Avibacterium paragallinarum-associated infectious coryza (IC) is an important threat in commercial poultry. Previous studies about the characteristics of A. paragallinarum are succeeded in revealing the course of IC disease, but whether and how resident microbes contribute to the infection remains unclear. To understand the role of commensal bacteria, we isolated 467 commensal bacteria, including 38 A. paragallinarum, from the respiratory tract of IC chicken. The predominant commensal isolates were Gram-positive bacteria belonging to Staphylococcus spp. , Enterococcus spp. , and Bacillus spp. . These isolates were closely correlated with the survival of A. paragallinarum. We examined and found that commensal bacteria aggravate A. paragallinarum-associated infections because certain commensal species  induced hemolysis and promoted the growth of A. paragallinarum in vitro. Notably, A. paragallinarum showed high resistance to routine antibiotics such as erythromycin , tetracycline  and carried diverse mobile resistance gene clusters. Overall, we found commensal bacteria especially Gram-positive bacteria facilitate the survival of multidrug-resistant A. paragallinarum to exacerbate infections, suggesting that novel strategies may diminish A. paragallinarum-associated infections by modulating the population dynamics of commensal bacteria. The scheme of interactions in this study.  A. paragallinarum and facilitate the survival.- Commensal bacteria isolated from respiratory tracts in IC chicken show correlative to the presence of A. paragallinarum commonly present in respiratory tracts of IC chicken.- The symbiotic relationship between commensals and A. paragallinarum isolates, against frequently-used antimicrobial agents in commercial poultry.- Mobile resistance gene clusters are distributed globally in Avibacterium paragallinarum, historically named Haemophilus paragallinarum  is an important threat to global animal health and causes huge economic losses, resulting in seriously decreased egg production and increased mortality in laying hens . The disllinarum . Such ba and healthy chickens (n = 9) were collected from commercial layer farms in Tianjin, Hebei, and Guangxi in China from 2019 to 2022. All samples were vaccinated against IC and with no antibiotic treatment after infection. The heads of infected and non-infected birds were disinfected with 75% ethyl alcohol before bacterial isolation. Swab samples collected from nasal cavity and infraorbital sinuses were cultured on blood agar [trypticase soya agar (TSA) supplemented with 5% filter-sterilized sheep] and TSA plus agar , respectively. TSA plus agar plates were used to isolate the A. paragallinarum and incubated aerobically at 5% CO2 and 37°C for 24–48 h. The colony morphology consisting with translucent light blue was selected as A. paragallinarum for further study. Commensals were cultured in blood agar plates at 37°C for 24 h and further purified in TSA agar. Afterward, all isolates were identified based on MALDI-TOF-MS identification and 16S rRNA gene sequence analysis. After confirming the taxonomic classification of these isolates, A. paragallinarum was freeze-dried with 10% skim milk and commensals were maintained in nutrient broth supplemented with 20% glycerol and stored at −80°C.Layer chickens with symptom of IC  Isolation rate (%) = the number of isolates of a specific genus or species/total number of isolates; (2) Satellitism rate (%) = the number of specific species or genus of bacteria that facilitate the growth of A. paragallinarum/total number of commensal isolates that facilitate the growth of A. paragallinarum; (3) Colocalization rate (%) = the number of chickens infected with a specific species or genus of bacteria and A. paragallinarum/the number of chickens infected with A. paragallinarum.According to the previous research , the rel6 CFUs/ml, and then recorded the transparent hemolysis radius after cultured for 12, 24, 36, 48, 60, and 72 h at 37°C. Meanwhile, hemolysis of supernatant from commensals was also determined according to the method described previously  = (ODSample − ODNegative)/(ODPositive − ODNegative) × 100%.To evaluate the hemolytic activity of commensal bacteria, isolates were spot on 5% sheep blood cells (RBCs) TSA agar after diluted to 10eviously  with minA. paragallinarum isolates to 13 antimicrobial agents . DNA was then fragmented to prepare the library and was sequenced using Illumina NovaSeq 6,000  in pair-end model. High-quality reads were de novo assembled by using SPAdes v3.12.0 (via BLASTn with detailed parameters (identity ≥70% and coverage ≥50%). Prophages and prophage-like elements were analyzed by Phigaro v2.3.0  to examine the correlation.The data was statistically analyzed by using GraphPad Prism 9. Tests of normal distribution were performed (Shapiro–Wilk test) and data were analyzed using one-way non-parametric  or one-way ANOVA with Tukey’s A. paragallinarum, as Firmicutes were the most prominent phylum. In URT of IC chickens, a total of 467 commensal and pathogenic bacteria were to be isolated, including the causative agents such as A. paragallinarum, G. anatis, Pseudomonas aeruginosa, and Rothia nasimurium . In contrast to the healthy group, URT microbiota in infected chickens was mainly composed of Gram-positive bacteria, which was dominated by three most prominent genera: Staphylococcus (33.19%), Enterococcus (16.49%), and Bacillus (16.27%) , 4.91% (23/467), 4.28% (20/467), and 3.43% (16/467), respectively. The abundance of minor bacterial genera (less than 2% isolation rate) ranging from 1.71 to 0.21% contained respiratory tract pathogens such as Streptococcus pluranimalium and P. aeruginosa, implying that these isolates might not be the causative agents of IC.A total of 554 culturable bacterial isolates were identified from the respiratory tracts of healthy chickens and IC chickens . The URTsimurium . Althoug(16.27%) . Each abectively . Intereslezensis , which wA. paragallinarum was associated with a part of commensals. To reveal the potential contribution of commensal to A. paragallinarum-associated infections, we determined the growth-promoting ability of commensal bacteria by measuring the radius of satellitic zone , the promotion effect was restricted within certain species. In this case, the ability of growth promotion was not shared by closely related species. Conversely, in two abundant genera (Bacillus and Staphylococcus), all isolates showed promotion capacity with average satellitic radii ranging from 5 to 9 mm  and colocalization  , the same positive correlations were observed as most of these isolates could support the growth of A. paragallinarum  . Of threllinarum , but sucfaecalis . The mosllinarum . In concA. paragallinarum are available after microbiota-mediated RBCs lysis  was composed of multiple ARGs such as cat, aph, and sul, counting as Tn10 variant (mef(B), implying that novel determinants could play a part in such resistance. As for antimicrobials of fluoroquinolones and β-lactams, we found two isolates showed extremely high MIC values of >16 μg/ml for enrofloxacin, and 28.94% (11/38) of isolates are ampicillin resistance while eight isolates (21.05%) were positive for β-lactam resistance genes including blaCTX–M–14, blaOXA–1, and blaROB–1  showed resistance to gentamicin with a MIC value of >16 μg/ml. The genetic environment of type II gene cluster composed of several aminoglycoside resistance genes  shared high sequence similarity for the multi-drug resistance plasmid pYMH5, which was homologous to broad host-range plasmid pLS88 from H. ducreyi. Notably, 13 of 38 A. paragallinarum isolates exhibited potentially resistant to colistin with MICs value of 32 μg/ml or greater, but colistin resistance genes were not found, indicating a novel mechanism contributed to this phenotype.In the context of the commensal microbiota, resistant  poultry . Majoritelements . The Tn1 variant . Except blaROB–1 . The genA. paragallinarum. All of