it is unclear whether ucOC is the active form in humans. Clinical and epidemiological studies have assessed the associations of total OC with diabetes. The standard assay of OC detects both the carboxylated and uncarboxylated forms, indicating total OC. A meta-analysis showed that a higher total OC level was associated with an increase in HOMA-B and a decline in the fasting plasma glucose level, HbA1c, HOMA-IR, and body mass index (BMI) . In addiLately, a prospective cohort study with large samples has reported the association of baseline OC levels and risk of incident diabetic kidney disease . Blood lIt was indicated that the blood OC levels are decreased in postmenopausal women with diabetes . AlthougSclerostin is mainly expressed in osteocytes and suppresses bone formation via the inhibition of the Wnt-low-density-lipoprotein receptor-related protein (LRP) 5/6 signaling pathway on osteoblasts . Sclerosstimulatory subunit of G-proteins (Gsα) in osteocytes, beige adipogenesis was increased in white adipose tissue with the suppression of Wnt-LRP5/6 signaling [uncoupling-protein 1 (Ucp-1), in white adipose tissue of wild-type mice by inhibiting Wnt-LRP5/6 signaling [Gsα knockout mice decreased high expression of Ucp-1 in white adipose tissue [On the other hand, it was shown that circulatory sclerostin induces browning of white adipose tissue by inhibiting Wnt-LRP5/6 signaling in an experimental model . Althougignaling . These mignaling . In addiignaling . Furthere tissue . In addie tissue . TherefoThe association of increases in blood sclerostin level with diabetic cardiovascular diseases has been reported. It was reported that the blood sclerostin level can become reliable diagnostic and prognostic biomarker of cardiovascular risk in patients with type 2 diabetes . AnotherLipocalin 2 is also termed 24p3 or neutrophil gelatinase-associated lipocalin (NGAL) . LipocalTo date, the relationship between bone-derived lipocalin 2 and diabetic complications have not been elucidated. In clinical studies, it was demonstrated that increased systemic levels are associated with severity of coronary artery disease and increased risk of incidence cardiovascular diseases ,148. FurAs an endocrine organ, bone has various effects on glucose metabolism and diabetic complications through bone-derived hormones. However, several issues remain to be clarified. There may be differences in the bone and glucose metabolism of mice and humans. Furthermore, in vitro experiments cannot mimic in vivo environments. In particular, the pathogenesis of diabetes is much more complicated in humans, involving multiple genetic and environmental factors, including social, educational, and economic status . TherefoThe functions of bone extend beyond supporting the body, protecting the internal organs and the central nervous system, and its contribution to hematopoiesis. Bone acts as an endocrine organ to secrete several hormones and regulate functions of other organs through the formation of a multi-organ network system. Especially, the involvement of several bone-derived hormones in glucose metabolism and diabetic complications has been identified. We believe that further investigations concerning the network system among multiple organs via bone-derived hormones may give us a new perspective that facilitates the prediction, prevention, and treatment of diabetes and its complications.


Pregnancy loss predominantly occurs during the first 3–4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers. In cattle, pregnancy loss predominantly occurs during the first three to four weeks of gestation due to fertilization failure or early embryonic losses during the period of luteolysis or maternal recognition of pregnancy that occurs from day 14 to day 24 (day 0 = day of estrus) , resultiISG15, OAS1, MX1 and MX2, has been found to increase on day 15, peaks on day 20 and declines on day 22, of which expression is closely associated with those of IFNT [Data has been accumulated that although IFNT is generally considered not escaping from the uterine lumen, IFNT has recently been found to upregulate the expression of interferon stimulated genes (ISGs) in the endometrium , luteal  of IFNT ,10,11.Based on these results, intensive research has been conducted on the development of pregnancy diagnosis methods in AI-cattle. These include CL examination by Doppler ultrasonography , ISG expDevelopment of a method effectively identifying pregnant cattle from NP ones, which go through AI or ET procedure, requires, (a) the method to be convenient, and to be able to be used in the field, and (b) the method to be able to identify pregnant or NP cattle before or on day 22, the length of regular estrous cycles. In this regard, pregnancy associated glycoproteins (PAGs) would be ideal to identify pregnant from NP ones. However, the pregnancy diagnosis with the PAGs method can be conducted on or after day 28. It would thus be desirable if serum components or secreted body fluids, which could be used to develop a method for pregnancy diagnosis, are identified.To develop a simple method for pregnancy diagnosis, which detects pregnancy status on days 20–22 and could be used in the field, we opted to study serum components in pregnant and NP heifers. During the period of maternal recognition of pregnancy, conceptuses go through elongation, attachment, and implantation to the maternal endometrium . These cA total of 20 heifers, which had regular estrous cycles and responded to the PGF2α treatment, were subjected to this study, from which 17 heifers went through the ET procedure while the remaining three did not. Among 17 ET heifers, 12 heifers became pregnant and five were found as NP heifers, 70% pregnancy rate .n = 3) cyclic, or days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment) (pregnant (n = 12) and NP (n = 5)) heifers, were subjected to metabolome analysis. These data were subjected to principal component analysis (PCA), and serum components were divided as either serum from heifers with or without ET, or heifers pregnant or NP (data not shown). Heatmap with serum on days 17, 20, and 22 was also partly divided into pregnant and NP heifers (p < 0.05) (l-lysine (AUC 0.82), and n-methylethanolamine (AUC 0.73) were detected (p < 0.05) (p < 0.05) L-alanine (AUC 0.92), 1-hexadecanol (AUC 0.9), and 2-aminoisobutanoic acid (AUC 0.83) as the amino acid contents that differed between pregnant and NP heifers  b. In day < 0.05) c. Furthe heifers d.n = 17) with those from the estrous cycle (n = 3). First, we compared serum metabolites among cyclic days 0, 7, 14, and 17. The heatmap and PCA showed that the estrous cycle data were divided into different cyclic days (We next compared the serum metabolomic data from ET heifers (lic days a. In thelic days b. Howevelic days c. Metabolic days d. Metabop < 0.05): angiotensinogen , cadherin-5 , fibronectin alpha chain , ADAM metallopeptidase with thrombospondin type 1 motif 13 , and histidine-rich glycoprotein (AUC 0.9)  c. FurtheAUC 0.9) d.p < 0.05): sorting nexin 5 , acetyl-CoA carboxylase alpha , cleavage and polyadenylation specific factor 6 , damage specific DNA binding protein 1 , and serum response factor binding protein 1 (SRFBP1)  c. To exa(SRFBP1) d. Howeve(SRFBP1) e. Moreov(SRFBP1) f.Using PCA and the heatmap with top 25 factors from the metabolome analysis, serum components were divided by pregnant at days 17, 20, and 22 from those of NP heifers. On each pregnant day sample, serum components were partly divided into pregnant and NP groups, some of which were higher in NP heifers than those of pregnant ones. Notably, serum metabolomic components from cyclic days 0, 7, 14, and 17 were distinctive due possibly to circulating P4, which also differed from those of days-17, -20, or -22 NP heifers. These data indicate that serum metabolomic components in NP heifers somewhat differ from those of cyclic ones, but those components were not sufficient enough to separate NP heifers from the pregnant ones during the periods being examined. In the proteome iTRAQ analysis, the heatmap with top 25 proteins separated into pregnant and NP heifers on day 20 or 22. Furthermore, ROC analysis identified five candidate proteins detecting heifers as not pregnant, of which serum SNX5 on day 22 had the highest AUC of 0.983. Using western blotting with day-22 serum, we detected more SNX5 in NP heifers than pregnant ones. These results indicate that peripheral blood on day 22, around two to three days after conceptus attachment to the maternal endometrium is initiated, reflects some differences between pregnant and NP heifers during the early pregnancy period, and suggests that high levels of SNX5 in peripheral blood on day 22 could predict pregnancy failure or those undergoing embryonic losses in ET heifers.SNX5 encodes a member of the sorting nexin family. Members of this family contain a phox (PX) domain, a phosphoinositide binding domain, and are involved in intracellular trafficking. SNX5 protein functions in endosomal sorting, the phosphoinositide signaling pathway, macropinocytosis, and micropinocytosis ,15,16. MFor the last decade and particularly recently, substantial data on pregnancy detection in cattle have become available: ISGs expression in PBMCs and PMNs , CL examIn conclusion, this study shows that metabolomic analysis of peripheral blood on days 17, 20, and 22 partly separates pregnant and NP heifers. The proteome iTRAQ analysis also separated serum protein components between pregnant and NP heifers on day 20 or 22. Furthermore, ROC analysis identified five candidate proteins