A. paragallinarum were carried VGs lpxC, manB/yhxB, and gmhA/lpcA, which involved in lipooligosaccharide (LOS) and exopolysaccharide biosynthesis. Endotoxin biosynthesis genes kdsA were restricted to group B A. paragallinarum, suggesting that kdsA acted as a marker gene in comparison to group A A. paragallinarum (gmhA/lpcA) were flanked by integrases and insertion sequences (Strikingly, resistance genes were almost flanked by mobile genetic elements. Several resistance genes associated with MGEs combined into a large gene cluster were globally distributed in this study as shown in llinarum . Notablyequences . The posA. paragallinarum and commensal microbiota may compromise therapeutic efficacies and aggravate persistent infections (A. paragallinarum, including Staphylococcus, Enterococcus, and Bacillus. These commensals are mainly composed of Gram-positive bacteria, consisting with previous observations that Gram-positive commensals create suitable niches for Gram-negative pathogens under resource-limited conditions (A. paragallinarum, especially for these co-existed Gram-positive bacteria in the respiratory tract.The resident microbiota plays a crucial role in eradicating the colonization of pathogens and rebuilding microbial defense systems to maintain homeostasis . Howeverfections . Besidesnditions . Hence, + (B. cereus, C. jeikeium, S. hyicus, S. aureus, and S. haemolyticus, can disrupt host barriers to accelerate the replications of A. paragallinarum in respiratory tract. For example, B. cereus is capable to produce diverse enterotoxins like Nhe and Hbl (A. paragallinarum. Similarly, toxins secreted by Staphylococcus spp. potentiate infections as well (A. paragallinarum.The hemolytic activity may play a crucial role in interspecies interactions. The lysis of RBCs by commensal subsequently releases intracellular contents particularly NAD+  to favor and Hbl , to prom as well . TherefoA. paragallinarum aggravate infections. A. paragallinarum show increasing resistance to antimicrobial agents that are recommended for the treatment of IC such as ampicillin and tetracycline (A. paragallinarum isolates show resistance to ampicillin. Nevertheless, tetB is present in all A. paragallinarum isolates and locates in the Tn10 transposon. The high level of tetracycline may promote the acquisition of mobile tetB gene in sensitive strains. Additionally, the genetic environment of Tn10 is similar to the ICEs in G. parasuis (tetB is undergoing within Haemophilus (mcr genes are detected in A. paragallinarum, indicating that additional undetermined resistant elements may contribute to such phenotype. Conclusively, the resistance characteristics emerging in A. paragallinarum enable them to better survival in vivo.The antimicrobial resistant acycline . We findparasuis , suggestmophilus . NotablyA. paragallinarum. Commensal bacteria particularly Gram-positive commensals show closely related to the presence of resistant A. paragallinarum and benefit the survival thereof. The commensal-mediated hemolysis further promotes the growth of A. paragallinarum and probably aggravates the infection. Therefore, targeting the population dynamics of commensal bacteria may be a promising therapeutic approach to treat IC.The composition and diversity of URT microbiota shift by the colonization of PRJNA836796 can found in the article/The draft whole genome sequence assemblies in the study are deposited in the GenBank repository, accession number This animal study was reviewed and approved by the China Agricultural University. Written informed consent was obtained from the owners for the participation of their animals in this study.JJZ: conceptualization and writing—review and editing. YSC: methodology, validation, formal analysis, investigation, and writing—review and editing. YFW: methodology, validation, and formal analysis. KZ and YQW: conceptualization, methodology, and funding acquisition. All authors contributed to the article and approved the submitted version.


BAK1, CHMP4A, CHMP4B, DHX9, and GSDME was established. Subsequent analyses demonstrated that the model could credibly classify patients as low or high risk and was an independent prognostic indicator for HCC. Abnormal expressions of the five genes were validated by biological experiments and new bioinformatics analysis. In conclusion, this study recognized and verified two heterogeneous pyroptosis subtypes and a predictable prognosis model for HCC. Our work may help facilitate the clinical management and treatment of HCC and understand the functions of pyroptosis in oncology.Hepatocellular carcinoma (HCC) is a worldwide malignant cancer with high incidence and mortality. Considering the high heterogeneity of HCC, clarifying molecular characteristics associated with HCC development could help improve patients' outcomes. Pyroptosis is a novel form of cell death and is noted to be implicated in HCC pathogenesis whereas its molecular feature in HCC is unclear. Thus, we intended to clarify the molecular characteristic as well as the clinical significance of pyroptosis for HCC. A systematic bioinformatics analysis was conducted among 40 pyroptosis-related genes based on The Cancer Genome Atlas, the International Cancer Genome Consortium, and the Gene Expression Omnibus databases. A total of 12 HCC-associated pyroptosis-related genes (HPRGs) were identified to be overexpressed in HCC tissues and significantly connected to patients' poor survival. Through consensus clustering based on the HPRGs' expression, we found patients could be stratified into two distinctive pyroptosis subtypes, PyLow and PyHigh. The PyHigh group owned a notable lower survival rate and a higher high-grade proportion compared with the PyLow subtype. Besides, patients' sensitivities to chemotherapeutic drugs also presented distinctive differences between the two subtypes. Indicated by pathway enrichment analysis and immune characteristic difference analysis, the distinctions between the pyroptosis subtypes may be related to tumor immunity. Further, a five-gene risk model composed of  Liver cancer is a worldwide malignant cancer with high incidence and mortality, representing the fourth contributing cause of cancer deaths . HepatocPyroptosis is a new type of proinflammatory programmed cell death, featured by cell swelling, lysis, and release of cellular proinflammatory contents . PyroptoHerein, we aimed to systematically investigate the molecular classification potential as well as prognostic and therapeutic values of pyroptosis for HCC patients. A comprehensive bioinformatics analysis was conducted based on The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases utilized as training and validation cohorts, respectively. A sum of 40 pyroptosis-related genes (PRGs) gathered from the Molecular Signatures Database (MSigDB) were analyzed . Based uhttp://xena.ucsc.edu/) and the ICGC database (including 260 HCC samples) (https://dcc.icgc.org/). After removing samples replicated, expression data lost, overall survival (OS) data lost, or survival time less than 30 days, only 343 HCC samples in TCGA and 228 HCC samples in ICGC were employed. Before analyzing, gene expression was converted to transcripts per million (TPM) [https://www.gsea-msigdb.org/gsea/msigdb/) [mRNA expression and clinical profiles of HCC cases were extracted from TCGA database through the UCSC Xena (including 50 adjacent and 374 HCC specimens) (on (TPM) . Genes rmsigdb/) . There wP values in the above analyses less than 0.05 were defined as HCC-associated PRGs (HRPGs).The Wilcoxon test was adopted to measure the expression difference of PRGs between normal and HCC samples. The log-rank test was conducted to estimate the prognostic effects of PRGs employing the “survival” package . Kaplan-k (2 to 9) clusters utilizing the “ConsensusClusterPlus” package [On the basis of the HPRGs' expression, HCC patients were classified into  package . The opt package . Differe package . t-SNE i package .https://www.cancerrxgene.org/) using