detecting pregnancy failure, of which SNX5 was detected as a potential target for discrimination between NP heifers and pregnant ones. These results indicate that peripheral blood on day 22 can be used for a pregnancy test, and suggest that high levels of serum SNX5 on day 22 could predict females undergoing embryonic loss in heifers.n = 24), exhibiting regular estrous cycles, were given a single injection of 0.75 mg prostaglandin F2α , Kyoritu Pharmaceutical Co., Tokyo, Japan) to synchronize their estrous cycles and the heifers exhibited behavioral estrus 40–48 h after the injection . Twenty heifers were then divided into two groups, ET (n = 17) and non-ET (n = 3). For ET processes, in vivo-fertilized day-7 embryos were collected from super-ovulated and artificially inseminated (AI) Japanese black cows . Single blastocyst derived from the superovulation/AI procedure was then transferred non-surgically into the uterine horn of Holstein heifers, ipsilateral to the corpus luteum, on day 7 of the estrous cycle [All animal procedures in this study were performed in accordance with the guidelines of the Committee for Experimental Animals at Zen-noh Embryo Transfer Center , with the approval of the Institutional Animal Care and Use Committee of Zen-noh Embryo Transfer Center . All animals used were raised and kept at this center throughout the course of this experimentation. This study was carried out in compliance with the ARRIVE guidelines. Estrous synchronization, superovulation, and embryo transfer (ET) processes were performed as previously described . Holsteius cycle .n = 3) were collected on days 0, 7, 14, and 17 of the estrous cycle. The diagnosis of pregnancy or non-pregnancy (NP), indicative of early embryonic loss, was conducted by real-time B-mode ultrasonography  on day 30 of gestation, from which blood samples were divided into pregnant or NP groups. After sorting of blood samples, albumin was removed from serum samples by ProMax Albumin Removal Kit .Blood samples were collected from ET heifers on pregnancy days 17, 20, and 22. In addition, blood samples from non-ET heifers .A serum metabolomics analysis was performed using GC/MS as described previously  with somA global analysis of proteins using iTRAQ analysis was performed as described previously . BrieflySerum samples were separated through SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes . After blocking with Block Ace reagent , membranes were incubated with goat polyclonal anti-SNX5 , rabbit polyclonal anti-SRFBP1 , rabbit polyclonal anti-DDB1 , rabbit monoclonal anti-CPSF6 , or mouse monoclonal anti-ACACA  antibody. The second western blot was conducted with rabbit polyclonal anti-SNX5  antibody. Immunoreactive bands were detected using enhanced chemiluminescence  after incubation with horseradish peroxidase labeled anti-mouse, rabbit, or goat IgG . Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2) . The whop-value < 0.05 was considered statistically significant. The p-value was used to evaluate the significance of the ROC curve or other analyses in this study.All experimental data represent the results obtained from three or more independent experiments each with triplicate assays. Data were expressed as the mean ± SEM. A


Recently, a mobile tigecycline resistance gene tet(X4) has been identified in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet(X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized.Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of tet(X4).Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet(X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet(X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type  in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet(X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet(X4) was found adjacent to an insertion sequence ISCR2 downstream and a catD gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet(X4)-positive isolates.In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet(X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet(X4) raises concern for the use of tetracyclines in animal farming, and the tet(X4) gene should be listed as primary gene for resistance surveillance.The identification of  However, the discovery of a series of carbapenems resistance genes, such as  clinics . Under sacycline . The ideEscherichia coli  from 1 rising to 32 mg/L and leads to treatment failure in carbapenem-resistant Klebsiella pneumoniae infections , has again decreased the promised prospect of using tigecycline in the clinic  gene is that it also causes the resistance to both omadacycline and eravacycline  in duck and goose farms belonging to different breeding patterns in Southeast China. Isolation ratio for tet(X4) gene was analyzed, and the genomic profiling was conducted for a comprehensive understanding of the genomic background of tet(X4) gene. Furthermore, conjugation assays were tested to determine the dissemination and transferability of the tet(X4) gene.Aquaculture is a diversified production sector with different production systems and practices. Although centralization-breeding factory are increasingly popular in China, one of the integrated farming systems (duck or goose–fish production) still plays a significant role in southeast China . In thistet(X), and the tet(X4) subtypes were further confirmed by full-length amplification and Sanger sequencing, as previously described  and 16S rRNA sequencing. Then, for multiple isolates separated from the same sample, enterobacterial repetitive intergenic consensus (ERIC)–PCR was performed for preliminary typing using previous primers , soil (259), sewage (228), and dust (18) samples, from 25 duck farms and 3 goose farms in six provinces  along with the southeast coast of China . All ducescribed . The bac primers , and diftet(X4)-positive strains, MICs of tigecycline, chlortetracycline, amikacin, gentamicin, meropenem, colistin, ceftazidime, cefotaxime, fosfomycin, ciprofloxacin, ampicillin, and sulfamethoxazole–trimethoprim were determined and interpreted according to the Clinical & Laboratory Standards Institute (CLSI) guidelines . DNA purity and concentration were determined using a NANODROP 2000c spectrophotometer. Whole-genome sequencing (WGS) was performed with the Illumina HiSeq 2500 system  using the paired-end 2% 150-bp sequencing protocol. The draft genome was de novo assembled using SPAdes version 3.12.0. The putative coding sequences of the flanking regions of tet(X4) were obtained using RAST1. Multilocus sequence typing (MLST), plasmid in-compatibility (Inc) groups, antibiotic resistance genes (ARGs), and mobile elements were analyzed by Center for Genomic Epidemiology2 and ISfinder3.Genomic DNA was extracted from tet(X4)-positive E. coli strains were classified by XbaI digested  pulsed-field gel electrophoresis (PFGE) according to the PulseNet protocol4 using a CHEF Mapper System . PFGE patterns were compared using BioNumerics version 6.6  under appropriate optimization (1.5%) and tolerance (1.5%) settings and a cutoff at 85% similarity to delineate PFGE clusters. MLST was performed by the primers and protocol specified on the E. coli MLST database website5.All tet(X4) gene locations were identified using S1 nuclease-PFGE and Southern blot analysis. Briefly, DNA from donor strains and the transconjugants harboring tet(X4) were extracted and embedded in agarose gel plugs and then treated with S1 nuclease (Takara), and the DNA fragments were separated by PFGE. Southern blot hybridization was then performed with DNA probes specific for the tet(X4) gene that was non-radioactively labeled with a DIG High Prime DNA labeling and detection kit  (The Germany) .tet(X4)-bearing plasmid, conjugation assays were performed using streptomycin-resistance E. coli C600 as the recipient strain. Briefly, overnight cultures of donor and the recipient strains were 1:1 mixed and incubated at 37°C for 16 to 20 h. After incubation, 10-fold serial dilutions were mixed in sterile saline, and 100-μL samples were spread onto LB agar plates containing 4 mg/L tigecycline and 1,500 mg/L streptomycin. The tet(X4)-positive transconjugants were confirmed by PCR and ERIC-PCR (To investigate the transferability of ERIC-PCR . SusceptE. coli strains were identified harboring tet(X4) gene, among which nine were isolated from feces, six from soil, and one from sewage (tet(X4)-positive strains were detected from three provinces: Shandong, Jiangsu, and Guangdong -positive E. coli strains were resistant to tigecycline showing MICs over 16 mg/L (8 mg/L of FDA breakpoint)  . In addiakpoint) . The MICamikacin .tet(X4)-positive isolates and tet(X4)-bearing plasmids. The distinct tet(X4)-positive isolates belonged to seven sequence types (STs): ST3997 (n = 8), ST2325 (n = 1), ST48 (n = 1), ST3944 (n = 1), and ST746 (n = 1) and another two new STs labeling as New-1 (n = 3) and New-2 (n = 1) in tet(X4)-positive E. coli isolates from Guangdong province belonged to a new ST type (We further characterized the molecular structures of the  ST type .tet(X4) genes were successfully transferred to E. coli C600, and S1-PFGE of both donors and transconjugants suggested that these genes were located on plasmids. The tigecycline MICs of the transconjugants were increased by at least 32-fold over the C600 recipient strains (tet(X4) genes were mostly located on plasmids in sizes of 200, 230, or 330 kb (tet(X4) genes were confirmed by product enhanced reverse transcriptase testing of the corresponding transconjugants. During the mating assay, the cotransfer of IncHI1 and IncX1 plasmids was observed for YC101-1, YC113, YC102, and YCS29-1 as donors.In the mating tests, 11  strains . Furtherr 330 kb . Differetet(X4) gene was located primarily within three similar contigs in size of 2,700 to 9,553 bp bearing the core gene arrangement catD-tet(X4)-terlS-ISCR2-orilS (E. coli (1R and IS26 were identified at the upstream of tet(X4) gene in IncHI1 plasmid from 11 isolates and IncX1 plasmid from three isolates, respectively. This indicated that the tet(X4) genes are highly active and possibly will further transfer to other plasmids or isolates. For another two isolates, only orilS was found at the upstream of tet(X4) gene. Additionally, resistance genes of tet(A), aph, aadA, aadA2, blaCTX, blaTEM–1B, blaOXA, blaSHV, oqxAB, qnrS1, fosA3, mdfA, lnu, erm, cmlA1, floR, sul, and dfrA were detected in these tet(X4)-positive isolates as well -positive isolates were mostly reported in animals such as pigs, chickens, cows, and ducks. In our study, in the region of Southeast China, 0.7% (9/1211), 2.3% (6/259), and 0.4% (1/228) of studied fecal, soil, and sewage samples possessed tet(X4)-positive E. coli strains originated on duck farms. Although the detection ratio of tet(X4) in humans was low (0.07%), tet(X4) has been previously identified in pigs and chickens at slaughters, from soil and dust in animal farms (tet(X4) in animals may be due to the high-level use of tetracycline derivatives for livestock (tet(X4) gene possibly resulted from the evolution of tetX family members driven by the historical selective pressure due to the large-scale use of tetracyclines and had become the most worrisome resistant determinant to data. In addition, the importance of animals in the dissemination of tigecycline-resistance is only becoming recognized, and it might be just a matter of time when the tet(X4)-producing pathogens become widespread in public.For now, the al farms  . Among STs, ST3977 was the most prevalent type in Jiangsu province and has been reported harboring blaNDM–1 and mcr-1 genes in animals  isolates. In accordance with the antimicrobial susceptibility tests, the WGS analysis indicated that more than 20 different ARGs were carried in these isolates. For instance, we identified tet(X4), tet(A), aph(3’), aph(4), aph(6), aacA, blaCTX, blaOXA, blaTEM, oqxAB, qnrS1, fosA3, mdfA, lnu(G), floR, ARR-2, and sul2 genes in all the isolates from Jiangsu province. Notably, eight E. coli strains were detected cocarrying blaCTX–M–55 and blaCTX–M–14 genes, indicating the widespread and the diversity of ESBL resistance. Together with the similar PFGE patterns, it is possible that one epidemic clone has been disseminated in this region. The scenario is alarming as these isolates can act as an abundant reservoir spreading ARGs to both environment and humans.Whole-genome sequencing analysis showed that 16  animals . ST48 ha animals , indicattet(X4) strains from both patterns A and C breeding system. Duck farming in pattern A system often has high density of animals, and the duck–fish production system (pattern C) is much more economical than most traditional crop farming and poultry husbandry systems, because it is based on the concept that “there is no waste,” and “waste is only a misplaced resource that can be a valuable input for other component” (tet(X4) with the different breeding patterns. However, it is concerning that among all the tet(X4)-positive isolates, 45% (5/11) of the samples were isolated from environments in Shandong and Jiangsu provinces. Additionally, AMR genes including tet(X4) were identified in isolates recovered from sewage, river, and soil of ponds in this study, indicating the possible dissemination of AMR genes in environment.We detected mponent” . There wtet(X4)-positive E. coli isolates from duck farms in southeast China. Notably, this is the first study to report the development of diversity in the population of tet(X4)-positive E. coli isolates from ducks. WGS analysis further determined tet(X4) coexisted with other ARGs mediating multiple-drug resistance to chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin. The detection of tet(X4)-bearing IncHI1 and IncX1 plasmids in isolates from feces, soil, and even sewage samples enlarges our understanding of the dissemination of tigecycline-resistant genes as (i) tet(X4)-bearing IncHI1 and IncX1 plasmids were highly transferable and (ii) environmental-isolated strains could pose a greater threat to public health. It is absolutely the time to add the tet(X4) gene into the resistance surveillance.In conclusion, we identified 16 PRJNA743422.The 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: NCBI SRA BioProject, accession no: Y-HL, X-PL, and JS conceived of this study. YY designed the experiment and drafted the manuscript. YY and XK revised the manuscript. YY and C-YC analyzed the sequencing data. C-YC, CC, and XK performed the experiments. M-GW collected all the samples. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: COVID-19 pandemic has proved to be an unrelenting health threat for more than a year now. The emerging amount of data indicates that vitamin D, zinc and selenium could be important for clinical presentation of COVID-19. Here, we investigated association of genetic variants related to the altered level and bioavailability of vitamin D, zinc and selenium with clinical severity of COVID-19.Methods: We analyzed variants in genes significant for the status of vitamin D , zinc (PPCDC rs2120019) and selenium (DMGDH rs17823744) in 120 Serbian adult and pediatric COVID-19 patients using allelic discrimination. Furthermore, we carried out comparative population genetic analysis among European and other worldwide populations to investigate variation in allelic frequencies of selected variants.Results: Study showed that DHCR7/NADSYN rs12785878 and CYP2R1 rs10741657 variants were associated with severe COVID-19 in adults ; carriers of DHCR7/NADSYN TG+GG and CYP2R1 GG genotypes had 0.21 and 5.9 the odds for developing severe disease, OR 0.21 (0.05–0.9) and OR 5.9 (1.4–25.2), respectively. There were no associations between selected genetic variants and disease severity in pediatric patients. Comparative population genetic analysis revealed that Serbian population had the lowest frequency of CYP2R1 rs10741657 G allele compared to other non-Finish Europeans , suggesting that other populations should also investigate the relationship of CYP2R1 variant and the COVID-19 disease course.Conclusion: The results of the study indicated that vitamin D related genetic variants were implicated in severe COVID-19 in adults. This could direct prevention strategies based on population specific nutrigenetic profiles. COVID-19 pandemic has proved to be an unrelenting health threat for more than a year now. More contagious and more deadly SARS-CoV-2 variants are a major concern, especially because effective, causal therapy is still unavailable, and vaccination coverage rates are lower than anticipated. Moreover, protection from vaccination is likely to last only for a season or two. One of the strategies to save lives is to identify groups at risk of severe COVID-19 disease and to implement measures aimed at those groups. The elderly, male, obese, chronic disease patients, patients with malignancies, immunocompromised, dark-skinned, socioeconomically disadvantaged and tobacco users suffered the most death toll from COVID-19 , 4.Another strategy directed toward reducing severe COVID-19 symptoms relies on high dose vitamins and trace elements (micronutrients) supplementation. Among micronutrients important for adequate immune function, vitamin D, zinc and selenium are of particular importance for coping with viral, respiratory infections, such as COVID-19 . AdequatAlthough important for various processes in our body, a lot of people worldwide have inadequate status of vitamin D, zinc or selenium. There is a pandemic of vitamin D deficiency, especially in higher latitudes due to inadequate sun exposure necessary for the synthesis of this vitamin . SeleniuDHCR7/NADSYN1, GC and CYP2R1, respectively  identified variants located near genes involved in synthesis, transport and metabolism of vitamin D, namely ectively –21. Varian genes , have bean genes –26. GWASan genes , as wellan genes  with a lher GWAS . Zinc nuCDC gene .DHCR7/NADSYN1 rs12785878, GC rs2282679, CYP2R1 rs10741657, VDR rs2228570), zinc (PPCDC rs2120019) and selenium (DMGDH rs17823744) as risk factors for severe forms of COVID-19. Furthermore, we attempted to investigate the inter-population diversity of the selected genetic variants. Inter-population nutrigenetic variability could suggest different relevance of nutrigenetic factors in populations exposed to different nutritional habits and specific environmental factors. These data should be taken into account when evaluating the possible relationship of nutrigenetic variants with severe COVID-19 forms in other populations struck by the pandemic.Building on this premise, we hypothesize that genetic variants linked to suboptimal level or decreased bioavailability of vitamin D, zinc and selenium, may also serve as markers of disease course in COVID-19 patients, influenced by the level of these micronutrients. Therefore, we evaluated genetic determinants of vitamin D (Present study included 120 patients with diagnosis of COVID-19 (adult and pediatric) who were treated in tertiary healthcare institutions, the Clinic of Pulmonology, Clinical Center of Serbia and Children's Hospital for Lung Diseases and Tuberculosis, Medical Center “Dr Dragiša Mišović,” Belgrade, Serbia, between April and June of 2020. For all patients, a COVID-19 infection was defined as a positive SARS-CoV-2 laboratory test . From each patient, peripheral blood sample was collected for DNA isolation and further genetic analyses.Clinical data were assessed using electronic health records. From a total of 120 COVID-19 patients included in the study, clinical data were available for 115 patients, 73 adults and 42 pediatric.Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. 2020. Available at: http://www.covid19treatmentguidelines.nih.gov), as follows: mild – patients with symptoms such as fever, fatigue, cough, myalgia, and headache, but without dyspnea or pneumonia; moderate- those with evidence of pneumonia based on imaging showing up to 50% of lung involvement, who had blood oxygen saturation >93% on room air; severe- patients who demonstrated pneumonia with >50% of lung involvement on imaging or had blood oxygen saturation level equal to or <93% on room air and required supportive oxygen therapy.Adult COVID-19 patients were categorized into mild, moderate or severe clinical course according to the National Institutes of Health (NIH) Definition of COVIID-19 Disease Severity  demonstrated mild form of the disease. Therefore, for the purpose of this study, we classified pediatric patients into 2 groups – This study was approved by the Ethics Committee of the Institute of Molecular Genetics and Genetic Engineering University of Belgrade . Informed consent was obtained from each participant or their parents/legal guardians. The study was performed in accordance with the Declaration of Helsinki.Genomic DNA was isolated from the whole blood samples using QIAamp DNA Blood Mini Kit (Qiagen) and stored at −20°C until analysis.DHCR7/NADSYN1 rs12785878, GC rs2282679, CYP2R1 rs10741657, VDR rs2228570, PPCDC rs2120019 and DMGDH rs17823744 were analyzed using TaqMan SNP Genotyping Assays (Thermo Fisher Scientific) , according to the manufacturer's instructions with modifications. A total volume of 8 μl PCR reaction contained 36 ng of DNA, 1x Taqman Genotyping Master Mix and 1x TaqMan SNP Genotyping Assays (Thermo Fisher Scientific). Genotyping was performed on a real-time PCR system . Genotype calling was implemented with built-in 7500 System Software v1.3.1 (Thermo Fisher Scientific).Genetic variants Statistical analyses were performed in software SPSS v23.0 and R v.3.6.2.Differences in demographic and clinical data between COVID-19 mild/moderate/severe subgroups were examined using adequate statistical tests for continuous and discrete data .For genetic data, Hardy-Weinberg equilibrium was examined to assess the performance of the genotyping assays. Differences in genotype and allele counts distribution for each variant between COVID-19 subgroups were tested using the Chi-squared test. Impact of each genetic variable on severity of the COVID-19 was estimated by odds ratio with 95% confidence interval using a logistic regression model in order to control for confounding factors, such as age and gender.Frequencies of the analyzed variants were compared between group of Serbian COVID-19 patients and other populations using two openly available variant databases, 1,000 Genomes Project (1KGP) and Genome Aggregation Database (gnomAD) v.3.0 , 31. ChiWe examined the level of population genetic variability at each genetic loci using the maximal global difference in risk allele frequency (delta AF), which was calculated by subtracting the maximum and the minimum allele frequency across analyzed populations.All tests were bi-directional. The threshold for statistical significance was defined using appropriate multiple comparisons correction methods, such as Bonferroni or Benjamini-Hochberg false discovery rate (FDR).In the selected study group, 73 were adults while 42 were pediatric patients. Among adults 65.8% were females and the median age was 43 (range 20–82); among pediatric patients 38.1% were females and median age was 9 (range 0.3–17). Demographic and clinical descriptions of COVID-19 patients are presented in n = 35), moderate (n = 21) or severe (n = 17) disease groups. Age distribution of the patients significantly varied between groups; gradual increase in participants' age has been observed across mild, moderate and severe groups (p < 0.001). In the severe group, a significantly higher number of patients suffered from hypertension, diabetes and were using ACE inhibitors, compared to mild and moderate groups . The highest percent of lymphopenia and thrombocytopenia events were observed in the severe group, 93.8 and 56.3%, respectively. Also, patients with severe COVID-19 had higher levels of CRP, compared to patients with moderate and mild disease (p < 0.001).In the group of adult COVID-19 patients, 10 (13.7%) required supportive oxygen therapy and 3 (4%) had COVID-19 related death outcomes. Examining available clinical data, patients were categorized into mild (n = 20) and symptomatic (n = 22). There was no significant difference in distribution of age, gender, nor in the presence of comorbidity between the two groups, except past respiratory diseases. The level of CRP was significantly higher in COVID-19 symptomatic compared to asymptomatic pediatric patients (p = 0.003).Pediatric COVD-19 patients were categorized into two groups – asymptomatic , associated with lower levels of 25OHD were 4.9 times less likely (or had 0.21 the odds) to develop a severe form of COVID-19, compared with carriers of TT genotype. When adjusted for age and gender, observed association was still significant . Association has also been observed between severe COVID-19 and CYP2R1 GG genotype, previously related to decreased levels of 25OHD. In the logistic regression model, CYP2R1 GG carriers had 5.9 times higher odds to develop severe disease, adjusted for age and gender . Additionally, we applied adjustment for obesity in multivariate logistic regression models, showing that DHCR7/NADSYN1 rs12785878 and CYP2R1 rs10741657 variants remained significant predictors of severe COVID-19 in adults . Our study did not show association of GC and VDR variants with higher risk of severe COVID-19 in adults.We compared the distribution of analyzed genotypes between different COVID-19 severity groups. In the group of adult patients, two outcomes were considered in the logistic regression model – first included both mild and moderate disease, and second was severe disease . LogistiDMGDH A allele, which has been related to lower levels of selenium, in our study were less likely to present severe form of COVID-19 . However, this association was not significant after correction for age and gender. Regarding zinc-related PPCDC variant, we observed no significant association with severe COVID-19 outcome in adult patients.Homozygous carriers of In pediatric COVID-19 cases, we applied a logistic regression model to predict two outcomes – asymptomatic and symptomatic disease, using genotype data . No signDHCR7/NADSYN1 allele G between Italian compared to Serbian population . Difference was also observed in the frequency of CYP2R1 rs10741657 allele G between Serbian (0.58) and Spanish (0.69) as well as Italian population (0.66) . Serbian population had the lowest frequency of the CYP2R1 rs10741657 G variant among European non-Finish populations, while Spanish and Italians demonstrated the highest. However, noted differences were not statistically significant. As expected, significant differences were mostly seen between Serbian and African, South and East Asian, American Latino/Admixed, and Ashkenazi populations.Finally, we aimed to compare allele frequencies of selected genetic variants in Serbian COVID-19 study group with their frequencies in other populations. Therefore, we retrieved variant site data from the two genetic databases, 1KGP and gnomAD. Distributions of alleles frequencies (AF) associated with decreased levels of selected micronutrients in European and worldwide populations have been presented in DHCR7/NADSYN1 rs12785878 variant (dAF 0.56). The lowest dAF was detected for CYP2R1 rs10741657 and DMGDH rs17823744 variants .The level of population genetic variability at each selected loci was assessed using delta AF (dAF), representing the difference between maximum and minimum AF value across analyzed populations. The highest dAF was observed for the In this study, we focused our attention to genetic variants related to altered level, bioavailability or mechanism of action of vitamin D, zinc and selenium, aiming to investigate their association with different COVID-19 presentation in children and adults. Serbia, like much of Europe and the world, faces micronutrient deficiency/insufficiency which is in part influenced by genetic variability. The prevalence of suboptimal levels of vitamin D and selenium in Serbian population is estimated to be around 50% , 33, whiDHCR7/NADSYN1 rs12785878 and CYP2R1 rs10741657 variants with severe form of COVID-19 in adult patients. In the pediatric COVID-19 group, we did not detect any associations between analyzed genetic factors and disease severity.Results of our study showed association of DHCR7 gene on chromosome 11q12. DHCR7 codes for 7-dehydrocholesterol reductase, a key enzyme in cholesterol biosynthesis and vitamin D cutaneous production. Particularly, the activity of DHCR7 enzyme decreases the level of vitamin D precursor availability, 7-dehydorcholesterol, shunting it in the direction of cholesterol biosynthesis  in European populations  gene. Variant rs10741657 located near the CYP2R1 gene was linked by several studies to 25OHD serum concentrations ; G allele frequencies ranged from 0.58 in Serbian, which was the lowest among European populations, to 0.73 in African population. The highest frequency of the G allele among Europeans was observed in Italian and Spanish populations . Recent study that reported negative correlations between mean levels of vitamin D in various European countries with COVID-19 cases per million people, suggested that Spain, Italy and Switzerland are the most vulnerable countries since they have very low levels of vitamin D in the aging population  enzyme which hydroxylates carbon 25, producing the intermediate 25-hydroxyvitamin D3, or 25OHD, the major circulatory form of the vitamin D . This entrations , 38, witpulation . Our resA role of vitamin D in the pathogenesis of COVID-19 has been extensively studied since the beginning of the pandemic. Calcitriol  has an important role in regulating renin angiotensin system by enhancing the expression of ACE2, which is the main target of SARS-CoV2 cell entry , 8. AlsoGC gene that encodes vitamin D binding/transport protein (DBP), and the prevalence and mortality rates of COVID-19, pointed out to rs7041 variant  on vitamin D and COVID-19 in individuals of European ancestry, genetically increased 25OHD concentrations did not protect against COVID-19 susceptibility, but increased the odds for hospitalization and severe disease , GPX4 and selenoprotein P (SELENOP) with response to selenium supplementation  .NK: conceptualization, investigation, statistical analysis, writing - draft preparation, and editing. AS: methodology, investigation, and writing – draft preparation. KK: investigation, methodology, and writing – draft preparation. VG: data analysis and interpretation, investigation, and writing – draft preparation. BZ: methodology, investigation, writing — review, and editing, VS-T, MS, ZZ, OO, GS, and LL: methodology, sample collection, and clinical data analysis. SP: concept and design of the study, writing –review, and editing, BS: concept and design of the study, statistical analysis, results interpretation, drafting, and review of final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


OLEOSIN genes and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs were enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, such as PUX10 and CDC48A. All mRFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes, this suggests that during bud maturation, OLEOSINs were replaced by LDIP/LDAPs at enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), “myosin XI tail-binding” RAB GTPase C2A, an LB/PD-associated γ-clade 1,3-β-glucanase, and various organelle- and/or PD-localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles, and deliver a diverse cargo to PD.Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ-clade 1,3-β-glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes, and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific  Plasmodesmata (PD) are notoriously difficult to investigate, and understanding PD functioning from the composition is fraught with difficulties. Both composition and architecture of PD are subject to regulation by cells that share them. Rather than functioning autonomously, they are subject to control by cells that construct, maintain, and operate them. Consequently, PD composition and function are context-dependent, differing between tissues in dependence on developmental and metabolic cellular states. Filtering out commonalities while recognizing the unique aspects of PD at specific locations and conditions, therefore, remains a challenge.Information on PD composition and function has been gathered using a variety of plant and tissue systems and approaches. Frequently, investigations are focused on PD (ultra)structure and/or localization of suspected PD proteins by immunochemistry or transgenic expression of fluorescently tagged proteins, microinjection studies, and proteomic and lipidomic studies . DSCs act as circuit breakers that interrupt the symplasmic circuitry of the SAM, preventing electrical and metabolic coupling and exchange of transcription factors and other regulatory molecules, arresting the SAM in a dormant state , possibly proteins that become integrated into the fabric of PD, and non-cell-autonomous signals  sections of meristems of growing plants (APs), developing terminal buds (DEBs), and dormant terminal buds (DOBs) . In all OLEOSINs were upregulated  and the mitochondrial gene SDP6, which is required for post-germinative seedling growth in Arabidopsis  as well as proteins with amphipathic stretches (class II) and peripherally and transiently associated proteins  showed the presence of at least six putative ubiquitination motifs  and the segregase Cell Division Control Protein 48A (CDC48A)   (Deruyff(CDC48A) . The bud(CDC48A)  (Table 1(CDC48A) . In addi(CDC48A) , and 26S(CDC48A) . AlthougThe question remained how LBs retained their structural integrity during their enlargement  while OLP. trichocarpa homologs of PUX10 and CDC48A, six LDAP , and one LDIP homolog  during dormancy development, was little expressed in apices , while LDAP1 was not detected . In contPopulus LDAPs can localize to LBs was demonstrated in leaf epidermal cells of N. benthamiana using a binary vector expressing the 35S promotor driven OLE6-eGFP fusion protein and binary vectors expressing LDAP1a-mRFP, LDAP1b-mRFP, LDAP2a-mRFP, LDAP3a-RFP, and LDAP3b-mRFP. The vectors were transformed into Agrobacterium tumefaciens and infiltrated into leaves for 2 days and investigated with CLSM. Captured images (Leica Application Suite X software) showed that all mRFP-tagged LDAPs were exclusively localized to OLE6:eGFP-tagged LBs  that is commonly used to remove proteins that might associate with the monolayer during LB isolation, or are commonly considered contaminants. As argued above, this would also remove the peripherally associated proteins that hitch a ride to the PD. In buds, cytoplasmic proteins might associate with LBs when molecular crowding is reduced , LDPI, LDAP3a, LDAP3b, LDAP1a, LDAP1b, Oil Body Lipase1 , GPAT8 , and the 1,3-β-glucanase enzyme GH17_44 . Like OLAs LBs move in the actomyosin system motorized by myosin XI-k/1/2 , which is a C2 tethering protein, Syntaxins (SYP71) and other SYPs, the vesicle/protein transporter ADP-ribosylation factor 1 (ARF1), and the endosomal protein Guanine Nucleotide-Exchange (GEF), which recruits ARF1 to vesicles . MoreoveOther identified proteins potentially reflect organellar interaction, protein folding and unfolding at organelles and PD, lipid metabolism, storage, detoxification, and desiccation stress. Unfolding/folding proteins in the bud LB fraction included the chaperones/Heat Shock proteins Hsp-90-4, Hsp90-6, Hsp90-7, the Hsp70 proteins BIP2 and BIP3, and homologs of the cpn60 chaperonin TCP-1/CCT8. Of the TCP-1 chaperonin family proteins, 10 members were present . ProteinP. trichocarpa   and various 26S components, the ubiquitin-activating enzyme UBA1, the crucial 19S ATPase subunit RPT2A, and several deubiquitinating proteases, which are required for insertion of proteins into the 20S core . While RAB-C2A specifically localizes to peroxisomes  in maize, which requires chaperon-assisted unfolding in the donor cell and chaperonin-assisted refolding in the destination cell fusion. Chaperones transport, fold and unfold proteins during autophagy, and function at peroxisomes, mitochondria, chloroplasts, and ER  clone T89 plants were micro propagated, moved to a glass house, and grown under an 18-h long day , and homozygous lines were obtained as previously described by Veerabagu et al. , 200 μM MG132, or 40 μM E64d , developing buds  and dormant buds  were fixed overnight in 2% (v/v) glutaraldehyde and 3% (v/v) paraformaldehyde at 4°C in 100 mM phosphate citrate buffer  . Per phovia LR reaction using the respective LDAP entry clones and the destination vector pB7WGR2  preparations derived from dormant buds were used as a template to amplify all the LDAPs. The LDAP entry clones were obtained by BP reaction in pDONR207 and verified by sequencing. The binary constructs for mRFP fusion protein expression under the control of the 35S promoter were obtained Dormant buds were collected from short-day (9 weeks) exposed plants and used for LB purification and repeated in three independent experiments (Bio 1–3). Freshly harvested buds weighing 5–8 g were kept on ice until all the hard bud scales were carefully removed. The material was rinsed thrice in cold distilled water and then processed for LB extraction and purification. For LB purification, we used a previously described method  at a dilution ratio of 1:2000, Hevea brasiliensis anti-SRPP1 (LDAP1) monoclonal antibody  at a dilution ratio of 1:1000, and anti-eGFP monoclonal antibody  at a dilution ratio of 1:2000. Goat anti-rabbit IgG H&L (HRP) antibody phytoAB-PHY6000 at a dilution ratio of 1:5000 was used as a secondary antibody for OLE1. Goat anti-mouse IgG H&L (HRP) (Abcam-ab205719) at a dilution ratio of 1:5000 was used as a secondary antibody for both LDAP1 and eGFP antibodies. Immunoblot signals were detected with the Clarity Western ECL kit  and imaged with a Chemidoc  digital imager.To investigate the production of LDAP1 and OLEOSIN in Arabidopsis seedlings . The mascot was searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine, and acetylation of the n-terminus were specified in the Mascot as variable modifications.In short, the LB pellets were rinsed with methanol and air-dried. Subsequently, proteins were extracted in appropriate volumes of 2x Laemmli buffer. The third biological