the half-maximal inhibitory concentration (IC50) predicted by the “pRRophetic” algorithm as an indicator [P < 0.05 was considered meaningful.Survival rate differences between the pyroptosis subtypes were estimated by the log-rank test. The chi-square test was applied to determine the associations between the pyroptosis subtypes and clinical characteristics. In this part, patients who lost information of age, gender, grade, or stage were removed. Finally, 317 and 209 samples were, respectively, left in TCGA and ICGC cohort. Drug sensitivity of chemotherapeutic drugs for HCC patients was assessed based on the Genomics of Drug Sensitivity in Cancer (GDSC) database (ndicator . The Wil2 (meanPyHigh–meanPyLow) (logFC) and P values calculated by the Wilcoxon test. P values were adjusted by the false discovery rate (FDR) method [P < 0.05 was considered meaningful.Expression differences between the subtypes were evaluated with log) method . Genes w) method . P < 0.0http://cis.hku.hk/TISIDB/index.php) [https://www.immport.org/home) [P < 0.05 was regarded meaningful.Tumor microenvironment (TME) components including StromalScore, ImmuneScore, and ESTIMATEScore were obtained by the ESTIMATE algorithm . A list dex.php) . Cytokinrg/home) . Patientn = 221), GSE76427 (n = 95), and GSE10143 (n = 80), were also downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) to test the model genes' performance. Univariate and multivariate Cox proportional hazards regression analyses of the risk score and conventional clinical features were carried out to test whether the risk score was an independent prognostic factor for HCC patients. Only variables with P < 0.05 in the univariate Cox analysis in both the databases were included in the multivariate Cox analysis.The predictive risk model based on the HPRG expression was constructed by the least absolute shrinkage and selection operator (LASSO) penalized regression analysis employing the “glmnet” package , 32. Thehttp://www.ncbi.nlm.nih.gov/geo/) and then tested by the Wilcoxon test. The number of HCC samples and normal samples is 268 and 243 in GSE25097, 240 and 193 in GSE36376, and 95 and 39 in GSE45436. In qRT-PCR analysis, total RNA was extracted using RNAiso Plus (TaKaRa) and reverse transcribed into cDNA with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) followed by qPCR detection using the SYBR Green qPCR Master Mix (Bio-Rad). Primers are listed in Table t-test. Protein expression differences were investigated by comparing their staining level differences based on the immunohistochemistry analysis results from the Human Protein Atlas database  [Expression differences of the model genes were validated through expression difference analysis based on GEO database, quantitative real-time PCR (qRT-PCR) analysis, and immunohistochemistry analysis. In expression difference analysis, mRNA expression profiles were extracted from three GEO datasets  , 34.All analyses were carried out in the R 4.0.4 software except qRT-PCR data which was analyzed in the GraphPad Prism 8.0.1 software. All packages and parameters applied in the ICGC cohort were the same as those in TCGA cohort.P < 0.05, P < 0.05, Figures The intention of this study is shown in k = 2 there was the flattest middle segment of the CDF curve  for HCC patients were tested between the two subtypes . ApplyinP < 0.05, P < 0.05, DEGs between the two subtypes were identified based on differential expression analysis. Then, enriched pathways among these DEGs were determined through the KEGG analysis. In differential expression analysis, 1119 genes were expressed aberrantly in TCGA cohort, among which 1063 genes were overexpressed in the PyHigh subtype Table . In the P < 0.05, Figures P < 0.05, Figures Immune characteristic differences between the pyroptosis subtypes were compared on three levels: TME components, TIL abundance, and CRG expression level. In TME component analyses, the PyHigh subtype harbored notably higher ImmuneScore and ESTIMATEScore than the PyLow subtype in both TCGA and ICGC databases  + (0.0227 × CHMP4AExp) + (0.0006 × CHMP4BExp) + (0.0044 × DHX9Exp) + (0.0406 × GSDMEExp). Based upon the median risk value, samples were separated into low- or high-risk groups. Survival analysis indicated that the high-risk group was markedly correlated to the lower OS rate of patients  which cooccurred in the HCC datasets with survival information, GSE14520, GSE76427, and GSE10143. Results showed that high expression of GSDME and DHX9 in GSE14520 was significantly associated with patients' poorer OS (log-rank test, P = 0.016 and P = 0.017, respectively, Figures P > 0.05, Figures To evaluate the joint effect of the 12 HRPGs on patients' survival, we established a multigene prognostic model by conducting the LASSO penalized regression analysis in TCGA cohort. Consequently, a 5-gene signature was identified according to the optimal value of  Figures . Each paP = 0.008 and P = 0.020 in TCGA and the ICGC cohort, respectively, P = 0.007 and P = 0.033 in TCGA and the ICGC cohort, respectively, To test the genetic selection consistency, we also performed elastic net regression analysis among the 12 HPRGs as suggested by a published article . As a reP < 0.05, Figures t-test, P < 0.05, Figures To verify the five model genes' abnormal expressions, we preformed several different analyses. In mRNA expression difference analysis based on GEO databases, all the model genes were significantly overexpressed in HCC tissues in GSE25097, GSE36376, and GSE45436 datasets (Wilcoxon test, Tumor-promoting inflammation is one of the ten well-known characteristics of cancer . As a ne2O3 on HCC cells was reported closely related to caspase-3-dependent GSDME pyroptosis [Usually, pyroptosis is reckoned to be induced by caspase-1-dependent or caspase-1-independent ways with GSDMD as the effector. In recent years, a novel avenue, namely, the caspase-3-GSDME axis, has been identified. In the pathway, activation of caspase-3 could induce GSDME cleavage and thus result in pore formation and ultimately trigger pyroptosis . Accordiroptosis . In anotroptosis . Based oPyroptosis is a double-edged sword for tumors . It can In terms of mechanism, pyroptosis affecting HCC patients' prognosis and progress are inseparable from tumor immunity. In our study, several results from multiangle have implicated it. First, several immune-relevant biological functions, such as viral protein interaction with cytokine and cytokine receptor, were remarkably enriched. Second, TME components, TIL inflation level, and CRG expression conformably harbored more abundant in the PyHigh group in both the databases. It is well acknowledged that hepatitis B and C viruses are the two crucial risk causes for HCC . They caIn the drug sensitivity test, 4 common chemotherapy drugs, Axitinib, Doxorubicin, Erlotinib, and Pazopanib, presented noticeably different sensitivities to the two subtypes. A previous study found that Erlotinib could elicit GSDME-modulated pyroptotic tumor cell death in lung cancer . OverexpNevertheless, there are some limitations of our research. For example, only the most applied mRNA expression data for tumor classification was included in HCC subtyping. Other omics data, such as gene mutation, which is also important to tumor heterogeneity, should be considered in the future. Secondly, some model genes' expression showed no significant association with patients' survival in some GEO datasets. Actually, since gene's expression was affected by various confounding factors, usually several genes combined together rather than single gene alone were utilized to conduct predictive model . The risIn summary, we have recognized and verified two heterogeneous pyroptosis subtypes and a multigene prognostic model for HCC patients. Mechanically, pyroptosis playing an effect on HCC development is closely related to tumor immunity. These results may help guide HCC clinical management and deepen the understanding of pyroptosis.