replicate received an additional wash, which further purified the sample. The extracted proteins were loaded on a 10% Bio-Rad mini protein gel  and allowed to run ~1.5 cm into the gel. The protein gel was stained with Coomassie Brilliant Blue  for an hour and destained overnight. The region of the gel showing stained proteins was isolated and excised into six fractions. The gel pieces were reduced (DTT) and alkylated (iodoacetamide) before overnight digestion with trypsin. After digestion, the peptides were extracted from the gel by sonication in a 0.1% TFA solution and cleaned up by a reversed-phase (C18) spin-tip procedure. Eluted peptides were dried and re-dissolved in a loading solution . Samples were analyzed by LC-MS/MS, with Ultimate™ 3000 RSLC nano system coupled with Q Exactive hybrid quadrupole-orbitrap . Thermo raw files were converted using the msfileconvert module of the Proteowizard (v 3.0.7076) software suite. All MS/MS samples were analyzed using Mascot . The mascot was set up to search the via the PRIDE*  tool from the SUBA toolbox and presented in Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at >99% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm . The MEGA6 program (http://www.megasoftware.net/) was used for ClustalW multiple sequence alignments and phylogenetic analysis, with maximum likelihood method and Poisson correction model. The Populus and Arabidopsis proteins of the phylogenetic analyses are presented in One-way ANOVA followed by Fisher's and Tukey's multiple comparisons tests were performed using GraphPad Prism Version 8.0.0 (https://www.ebi.ac.uk/pride/archive/projects/PXD020099.The original contributions presented in the study are publicly available. This data can be found here: MV, PR, LP, and CvdS designed the study. MV, PR, LP, and MS performed the experiments. MV and PR analyzed the data. MV, CvdS, and PR interpreted the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Komagataeibacter genus but belonged to a different clade within the genus. Comparative genomic analysis revealed cross-strain distribution of duplicated sequences in Komagataeibacter genomes. It is particularly interesting that FNDCR1 has many duplicated sequences within the genome independently of the phylogenetic clade, suggesting that these duplications might have been obtained specifically for this strain. Analysis of the cellulose biosynthesis operon of the three determined strain genomes indicated that several cellulose synthesis-related genes, which are present in FNDCR1 and FNDCR2, were lost in the FNDCF1 strain. These findings reveal important genetic insights into practical nata de coco-producing bacteria that can be used in food development. Furthermore, our results also shed light on the variation in their cellulose-producing abilities and illustrate why genetic traits are unstable for Komagataeibacter and Komagataeibacter-related acetic acid bacteria.We determined the whole genome sequences of three bacterial strains, designated as FNDCR1, FNDCF1, and FNDCR2, isolated from a practical nata-de-coco producing bacterial culture. Only FNDCR1 and FNDCR2 strains had the ability to produce cellulose. The 16S rDNA sequence and phylogenetic analysis revealed that all strains belonged to the  Numerous bacteria produce cellulose extracellularly. Bacterial cells are often entrapped in the cellulose fiber, forming pellicle at the air–liquid interface. Depending on the bacterial strain and culture conditions, cells consume as much as 10% of the total energy budget for cellulose biosynthesis . AlthougKomagataeibacter genus , which are kinds of α-proteobacteria and obligately aerobic and acidophilic bacteria, are the most preferably employed practically because the bacteria of this genus have the exceptionally high capability for producing cellulose . Among AAB, the species of ellulose .However, the productivity of nata-de-coco by these bacteria is highly unstable; AAB are well known to be genetically unstable and to lose cellulose producing ability frequently in the absence of selection pressures . The genKomagataeibacter medellinensis NBRC 3288 strain  was isolated from rice vinegar as a cellulose-producing strain in 1954, but complete loss of its production capability was reported in 1999 (bcs operon) of this strain in 2015. In Komagataeibacter bacteria, the bcs operon is typically organized as bcsZHABCD-bglX or bcsABXYC. The genes in these operons encode cyclic dimeric (3′ → 5′) guanosine monophosphate (c-di-GMP) -regulated cellulose synthase , an outer membrane porin protein (BcsC), putative periplasmic protein (BcsD), cellulose-complementing protein A (BcsH), probable cellulose deacylase (BcsX), probable cellulose acylase (BcsY), endo-β-1,4-glucanase (BcsZ), and β-glucosidase (bglX). Moreover, the genes play a central role in cellulose biosynthesis  that had been isolated from a practical culture of nata-de-coco in Japan. This report is the first of a study of whole genome sequences of practical nata-de-coco producing bacteria. All of the strains were bacteria of 2HPO4, 1.5 g/L citric acid, 0.1% (v/v) acetic acid, and 10 g/L sucrose or glucose. For preparation of HS-agar plates, agarose was added at 20 g/L.The nata-de-coco producing bacteria were isolated from a nata-de-coco production facility in Japan. The FNDCR1 strain was stocked in 2017 as a strain that had shown good nata-de-coco productivity from those stored in a nata-de-coco production facility in Japan. The FNDCF1 strain was isolated in 2016 when the productivity loss occurred in the facility. FNDCR2 is a strain that was isolated in 2017 after 20 passages of culture from a stock solution stored at the facility for producing nata-de-coco. In this study, all strains were single colony isolated in 2020, followed by cellulose productivity experiments and whole genome sequencing. Unless stated otherwise, cells were cultured at 28°C using Hestrin–Schramm (HS) medium: 5 g/L yeast extract, 5 g/L peptone, 2.7 g/L NaAfter fermentation, the cellulose amount was quantified according to the method described in an earlier report, with modifications . In brieAspergillus niger; Fujifilm Wako Pure Chemical Corp., Osaka, Japan) was added at 1 mg/mL and incubated for 1 h at 28°C before harvesting. The purified DNA was subjected to sequencing with PacBio RS II and Illumina Hiseq 2,500 systems . The Hiseq reads were obtained with paired-end 100 base pairs (bp) sequencing. The numbers of reads over ≥ Q30 were 47,239,534, 47,458,318, and 44,626,206 bp, respectively, for FNDCR1, FNDCF1, and FNDR2 strains. In the PacBio reads, the numbers of subreads and mean subread lengths were 145,440 and 6,655 bp, 149,926 and 7,350 bp, and 175,664 and 9,362 bp, respectively, for FNDCR1, FNDCF1, and FNDR2 strains. The raw sequencing reads have been deposited in NCBI SRA under their accession numbers SRR15903111-SRR15903116.Whole-genome DNA was isolated using the neutral phenol—chloroform—isoamyl alcohol method  with one1 Both the trimmed Hiseq and PacBio reads were used to construct the genomes using the Unicycler software .me maps3 . The ass4 Phylogenetic classification was then performed from the 16S rRNA gene sequences using the Alignment, Classification and Tree Service5 of SILVA .Komagataeibacter genus) was obtained from NCBI Genbank6 using NCBI-genome-download v.0.3.0.7 To ensure the genome assembly levels, the downloaded genomes were limited to “complete” whole genomes.Based on the classification results, the public genome of an assigned genus . The assembled genomes of FNDCR1, FNDCF1, and FNDCR2, respectively, showed high completeness of 99.5, 99.6, and 99.7%. All the strains were found to harbor multiple circular plasmids in addition to circular chromosomes –4. ThereKomagataeibacter with > 99% similarities  were mutually similar. Also, pKFR1-4 was similar to pKFR2-2 throughout the plasmids. Moreover, pKFF1-4, pKFR1-4, pKFR2-1, pGX4 (K. xylinus E25), and pGX3 (K. xylinus E25) shared partial homology: an approximately 3 kb part of pKFR1-7  was highly homologous to the part of pKFR1-6 and pGX1 (K. xylinus E25). In addition, a consensus sequence of approximately 3.7 kb in pKFR1-1, pKFR1-2, pKFR1-3, pKFR1-4, pKFR1-5, pKFR1-6, pKFR2-1, and pKFR2-2 was identified. On this sequence, only transposase and resolvase were coded. Therefore, the same transposable elements were present on these plasmids.The sequence of each plasmid was compared to those of all other plasmids presented herein, as well as those of plasmids found previously in 5 strain . ActuallTaking these findings together, one can infer that the genome structure of FNDCR1 and FNDCR2 strains are complex, leading us to predict that the genomes of these strains are genetically unstable.bcsABCD genes are the most important genes: with other accessory genes, they are involved in efficient biosynthesis (bcs and other genes from the gff3 annotation file created using the prokka and RAST programs (Komagataeibacter bacteria harbor two bcs operons (type I and II). Typically, the type I and II operons, respectively, comprise bcsZ-bcsH-bcsAI-bcsBI-bcsCI-bcsDI-bglX, and bcsAII-bcsBII-bcsX-bcsY-bcsCII   .bcs operon was found in the FNDCF1 strain. The sole bcs operon of the FNDCF1 strain was a fused one of type I and II operons, respectively. Furthermore, no bcsCI, bcsAII, or bcsBII gene was found. A premature stop codon appeared in the C-terminal part of bcsCII in the FNDCF1 strain  and intragenic suppressor mutations (reversion of frameshift) in the bcsBI gene  domain  domain . In fact) domain . AdditiopG level .Komagataeibacter genus. Phylogenetic tree analysis