Background: Primary serine deficiency disorders have a broad range of the phenotypic spectrum. As an inborn error of metabolism, individuals with severe phenotype may be easily recognized with Neu-Laxova syndrome. However, late-onset mild phenotypes may be underdiagnosed and will lead to disastrous consequences due to treatment delays.Materials and Methods: Clinical features of patients with serine deficiency disorders were summarized in two unrelated patients. Skin and sural nerve biopsies were conducted on the patients. Whole exome sequencing (WES) was performed in the index patients. Sanger sequencing was used to analyze family cosegregation.Results: Patient 1 was a 19-year-old male presenting with infancy-onset ichthyosis and juvenile-onset neuropathy. Patient 2 was a 17-year-old male manifesting childhood-onset ichthyosis and juvenile-onset neuropathy. Except for nystagmus, no other developmental or neurodegenerative disorders were found in the patients. Electrophysiological studies indicated a severe sensorimotor axonal neuropathy with a possible demyelinating component. High-dose oral L-serine and glycine completely alleviated skin lesions and only slightly improved neuropathy symptoms. Skin biopsies showed typical features consistent with ichthyosis and severe loss of unmyelinated axons. Sural biopsies revealed a severe loss of axons and a few thinly myelinated fibers. WES found the same homozygous variant c.43G > C (p.A15P) in the PSAT1 gene, which was cosegregated in the two families.Conclusions: The skin and nervous system may be the main affected targets in serine deficiency disorders. Our patients show a more simple and mild phenotype of PSAT1-related serine deficiency disorder. The pathological changes and regenerative ability of skin and peripheral nerves determine their response to serine supplements. PHGDH gene), phosphoserine aminotransferase , and phosphoserine phosphatase  (Serine deficiency disorders include a group of inherited metabolic diseases caused by a defect in one of the three synthetases in the L-serine biosynthesis pathway . L-serinPH gene) . The firPH gene) .Primary defects in all three enzymes of serine biosynthesis have been associated with a broad range of the phenotypic spectrum . CurrentPSAT1 was subsequently investigated as a candidate gene for serine metabolic dysfunction, and mutations in PSAT1 were found in six individuals with Neu-Laxova syndrome . The rest of the specimens were fixed by 4% formalin solution, embedded in paraffin, cut into 4-mm thick sections, and stained with hematoxylin and eosin (H & E). Immunostaining with neurofilament (NF) and myelin basic protein (MBP) antibodies was performed to observe the axon and myelin sheath, respectively.PSAT1 mutation in the patients and their available family members.Genomic DNA was extracted from peripheral blood samples. Whole exome sequencing (WES) was commercially supported by Running Gene Inc. . In brief, targeted exon enrichment was performed using Sure Select Human All Exon V5 (Agilent Technologies). The exon-enriched DNA libraries were subjected to paired-end sequencing with the Hiseq2000 platform . The sequenced data were aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner (BWA) . DuplicaThe individual was a 19-year-old male from a nonconsanguineous family and had a healthy sister 8 years older than him. The patient was born with birth weight, length, and head circumference within the standard percentile. He also had normal developmental milestones. Flaky and scaling skin on the trunk and limbs had been noticed since infancy, which obviously deteriorated in winter and slightly relieved in summer or hot weather. At age 16, the patient found himself walking on his toes and his heels could not touch the ground. He complained of frequent stumbles on uneven ground and numbness in distal lower limbs. One year later, he noticed high arches on his feet. The sensory disturbance gradually developed upward. He also had difficulty walking quickly and keeping on steady. At age 19, the patient had muscle weakness in both thumbs that showed difficulty in straightening, writing, and picking up food. He also showed difficulty in climbing the stairs and standing up after squatting.The patient at age 19 had a normal cognitive function with 30 scores on the mini-mental state examination (MMSE). The body mass index (BMI) was 14.2 . Physical examination revealed generalized xerotic skin with fine scaling and areas with a shiny and atrophic texture. Ichthyosiform scaling was prominent in his upper forearms, upper chest, abdomen, and thighs . The hanBlood count, blood biochemistry, serum creatine kinase (CK), inflammatory tests, paraneoplastic antibody panel, thyroid hormones, serum vitamin B12, and folic acid were within normal limits. Immunofixation electrophoresis was negative. The panel of anti-ganglioside antibodies including GQ1b, GT1b, GD1b, GD1a, GM2, and GM1 was negative. Cerebrospinal fluid (CSF) results were normal. Blood acylcarnitine and urine organic acid analysis were normal. Plasma L-serine was 66.6 μmol/L  at 12 h after fasting and other amino acids were within normal limits. Brain magnetic resonance imaging (MRI) and MR angiography (MRA) were normal. An electroencephalogram (EEG) showed no abnormalities. A pure hearing threshold examination revealed a decrease in perception of low-frequency sounds (125–500 Hz). Video-head impulse test indicated that the patient had impairments of bilateral horizontal and right posterior semicircular canals. Complete ocular examinations were normal.The patient is a 17-year-old male from a consanguineous family. He was born with normal head circumference, weight, and length. At age 4, the patient showed reddish and flaky skin on the plantar and toes, which gradually developed upward thighs and hands. The skin lesions were aggravated in winter and spring and mildly alleviated in summer and autumn. At age 16, the skin of the face, neck, and distal limbs is significantly reddened, desquamated, and locally ulcerated. Meanwhile, the patient gradually felt weakness in both the lower limbs and could not walk on tiptoe, but could walk with heels. Recently, he showed difficulty in climbing the stairs and standing up after squatting. He also felt numbness in his feet. Nail malnutrition, fingertip, and plantar ulcers were found.The patient at age 17 had a normal cognitive function with 30 scores of MMSE. The BMI was 22.5. The skin was red and mildly swollen on the face, hands, and feet. Ichthyosiform scaling was prominent in his neck, abdomen, and feet . Muscle Blood count, blood biochemistry, serum CK, inflammatory tests, paraneoplastic antibody panel, thyroid hormones, serum vitamin B12 and folic acid were within normal limits. Immunofixation electrophoresis was negative. The panel of antiganglioside antibodies was negative. The protein level of CSF was 534.4 mg/L . Blood acylcarnitine and urine organic acid analysis were normal. Plasma L-serine was 32 μmol/L at 12 h after fasting, and other amino acids were within normal limits. Brain MRI and EEG were normal. A pure hearing threshold examination revealed no abnormalities. Fundus examination showed mild atrophy in both perioptic discs.In patient 1, motor nerve conduction velocity (MNCV) was not elicited in the bilateral median and peroneal nerves. MNCV of ulnar and tibial nerves showed a severe reduction of compound muscle action potentials (CMAP) and a moderate decrease in conduction velocity. Sensory NCV (SNCV) showed that NCV and sensory nerve action potentials (SNAP) were decreased in the upper limbs, and no responses in the bilateral superficial peroneal nerves . A needlSkin pathological findings in patient 1 showed severe laminar to compact orthokeratotic hyperkeratosis extending in the follicular infundibula and decreased granular layer with follicular plugging . ElectroSural nerve biopsy in patient 1 showed a severe loss of axons on NF immunostaining . Toluidip.A15P) in the PSAT1 gene in both patients 1 and 2 (http://gnomad.broadinstitute.org). A mutation  at the same site leading to substitution for other amino acids has been confirmed to be pathogenic  and glycine (300 mg/kg/d). Three months later, the skin symptoms showed significant improvement with almost normal skin color . The patPSAT1-related serine deficiency disorder have been reported in other 29 individuals including 13 males, 15 females, and one unidentified gender died in utero , c.44C > T , c.delG107 , c.129T > G , c.296C > T , c.299A > C , c.432delA , c.497C > T , c.1A > G , c.536C > T , and c.del1023_1027insAGACCT .In addition to the two patients in this study, in utero  , one by PHGDH, and one by PSPH (p.R491Y) and c.1273G > A (p.V425M) in the PHGDH gene (p.V44G) and c.421G > A (p.G141S) in the PSPH gene (p.T156M) and c.43G > C (p.A15P) in the PSAT1 gene , respectively  dependent homodimer that is highly conserved from bacteria to humans . All mut pathway . The var pathway . The Ala pathway , and subresidues . Functiof serine . Collectntration . Our pathenotype . Therefohenotype . The milhenotype . Of notePSAT1-related serine deficiency is potentially treatable by oral L-serine alone or combined with glycine with varying degrees of success ( success . At pres success . Our patPSAT1-related serine deficiency disorder. The pathological changes and regenerative ability of skin and peripheral nerves determine their response to serine therapy. Therefore, early diagnosis and treatment are vital to the prognosis of PSAT1-related neuropathy as a treatable disease.In summary, serine deficiency disorders include a broad clinical spectrum, and the skin and nervous system may be the main and consistent target organs. Our patients presented with infancy- or childhood-onset ichthyosis and juvenile-onset peripheral neuropathy, which expands the clinical phenotype of


Klebsiella variicola from Klebsiella pneumoniae complex using updated MALDI-TOF databases, whereas many laboratories still report them as K. pneumoniae or K. pneumoniae complex.Modern laboratory methods such as next generation sequencing and MALDI-TOF allow identification of novel bacterial species. This can affect surveillance of infections and antimicrobial resistance. From 2017, increasing numbers of medical microbiology laboratories in Switzerland differentiated K. variicola and the Klebsiella pneumoniae complex affected the ANRESIS surveillance database.Our study explored whether separate reporting of K. variicola and non-K. variicola-K. pneumoniae complex isolates reported by Swiss medical laboratories to the ANRESIS database (Swiss Centre for Antibiotic Resistance) from January 2017 to June 2022.We analysed antibiotic susceptibility rates and specimen types of K. variicola compared with species of the K. pneumoniae complex other than K. variicola in all six antibiotic classes tested. This can lead to underestimated resistance rates of K. pneumoniae complex in laboratories that do not specifically identify K. variicola. Furthermore, K. variicola strains were significantly more often reported from blood and primarily sterile specimens than isolates of the K. pneumoniae complex other than K. variicola, indicating increased invasiveness of K. variicola.Analysis of Swiss antimicrobial resistance data revealed increased susceptibility rates of K. pneumoniae complex can improve our understanding of its taxonomy, susceptibility, epidemiology and clinical significance, thus providing more precise information to clinicians and epidemiologists.Our data suggest that refined differentiation of the  What did you want to address in this study?Klebsiella pneumoniae as an example.Our previously known microorganism species classification is increasingly refined by novel technological methods. In this study, we investigated how this technological leap affects antibiotic resistance surveillance data using What have we learnt from this study?Klebsiella pneumoniae species complex into individual species was inconsistent across laboratories. This affects antibiotic resistance monitoring: resistance rates of the species complex as a whole appear lower in those laboratories where Klebsiella variicola, one of the less resistant species in the complex, is still included.The introduction of further differentiation of the What are the implications of your findings for public health?Harmonisation of resistance monitoring requires early identification of such inhomogeneous data and agreement on a common approach, otherwise resistance data may differ across countries (and even within countries) for methodological reasons.Staphylococcus argenteus, Gemella taiwanensis, Actinotignum spp [Macrococcus spp [Corynebacteriae and Enterobacterales have been reclassified considerably, with new species and subspecies [Citrobacter europaeus strains and multi-resistant Enterobacter bugandensis isolates with the potential of increased virulence have been described [DNA sequencing methods refine phylogenetic population structures and allow identification of novel species. Novel species may show distinct resistance rates and clinical significance, revising our understanding of epidemiology, ecology, pathogenesis and antimicrobial susceptibility ,2. Exampgnum spp , Macrocoescribed . A recenescribed .Klebsiella pneumoniae complex is highly diverse: as a member of Enterobacterales, this complex of closely related species occupies various ecological niches. Several virulence and resistance factors have been described in strains causing a variety of infections in humans and animals [K. pneumoniae complex has identified four species with a total of seven subspecies [K.quasivariicola’. The  animals . To datebspecies . Recentlbspecies : ‘K.quaKlebsiella pneumoniae sensu stricto has been isolated from asymptomatic humans, but is also a frequent cause of serious invasive infections associated with strains carrying large varieties of virulence or resistance accessory genes [Klebsiella species are considered less virulent and resistant, although recent studies applying refined species discrimination methods revealed virulence and antibiotic resistance genes also in these species [K. variicola has been recovered from a wide range of environments and hosts [ry genes . Other K species . For exand hosts . It is and hosts . Moreovend hosts , increasnd hosts  and antind hosts .K. variicola from other species of the K. pneumoniae complex. An increasing number of laboratories has started reporting K. variicola separately and report the remaining species of the K. pneumoniae complex as K. pneumoniae or K. pneumoniae complex. Thus, K. pneumoniae or K. pneumoniae complex can include different sets of species depending on the reporting policies of individual laboratories. For antimicrobial resistance surveillance databases, inconsistent reporting practices may affect trend analyses of resistance rates if novel species show differential antimicrobial susceptibilities.Increasing antimicrobial resistance rates are a concern for public health . Many coK. variicola isolates, the aim of this study was to investigate whether differentiation of K. variicola from other species of the K. pneumoniae complex (we refer to those as non-K. variicola-K. pneumoniae-complex (non-kv-kpc) in this article) may affect the ANRESIS surveillance database. The Swiss Centre for Antibiotic Resistance (ANRESIS) has hosted the Swiss national antimicrobial resistance surveillance database since 2004 and collects antibiotic resistance data of all bacterial species isolated and reported from various invasive and non-invasive specimens  of up to 37 medical diagnostic laboratories (in 2020). Reported isolates were recovered from patients treated in different facilities, ranging from tertiary hospitals to general practices. The contributing laboratories are widely distributed across Switzerland, representing 89% of all hospitalised patients . With thK. variicola and non-kv-kpc isolates from the ANRESIS database for all laboratories differentiating K. variicola from K. pneumoniae complex, starting from 1 January 2017 when K. variicola became available in the MALDI-TOF database for routine diagnostics, or as soon as individual laboratories began K. variicola differentiation, until 13 June 2022. Species identification and antimicrobial susceptibility tests (AST) were performed at local laboratories following the guidelines from European Committee on Antimicrobial Susceptibility Testing (EUCAST) (36 laboratories) or the Clinical and Laboratory Standards Institute (CLSI) (one laboratory until 2020). The individual laboratories used various AST methods, such as automated minimum inhibitory concentration (MIC) tests or disc diffusion tests measuring inhibition zones manually or through an automated process. All reporting laboratories are accredited by the Swiss Accreditation Service (SAS). We extracted susceptibility rates and specimen types of Resistance categories were defined resistant if categorised R (resistant) by reporting laboratories, and non-resistant if categorised S (susceptible) or I , according to EUCAST's revised susceptibility categorisation that has been introduced gradually in Swiss laboratories since 2019 . For thiK. variicola and non-kv-kpc using Fisher's exact test. Repeated testing was accounted for by applying sequential Bonferroni corrections. All statistical tests were conducted in R v4.2.1 [Invasive strains were defined as isolates obtained from blood or primarily sterile specimen types, such as cerebrospinal fluid (CSF) and tissue specimens. We compared susceptibility rates and proportions of invasive strains of R v4.2.1 .K. variicola increased from four of 30 laboratories reporting antibiotic susceptibility data to ANRESIS in 2017 to 18 of 35 laboratories in June 2022. Klebsiella variicola was less frequently isolated than non-kv-kpc isolates: from 1 January 2017 to 13 