of the conserved core genes indicated that the three strains belong to mutually different clades. None of them matches any known strain. This finding suggests that the present strains are novel strains that have not been reported in the literature as Komagataeibacter strains. Duplicated genome regions as their genomic signature were also specifically examined. In fact, FNDCR1 and FNDCR2 have many duplicated sequences within their own genomes. By contrast, FNDCF1 has only a few duplicates in its own genome. It is particularly interesting that FNDCF1 has far fewer mobile genetic elements in its genome than the other two cellulose-producing lineages (FNDCR1 and FNDCR2), which might be attributable to the cross-strain variation in the sequence duplication among Komagataeibacter strains. After applying correlation analysis between the length of these homologous duplicated sequences and the number of mobile genetic elements, duplicates with minimum length of 1,000 bp were found to be positively correlated with the number of mobile genetic elements. Nevertheless, no correlation was found for duplicates with minimum length longer than 2,000 bp. This finding suggests that mobile genetic elements are related with transposition in the genome, but they might be subject to negative selection in their duplicated sequence lengths.In the phylogenetic classification based on 16S rRNA genes, all three isolated strains were assigned to Acetobacter pasteurianus 386B strain is exploited frequently. Its low number of mobile genetic elements in the genome and absence of complete phage genomes makes this strain more genetically stable than other A. pasteurianus strains  can be found below: KI and NN designed the study, collected the data, conducted data analyses, and drafted the manuscript. HK, TI, and KK reviewed the manuscript. All authors read and approved the manuscript.HK was employed by company Research and Development Department, Fujicco Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The tumor suppressor p53 is activated upon multiple cellular stresses, including DNA damage, oncogene activation, ribosomal stress, and hypoxia, to induce cell cycle arrest, apoptosis, and senescence. Mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, serves as a central regulator of cell growth, proliferation, and survival by coordinating nutrients, energy, growth factors, and oxygen levels. p53 dysfunction and mTOR pathway hyperactivation are hallmarks of human cancer. The balance between response to stresses or commitment to cell proliferation and survival is governed by various regulatory loops between the p53 and mTOR pathways. In this review, we first briefly introduce the tumor suppressor p53 and then describe the upstream regulators and downstream effectors of the mTOR pathway. Next, we discuss the role of p53 in regulating the mTOR pathway through its transcriptional and non-transcriptional effects. We further describe the complicated role of the mTOR pathway in modulating p53 activity. Finally, we discuss the current knowledge and future perspectives on the coordinated regulation of the p53 and mTOR pathways. MDM2 (p21 (NOXA (PUMA (RPS27L (0-13RRRC (A/T) (A/T)GYYY  is normally located near the transcription start site , an evolutionarily conserved serine/threonine protein kinase, is a member of the phosphoinositide-3-kinase (PI3K)-related kinase (PIKK) family, along with ATM, ATR, DNA-PK, and SMG-1 . While aIn mammalian cells, mTOR forms two complexes with distinctive structures and functions, namely mTORC1 and mTORC2. mTORC1 is composed of mTOR, Raptor, PRAS40, and GβL, whereas mTORC2 consists of mTOR, Rictor, mSin1, protor, and GβL . AlthougUpstream signals regulate mTORC1 activity mainly through two mechanisms: by direct control of mTORC1 components or by the supervision of Rheb GTPase, which interacts with and activates mTORC1 . Tuberou1) Growth factors: Growth factors, such as insulin and insulin-like growth factor 1 (IGF-1), promote cellular anabolic processes by activating mTORC1. The binding of insulin/IGF-1 to its receptor recruits insulin substrate 1 (IRS1) to the receptor and activates PI3K, which phosphorylates PIP2 to PIP3. PIP3 recruits AKT to the plasma membrane, where it is fully activated by direct phosphorylation of Thr308 by PDK1 and Ser473 by mTORC2. During growth factor signaling, AKT , along w2) Glucose and energy: Glucose deprivation decreases glycolytic flux and inhibits mTORC1 by lowering ATP levels. LKB1 and AMPK are two major upstream kinases of the mTOR pathway and are involved in monitoring the levels of glucose and energy (ATP and AMP) . In resp3) Hypoxia: Under hypoxic conditions, HIF-1α promotes the expression of REDD1 to inhibit mTORC1 by activating the TSC1-TSC2 complex . Mechani4) Amino acid: Amino acid signaling recruits and activates mTORC1 to the lysosomal membrane, where a pool of Rheb resides. In this process, the Rag GTPase heterodimer (consisting of RagA/B and RagC/D), which is activated by amino acids, serves as a scaffold for mTORC1 via interacting with Raptor .As a negative regulator of IRS1, mTORC1 also acts upstream of the PI3K-AKT pathway to inhibit mTORC2. This negative feedback loop is initiated through multiple mechanisms. First, S6K, a substrate of mTORC1, decreases the activity and protein levels of IRS1 by phosphorylating it . Second,To maintain normal cell growth and proliferation, it is important for cells to coordinate stimulatory signals  and inhibitory stresses (such as DNA damage and hypoxia). The tumor suppressor p53, a stress-induced transcription factor, can inhibit cell growth and proliferation via its target genes, such as the cyclin-dependent kinase (CDK) inhibitor p21, which serves as a cell cycle inhibitor . More reREDD1 (LKB1 (AMPKβ (a regulatory subunit of AMPK)  , and TSCK)  cancer cells (such as U2OS cells), p53 stimulates the transcription of mTOR and Rictor, two important components of mTORC2, to activate AKT and inhibit apoptosis . RecentlThe discovery of microRNAs (miRNA or miR) has added another layer of complexity to the regulation of the mTOR pathway by p53. miRNAs are a class of endogenously expressed small non-coding RNAs (17–24 nucleotides) that regulate the expression of multiple genes at the post-transcriptional level . miRNAs Accumulating evidence shows that a large number of miRNAs act opposingly on the mTOR pathway, which is often hyperactivated in cancers . Thus, p1) miR-100: miR100, a member of the miR-99 family , is negatively regulated by p53. p53 binds to the upstream sequences of miR-100 and suppresses its transcription in both mouse striatal cells and human cervical carcinoma HeLa cells . miR-1002) miR-101: miR-101 is downregulated in various cancers, including ovarian cancer, prostate cancer, hepatocellular carcinoma, bladder transitional cell carcinoma, gastric cancer, and non-small cell lung cancer, and is negatively associated with the progression and invasion of malignancies, such as prostate cancer . In humain vitro and in vivo (3) miR-145: miR-145, a direct target of p53, binds to the 3′-UTR of c-MYC and inhibits its expression, thereby repressing cancer cell growth both  in vivo . Moreove in vivo . Further4) miR-149: miR-149 plays a dual role, that is controversial, either as a tumor suppressor or as an oncogene in different types of cancer . miR-1495) miR-155: miR-155 targets several components of the mTOR pathway, including Rheb, Rictor, and S6K2, by directly binding to their 3′-UTRs . By inte6) miR-199a-3p: miR-199a-3p is upregulated by p53 at the post-transcriptional level . miR-199trans-activating or trans-repressing downstream gene expression as a transcription factor. Interestingly, in recent decades, several studies have shown that in addition to its activity in the nucleus, p53 exhibits transcription independent functions in the cytoplasm (L) to release pro-apoptotic Bcl-2 proteins (such as Bax and Bak) to induce apoptosis  expression of nuclear p53 (disturbed NES by L348A and L350A) failed to inhibit autophagy; and 3) a point mutation (R175H) in p53 that induces a conformational change, abrogated the autophagy-inhibitory effect of p53  and activated mTOR (reflected by an increased phosphorylation of S6K) to suppress autophagy , induces MDM2 translation in a PI3K-AKT dependent manner. On the other hand, inhibition of mTORC1 by rapamycin downregulating MDM2, induces p53-dependent apoptosis, and sensitizes cancer cells to chemotherapy . Additiop21 following UV treatment  suppresses p53-mediated senescence, facilitates ROS-induced tumorigenesis, and has no effects on DNA damage response induced by radiation  . Howeverreatment . Third, reatment  and stabreatment . Particuadiation . Moreoveadiation . Additioadiation . Fourth,adiation . Finallyadiation . Rapamycadiation , indicatadiation .in vitro findings truly indicate those in vivo physiological and pathological conditions ? The answers to these questions will advance our current understanding of the manner in which the cross talk between p53 and mTOR pathways regulates tumorigenesis.In summary, the coordinated regulation of the tumor suppressor p53 and the mTOR pathway is critical for cells and organisms to maintain homeostasis in response to various stimuli. p53 controls the mTOR pathway at multiple levels: 1) p53 directly regulates several signaling mechanisms in the mTOR pathway; 2) miRNAs, downstream of p53, regulate the mTOR pathway at the post-transcriptional level; 3) cytoplasmic p53 may control the AMPK-mTOR axis to inhibit autophagy by protein-protein interaction. In contrast, the mTOR pathway regulates p53 activity mainly by monitoring the interaction between p53 and its E3 ubiquitin ligase, MDM2. Although some cross talk between p53 and the mTOR pathway has been addressed, many fundamental questions remain unanswered such as: 1) Are the other regulators or components of the mTORC1 complex, the transcriptional targets of p53? 2) How does cytoplasmic p53 activate AMPK and suppress mTOR? 3) What is the precise role of p53 in regulating mTORC2, which has been poorly studied? 4) Does mTOR or its downstream effectors directly phosphorylate other sites of p53 in addition to Ser15 and regulate p53 function under physiological or “stress” conditions? 5) Do the Interestingly, activating p53 and inhibiting mTOR may be an effective strategy for combating coronaviruses (CoVs) such as COVID-19-causing SARS-CoV-2. During viral infection and replication, mTOR is activated and promotes type-I interferon expression in the presence of MyD88, TLR9, and IRF-7. mTOR inhibitors suppress viral infection and replication in the early stages . Recent