June 2022, 14.1%  of the 70,114 isolates analysed were reported as K. variicola. All six antibiotic substances or antibiotic classes tested showed significantly higher susceptibility rates in K. variicola than in non-kv-kpc isolates. These differences remained after applying sequential Bonferroni corrections  and against β-lactams (maximum rate for amoxicillin-clavulanic acid: 9.1 and 4.7%), followed by ciprofloxacin (6.9 and 2.1%). Resistance rates against gentamicin, however, only showed a small difference with 2.8% for non-kv-kpc and 1.7% for K. variicola isolates, and carbapenem resistance rates were generally low (respectively 0.4 and 0.1%), resulting in broader confidence intervals.Non-kv-kpc and K. variicola isolates were significantly more often reported from blood and primarily sterile specimens than non-kv-kpc isolates, namely 8.7% K. variicola isolates  and 6.1% of non-kv-kpc isolates  .In addition to different resistance rates, K. variicola isolates of the ANRESIS database were significantly more susceptible to six antibiotic classes tested than non-kv-kpc isolates. This is consistent with Cuénod et al., a collaborative international study of several institutions published in 2021 [Klebsiella within the K. pneumoniae and K. oxytoca complex and genomic data on the abundance of antimicrobial resistance genes. The authors suggested that higher susceptibility rates in K. variicola may be linked to lower abundance of plasmids. Our data show that  in 2021 . That stK. variicola  tend to underestimate resistance rates of non-kv-kpc isolates. Similar concerns have been raised by a recent review focusing on genomic data of the K. oxytoca complex. The authors describe several resistance and virulence genes with the potential to create multidrug-resistant pathogens [K. pneumoniae isolates from six countries  varied between countries. Even within Europe, resistance rates vary considerably, increasing from north to south and from west to east, with Switzerland among the countries with lowest resistance rates [Differential susceptibility rates may affect surveillance data, as laboratories not differentiating athogens . Based oathogens . Howeverathogens , showingce rates . These dHigher resistance rates come with higher abundance of transferable resistance genes ,17, poteK. variicola, for example, may lead to an underestimation of resistance rates of other members of the K. pneumoniae complex and thus to less appropriate treatments of infections. This may affect possible positive effects of appropriately tailored antimicrobial treatments [Studies estimating global health burdens based on statistical modelling or machine learning rely on empirical data of mortality, resistance and virulence genes. These data are obtained from literature reviews, hospitals and surveillance initiatives. Up-to-date knowledge of taxonomy, virulence and antimicrobial resistance is thus crucial to avoid misleading conclusions about the clinical significance or resistance rates of some pathogens ,23. Loweeatments .K. variicola may be more invasive than non-kv-kpc. However, this association has to be confirmed by clinical and functional studies addressing underlying causes. A recent study by Cuénod et al. comparing different clinical outcomes of patients infected with different Klebsiella species did not confirm our data [Our data also indicate that our data , whereasour data .K. pneumoniae complex will increase our knowledge of species-specific susceptibilities and clinical significance, providing valuable information to clinicians and epidemiologists [K. variicola from the K. pneumoniae complex is possible not only by molecular methods such as sequencing of rpoB, fusA, gapA, gyrA, leuS genes [K. variicola have been included in commercial databases allowing identification of K. variicola. However, differentiation of K. variicola from the K. pneumoniae complex by MALDI-TOF is not yet accepted by all microbiological laboratories because the close relatedness of Klebsiella spp. prevents verification by 16S rDNA sequencing, a species identification technique used as gold standard by many laboratories. Rodrigues et al. and Cuénod et al. both independently developed MALDI-TOF databases which allow differentiation of all currently known species of the K. pneumoniae complex, but discriminatory precision of these two MALDI-TOF databases have not yet been validated using large datasets [K. pneumoniae complex.A growing body of data, including our study, suggests that discrimination of species within ologists ,6,9,17. uS genes ,25, but K. variicola isolates in our ANRESIS antimicrobial resistance database suggest that refined differentiation of K. pneumoniae complex can improve our understanding of its ecological niches, epidemiology, antibiotic susceptibility and clinical significance, thus providing more accurate information to clinicians and epidemiologists. In addition, refined species information has the potential to improve antibiotic prescribing by tailoring antibiotic treatments more appropriately. Higher susceptibility and invasiveness rates of


Mycobacterium avium complex (MAC) infections. M. chimaera, a newly identified MAC species, is distinguished from the others due to the reduced virulence. Previous cases of disseminated M. chimaera infection have been linked to cardiothoracic surgery. Reports of disseminated M. chimaera in patients without a history of cardiothoracic surgery are rare.Patients with adult-onset immunodeficiency syndrome due to anti-interferon-γ autoantibodies (AIGAs) are susceptible to disseminated M. chimaera. Further immunological evaluation disclosed the presence of neutralizing AIGAs, which possibly led to acquired immunodeficiency and disseminated M. chimaera infection.A 57-year-old Asian man, previously healthy, presented with fever, dry cough, exertional dyspnea, and decreased appetite. The delayed resolution of pneumonia despite antibiotic treatment prompted further imaging studies and biopsies from the lung and lymph node. The fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) demonstrated intense uptake in lung consolidations and diffuse lymphadenopathy. Cultures of the specimens obtained from sputum, blood, stool, lung tissue, and lymph node grew M. chimaera infection. Further immunological evaluation, including AIGAs, may be warranted in otherwise healthy patients who present with disseminated mycobacterial infection.We herein present the first case of adult-onset immunodeficiency due to AIGAs complicated with disseminated The online version contains supplementary material available at 10.1186/s12879-022-07656-0. Mycobacterium avium complex (MAC) and M. abscessus were most commonly reported [M. chimaera is a newly described slow-growing non-tuberculous mycobacterium (NTM) belonging to the MAC and distinguished from the other MAC species due to the relatively reduced virulence. M. chimaera has been reported to cause pulmonary infection, recently linked to infections after cardiothoracic surgery and outbreaks attributed to contaminated heater-cooler devices (HCDs) [M. chimaera infection include pulmonary diseases and extrapulmonary manifestations, including surgical site infection, prosthetic valve endocarditis, vascular graft infection, arthritis, osteomyelitis, and disseminated infection [M. chimaera infection has more commonly occurred in patients undergoing cardiothoracic surgery and rarely been reported in patients with immune dysfunction [M. chimaera infection.Adult-onset immunodeficiency syndrome due to anti-interferon-γ autoantibodies (AIGAs) is associated with susceptibility to selective infections . Previoureported . Dissemis (HCDs) , 4. The nfection . Althougfunction , 7. We hA 57-year-old Malaysian Chinese man, previously healthy, presented to our hospital with fever and exertional dyspnea for 1 week. One month before the current presentation, he developed dry cough and progressively decreased appetite. He had undergone appendectomy in his childhood but had no other systemic disease. There was no known family history of immunodeficiency or exposure to immunosuppressants before disease onset. On assessment, he was hemodynamically stable with the heart rate of 111 beats per minute, the blood pressure 106/60 mmHg, the respirations 18 breaths per minute, and the temperature 38.4 °C. Physical examination revealed decreased breath sounds on the left lung and a swollen lymph node in the right neck. Laboratory evaluations showed leukocytosis  with neutrophilia (89%), anemia (7.9 g/dL), thrombocytosis , elevated C-reactive protein (15.81 mg/dL) and elevated alkaline phosphatase (145 U/L). A chest radiograph and computed tomography (CT) demonstrated multifocal patchy consolidations, bilateral pleural effusion, and enlarged subcarinal lymph nodes , rifabutin , azithromycin , and ethambutol . Three days after initiation of anti-mycobacterial therapy, the low-grade fever, leukocytosis, and