Background: Congenital heart defects (CHDs) are the most common birth defects, and left heart hypoplasia (LHH) is a severe form of CHD and responsible for more than 20% cardiac deaths during the first week of life, however, its genetic causes remain largely elusive.Methods: Three families with fetal LHH were recruited. Genomic DNA from amniotic fluid or peripheral blood, and trio whole exome sequencing (trio-WES) and copy number variation sequencing (CNV-seq) were performed.Results: All the three couples had no family history, and mid-gestation ultrasound revealed LHH and other variable cardiovascular defects in the fetuses. Trio-WES revealed de novo pathogenic variations in KMT2D (p.Gly3465Aspfs*37) (NM_003482) and WDFY3 (p.Ser117Xfs*) (NM_014991), and CNV-seq identified a deletion of 150 kb encompassing NOTCH1. KMT2D and NOTCH1 previously have been reported to be associated with CHDs, however, WDFY3 is reported for the first time to be possibly related to CHD in human.Conclusion: Our study suggested that genetic component is an important risk factor for the development of LHH, and next generation sequencing is a powerful tool for genetic diagnosis in fetuses with CHDs and genetic counseling, however, more studies and data are need to establish the correlation of fetal phenotypes and genotypes. Congenital heart defects (CHDs) are a spectrum of diseases involving structural abnormalities of the heart and great vessels that affect about 1% of liveborn infants and occur in 10% of aborted fetuses . HypoplaNKX2.5, GJA1, NOTCH1, HAND1, FOXC2, FOXL1, IRX4, MYH6, and RBFOX2, in patients with HLHS  –7, as weith HLHS –15, and de novo loss-of-function variant in KMT2D, and WDFY3 (WD40 repeat and FYVE domain containing 3) gene, respectively, and a de novo heterozygous microdeletion variant in chromosome 9q34.3, encompassing NOTCH1, which aid prenatal genetic counseling and understanding of the pathogenesis of LHH.The aim of this study is to identify the pathogenic variants in 3 fetuses with LHH by using next generation sequencing, and we found a This study was approved by the Wuhan Children's Hospital  ethics committee and informed consent was obtained from the parents of the fetuses. We recruited 3 fetuses with LHH detected by ultrasound during middle gestation. The parents were healthy and non-consanguineous, and denied any adverse history. The maternal age range at diagnosis of LHH was 29–30 years, and the gestation age range at diagnosis was 23–25 weeks. Fetal samples were from amniotic fluid during the invasive prenatal diagnosis. A total of 2 ml of peripheral blood was collected in EDTA-containing tubes from the couples. Multidisciplinary consultation including genetic counseling regarding the risk of LHH and the chance of surgery as well as the benefits and limitations of whole exome sequencing (WES) and CNV sequencing (CNV-seq) were introduced to the couples.Genomic DNA was extracted from 20 ml of amniotic fluid or 2 ml whole blood according to the protocol of the Omega DNA Mini Kit. A NanoDropTM spectrophotometer was used for quality control (QC) of DNA purity and concentration.Genomic DNA was sheared by sonication and exome sequences were enriched by IDT xGen® Exome Researcher Panel v1.0, according to the manufacturer's protocol. DNA libraries were sequenced on Illumina Hiseq XTen with a method of pair-end 150 bp reads. Raw image files were processed by the Bcl To Fastq (Illumina) for base calling and raw data production. Low quality variants were filtered out using the quality score ≥ 20 (Q20) standard. The sequencing reads were then mapped to the human reference genome (hg19/GRCh37) with BWA. Single nucleotide variations (SNVs) and Indels were scored by GATK 3.8. All short variants were annotated with databases including 1,000 genomes, dbSNP, Exac, GnomAD, ClinVar, HGMD, and OMIM. Impacts of variants on protein functions were predicted using software packages Provean, SIFT, Polyphen2_HDIV, Polyphen2_HVAR, Mutationtaster, M-CAP, and REVEL. The pathogenicity of candidate variants was evaluated on the base of mutated frequency, conservation of amino acid change and structural or function domain of the protein as well as inheritance pattern. The pathogenicity of identified variants was assessed in accordance with the American College of Medical Genetics (ACMG) guidelines and Sherloc , 17.Generally, the variants detected by Trio-WES were analyzed according to ACMG standards and guidelines, and classified into pathogenic variants, likely pathogenic variants, uncertain significance variants, likely benign variants and benign variants. The former three types were further annotated in OMIM diseases database and filtered through family co-segregation according to genetic rules. Potential pathogenic genes were identified combining disease correlation and clinical observations. In addition, the incidental findings were disposed according to the document of ACMG about prenatal diagnosis . PathogeA total amount of more than 1.2 μg purified genomic DNA was broken into 200–300 bp fragments by sonication, followed by fragment DNA being sequentially repaired, tailed, ligated with an adaptor and amplified for library construction. The samples were then subjected to Illumina NovaSeq 6000 . Raw data were processed by the basecall analysis software fastq v0.18.1. The clean data were then mapped to human reference genome hg19) using BWA. The candidate CNVs were first filtered through normal frequency databases DGV, and, subsequently, were annotated based on scientific literature review and public databases including Decipher, ClinVar, ClinGen, ISCA, and dbVar. According to the ACMG, the candidate CNVs were classified into five categories: pathogenic, likely pathogenic, uncertain clinical significance, likely benign and benign , PMPCA exon 13(Forward: 5′-CCG TTC CCG TGC GTG TTA-3′ and Reverse: 5′-TTC CTG GCT TGC GGT GGT-3′), INPP5E exon 10(Forward: 5′-GCA CCA TCT GCT CCG TTT C-3′ and Reverse: 5′-TTC CTT CCT GGG ACG CTG-3′), NOTCH1 exon 1(Forward: 5′-GAG CGC AGC GAA GGA ACG AG-3′ and Reverse: 5′-CCT CTC TTC CCC GGC TGG CT-3′) and NOTCH1 exon 34 (Forward: 5′-GCA CAG GAG CGC ATG CAT CA-3′ and Reverse: 5′-GCT GGG CTT GCG GAC CTT CT-3′). The 36B4 gene was used as reference gene. q-PCR was performed on a ABI 7500 apparatus using SYBR Green Real-Time PCR Master Mix (Takara) according to the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 53°C for 15 s and 72°C for 34 s.Semi-quantitative qPCR was performed to confirm the presence of the microdeletion identified in case 3 by detecting the copies of DNA fragments within the region on both the fetus and the parents. Primers were designed to quantify the relative copies of KMT2D (NM_003482) and WDFY3(NM_014991), and a microdeletion involving NOTCH1 were detected in three fetuses, respectively. All the three variants were classified into pathogenic variants because they were LOF and de novo mutations, and absent in control population. The detailed clinical features and genetic information were shown in After performing genetic analysis, variants of two genes, de novo heterozygous variant  in the exon 36 of KMT2D gene (CD114940)  . At 23-wde novo 150 kb microdeletion at 9q34.3 , resulting in deletion of full-length of NOTCH1, INPP5E, and SEC16A as well as part of PMPCA  in the exon 6 of WDFY3 gene  in the KMT2D gene, resulting in a truncated protein, in Case 1. The KMT2D encodes a histone H3K4 methytransferases that is associated with transcriptionally active genes. Mutations in KMT2D are most frequently, up to 70%, found in patients with Kabuki syndrome , which is a rare, autosomal dominant multiple congenital anomaly syndrome characterized by recognizable facial features, global developmental delay, intellectual disability, short stature, and musculoskeletal abnormalities  followed by NOTCH1 (6.1%)  . NOTCH1,ided-CHD , 32. Deland HLHS . Our Cas parents . INPP5E *1) variant in WDFY3, thus, we propose novel candidate gene for early cardiac development supported by functional and phenotyping evidence in animal model  were loss-of-function variant and putatively lead to haploinsufficiency  ethics committee. The patients/participants provided their written informed consent to participate in this study.HX and ZA conceived the study. CL, WW, ZM, DZ, CJ, and ZY clinically analyzed the patients. LS and TL conducted genetic data interpretation. LS and HX performed literature review and prepared the manuscript. ZA, ZY, and HX revised the manuscript. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.