elevated alkaline phosphatase persisted; and therefore, moxifloxacin  was added to the treatment regimen. The results of antimicrobial susceptibility testing are summarized in Additional file Ceftriaxone was administered intravenously at a dose of 1000 mg twice daily for 10 days, but his clinical condition and chest radiographs did not improve. The delayed resolution of pneumonia prompted a sonography-guided lung biopsy, in which pathology disclosed granulomatous inflammation with the presence of acid-fast bacilli  infection, elevated immunoglobulin G (IgG) levels, and normal counts of lymphocyte subsets and monocyte. He had a high anti-nuclear antibody titer of 1:1280 with a fine speckled pattern, but did not meet the criteria of autoimmune diseases due to the absence of associated symptoms and specific extractable nuclear antigens. He tested positive for neutralizing AIGAs, with a plasma concentration of 901 μg/ml . The methodologic details on AIGAs enzyme-linked immunosorbent assay (ELISA) are shown in Additional file Mycobacterium chimaera has been shown to be of low virulence, but could cause pulmonary infection as a consequence of colonization to the airways in patients with immunodeficiency or pre-existing lung conditions [M. chimaera lung disease were reported to be suboptimal despite the combination use of long-term antimicrobial agents [M. chimaera infection had been scarcely described until 2013, when the first two cases were reported in patients with prosthetic valve endocarditis and bloodstream infection due to M. chimaera after open-chest cardiac surgery. Afterwards, more cases were identified in the prolonged outbreak, which was linked to a source of contaminated water in HCDs connected to the cardiopulmonary bypass. The increased use of contaminated HCDs has led to HCD-associated M. chimaera outbreaks globally since 2015, with more than 120 cases of M. chimaera infections reported following cardiac surgery in the literature [M. chimaera infection was remarkably high (50%) [M. chimaera infection [nditions . The trel agents . On the terature . With a gh (50%) . To evalM. chimaera infection attributable to their immunodeficiency , new MAC species (M. colombiense and M. mantenii) have been reported to cause disseminated infection in patients with AIGAs [M. chimaera, a recently identified species of MAC, has a lower virulence than the two original species of MAC [M. chimaera infection in a patient with AIGAs; nevertheless, systemic infection caused by M. chimaera among patients with AIGAs may be under-diagnosed due to inaccessibility to AIGAs ELISA and identification of MAC species. To reduce misdiagnosis, the commercially available interferon-γ release assay (IGRA) could be used in the clinical setting without readily accessible AIGAs ELISA. An indeterminate IGRA result may indicate the presence of neutralizing AIGAs in a patient with disseminated NTM infection [Despite geographically varied distribution of NTM species, the dominant NTM species resulting in disseminated infection among patients with AIGAs is MAC 30.7%) . In addith AIGAs –15. M. c0.7% . Innfection , 17.M. chimaera infection can also occur in patients with adult-onset immunodeficiency syndrome due to AIGAs. Early recognition and immunological evaluation in otherwise healthy patients who present with disseminated mycobacterial infection are essential to improving outcomes.The present case highlights that disseminated Additional file 1. Supplementary material.


Ornithobacterium rhinotracheale has been associated with respiratory disease in poultry, particularly turkeys, leading to significant economic losses. However, O. rhinotracheale is poorly studied, and a very limited number of complete genomes are available. Here, we report the complete genome sequences of three O. rhinotracheale strains, generated using a Nanopore-Illumina hybrid assembly approach. Ornithobacterium rhinotracheale has emerged as an important bacterial pathogen for domestic poultry and wild birds (–https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/10734/) to date (November 2022), three of which are from this study. Here, we report the complete genome sequences of three O. rhinotracheale strains, generated using a Nanopore-Illumina hybrid assembly approach  using the Gram-negative bacteria high molecular weight DNA extraction protocol. Nanopore libraries were prepared by multiplexing DNA extracts from the three isolates using the SQK-LSK109 and EXP-NBD104 kits  according to the manufacturer's protocol. Barcodes 1, 3, and 7 from the EXP-NBD104 kit were used.PRJNA224116). Reads generated with each sequencing platform, assembly statistics, annotation of the closed genomes, sequence typing, average nucleotide identity (ANI) values, and GenBank accession numbers are presented in Raw reads from Illumina sequencing were first trimmed for quality and sequencing adapters using Trimmomatic v0.33  with defO. rhinotracheale type strain DSM 15997 (accession number CP003283.1) were above the species cutoff value of 95 to 96%, while the ANI value for genome K223 was below the species cutoff value (89.49%). These findings support the suggestion by Alispahic et al. (O. rhinotracheale serotypes belong to a different bacterial species.It is noteworthy (as shown in c et al.  that somThe genomes are available in GenBank under the accession numbers shown in


A considerable fraction of microRNAs (miRNAs) are highly conserved, and certain miRNAs correspond to genomic clusters. The clustering of miRNAs can be advantageous, possibly by allowing coordinated expression. However, little is known about the evolutionary forces responsible for the loss and acquisition of miRNA and miRNA clusters.The results demonstrated that several novel miRNAs arose throughout grass carp evolution. Duplication and de novo production were critical strategies for miRNA cluster formation. Duplicates accounted for a smaller fraction of the expansion in the grass carp miRNA than de novo creation. Clustered miRNAs are more conserved and change slower, whereas unique miRNAs usually have high evolution rates and low expression levels. The expression level of miRNA expression in clusters is strongly correlated.This study examines the genomic distribution, evolutionary background, and expression regulation of grass carp miRNAs. Our findings provide novel insights into the genesis and development of miRNA clusters in teleost.The online version contains supplementary material available at 10.1186/s12864-023-09159-x. According to large-scale studies of microRNAs (miRNAs) in various species, miRNAs are widely dispersed, and many of them tend to be clustered together on the same chromosome in animals . Based oDrosophila, explains the vast flux of evolutionarily young miRNAs in multiple lineages [The origin of miRNAs can be traced back to early metazoans. Evolutionary forces such as duplication, inversion, mutation, amplification, and other forms of genetic drift that shape the genome may be the most influential factors in the origin and evolution of miRNA genes . During lineages .Populus miRNA clusters, 37.2% of miRNAs were derived from duplication events, while de novo formation resulted in 62.8% of Populus-specific miRNAs [Genome duplication and de novo formation are crucial mechanisms for miRNA cluster formation . Among Pc miRNAs . Yu et ac miRNAs  analyzedc miRNAs , > 30% oc miRNAs , 20. Typc miRNAs  analyzedc miRNAs .Pseudomonas aeruginosa clearance in macrophages by targeting NF-kB [Notably, miRNAs belonging to the same cluster indicate the possibility of co-regulation. Experimental evidence demonstrated that clustered miRNA loci form an operon-like gene structure and are transcribed from a shared promoter, resulting in a cooperative function  and sustng NF-kB . In contng NF-kB . The actng NF-kB . Thus, mCtenopharyngodon idella), a member of the Cyprinidae family, is one of the most important cultivated species in freshwater aquaculture. It's the highest output for a single species worldwide [C. idella. We performed a systematic, genome-wide survey of C. idella miRNAs to comprehend the genomic arrangement of miRNAs. We investigated the origin and evolution of miRNAs and determined the selection pressure on miRNAs. The expression profiles of miRNAs in various tissues were compared. The evolution, expression, and genomic data were merged to completely appreciate why miRNA genes were clustered and how this influences miRNAs' evolution, expression, and function.The grass carp  that can mature into 1,513 miRNAs  within introns, and 870 throughout the intergenic area. Consequently, there were 1.76 times more miRNAs in the intergenic area than in the introns of coding genes  of all grass carp-specific miRNAs, and 52 (73.24%) of all common miRNAs , the most prominent of which are anion transmembrane transporter activity, active transmembrane transporter activity, and active ion transmembrane transporter activity Fig. .Danio rerio) [C. remanei, Jovelin et al. demonstrated that natural selection continues to restrict the evolution of all sequence domains inside miRNA hairpins [The initial evidence of miRNAs in teleosts was identified in 2003 in zebrafish (o rerio) . Since mo rerio) . In raino rerio) . Overexpo rerio) . Howevero rerio) . In humahairpins . This woArabidopsis miRNA genes are conserved in monocot rice, showing a significant percentage of unconserved miRNA genes [Oryzias latipes, Gasterosteus aculeatus, and Chaenocephalus aceratus [In the present study, 1513 mature miRNAs were discovered, of which 1442 were specific to grass carp, demonstrating a high rate of gain for grass carp-specific miRNAs. Despite the high conservation of many miRNAs, many of the miRNAs are only found in one or two species that belong to a certain evolutionary branch. In contrast, others are present in at least three members of a particular evolutionary lineage . For exaNA genes . Due to NA genes –46. Due NA genes , 48. TheNA genes . Genomicaceratus . FinallyDrosophila genome originate de novo; about 60 de novo genes are known in the human genome, and at least 782 de novo genes are found in Arabidopsis [Drosophila species estimated that only ~ 30% of gene gains within clusters could be explained by duplication events, with 46.1% of the new miRNAs in clusters originating from de novo formation [The process of “birth” and the development of new genes has garnered considerable interest among scientists. De novo formation, gene duplication, exon rearrangement, gene fusion/splitting, and horizontal gene transfer are all possible mechanisms. De novo formations are usually considered so unlikely that they are impossible in terms of earlier evolutionary processes . In recebidopsis . Similarbidopsis  or de nobidopsis , 55. As bidopsis . A recenormation . Some miormation , 57, 58;ormation . A totalPopulus-specific recently evolved miRNAs, new miRNAs have a greater chance of targeting over-retained copies of salicylic species, illustrating the potential role of Populus-specific recently evolved miRNAs in local adaptation and functional innovation [Caenorhabditis species [New miRNAs, which always coincide with rapid evolution rates, are less conserved , have lonovation . Jovelin species . These fOur findings indicated the evolutionary dynamic and genomic architecture of the grass carp miRNA repertoire. Grass carp miRNAs can exist in clusters and common miRNAs are more likely to be enriched in miRNA clusters. Grass carp-specific miRNAs evolve at a faster rate with a lower expression levels. It is claimed that the clustered arrangement of miRNAs has a remarkable effect on their origin in evolution and expression control. This work, which included comparative genomics and bioinformatics methodologies, was a worthwhile effort that yielded fresh insights into the origins and development of miRNA clusters in grass carp.n = 15, average weight 40 g, average length 16 cm) were collected, including the brain, muscle, gill, intestine, and heart, and then these tissues were quickly frozen in liquid nitrogen and stored at -80 ℃. Embryos were obtained by artificial insemination spawning. According to the common carp model, the developmental phases of the embryo were split into seven stages and 24 periods, including the zygote, cleavage, blastula, gastrula, neurula, organogenesis, and hatching stages. In this work, we compared the differences in juvenile and embryonic development using hatching stages [The experimental fish used in the experiment were from the Center of Grass Carp Breeding . The tissue samples of grass carp juveniles  and the purity and integrity were evaluated with the Bioanalyzer 2100 system . The analyses that followed employed samples with an RNA integrity number (RIN) ≥ 7. The TruSeq Small RNA Sample Prep Kits  were used in accordance with the manufacturer's instructions to prepare 1 μg of total RNA for the creation of the small RNA library. Briefly, total RNA was ligated to adapters at each end. Then the adapter-ligated RNA was reverse transcribed to cDNA and performed PCR amplification. DNA fragments corresponding to sizes of 140–160 bp were recovered. DNA High Sensitivity Chips were used to evaluate library quality on the Agilent Bioanalyzer 2100 system. Small RNA transcriptome sequencing were conducted by OE Biotech Co., Ltd.  with the Illumina HiSeq X Ten platform.http://rfam.xfam.org/) [Initial reads were processed by removing poor quality reads, 5' adapter pollution reads, reads without 3' adapter, reads without insert fragment, reads containing poly(A) stretches, and reads less than 18 nt or longer than 30 nt. The qualified reads that passed the filters above were then aligned to grass carp genome, known miRNAs, repeat sequences, exon and intron sequences using Bowtie version 1.3.1  with theam.org/)  allowingam.org/)  to prediThe expression levels of miRNAs within each library were normalized to give the expression in transcripts per million mapped reads (TPM). Target prediction was performed by integrating three miRNA target prediction software packages, including miRanda v3.3a, RNAhybrid v2.1.2, and targetscan_70 [Ciona intestinalis, Branchiostoma floridae, Salmo salar, Danio rerio, Cyprinus carpio, Xenopus tropicalis, Anolis carolinensis, Gallus gallus, Ovis aries, Bos taurus, and Petromyzon marinus were downloaded from Ensembl (https://asia.ensembl.org/index.html). The miRNA sequences of all the other species except C. idella were downloaded from miRbase (https://www.mirbase.org/). The birth, death, and age of miRNA families were estimated based on a phylogenetic tree generated by RAxML(-m PROTGAMMAJTT -f a -# 100) [. The birth, death, and age of miRNA families and the ancestral gene contents were assessed using the COUNT software [http://www.timetree.org/).The genome sequences of  -# 100)  analysissoftware  followinA miRNA cluster was defined if neighboring miRNAs were located within 10 kb. Because ancient events of miRNA genesis might be challenging to determine, inherited miRNAs were not examined. We also omitted the initial miRNA in a cluster since it appeared as a single miRNA incapable of forming a cluster on its own. To identify clustered miRNA origin, the mature miRNAs were compared with each other by an all-against-all blast. Then, they were clustered according to sequence similarity, requiring alignment ≧ 15 nt, which covers the miRNA seed region ( position 2–8 nt) and mismatch ≦ 2. Consider clustered miRNAs that comply with the following requirements evolved by duplication. There are two types of miRNA duplication: cis-duplication between adjacent locations and trans-duplication between different chromosomal locations. MiRNAs that don't fit the criteria above are thought to evolve by de novo formation.https://www.ebi.ac.uk/Tools/msa/tcoffee/) with the default parameters. From these alignments, we calculated the sequence divergence between the miRNA precursors and their homologous sequences using KaKs_calculator 3.0 software [p. We then calculated the sequence divergence between the flanking sequences and their homologous sequences, marked as Kf, in the same way. The miRNA evolutionary rate was calculated as K = Kp / Kf.Select miRNA flanking sequences with the same length as the miRNA precursor. Then the alignments for precursors and flanking sequences were constructed using T-coffee .Collinear blocks harboring orthologous miRNAs between  MCScanX . The miRij be the average expression of gene i in tissue j. Then the tissue specificity of gene i is defined by:n is the number of tissues. Thus, if a gene is expressed in only one tissue, the score is 1; if the expression of a gene is the same in all tissues, the score is 0.To measure the tissue specificity, the tissue specificity score was compuCo-expression networks were constructed using the WGCNA package in R . NormaliAdditional file 1: Fig. S1. Detailed information of miRNAs collinear blocks. Collinear blocks between the two species are shown in blue.Additional file 2: Fig. S2. Co-expression networks of modules. (A) A co-expression network of the blue module. (B) A co-expression network of the green module. (C) A co-expression network of the grey module. (D) A co-expression network of the red module. (E) A co-expression network of the brown module. (F) A co-expression network of the yellow module. The orange-filled octagons refer to clustered miRNAs. Red links represent the co-expressed miRNA cluster.Additional file 3: Table S1. miRNA location in the grass carp genome. Table S2. The miRNA family of twelve species analyzed. Table S3. miRNA clusters in the grass carp genome. Table S4. Expression correlation between miRNA duplication of grass crap. Table S5. MiRNA evolution rate. Table S6. The colinearity blocks of protein between grass carp and zebrafish. Table S7. The colinearity blocks of miRNA between grass carp and zebrafish. Table S8. Tissue-specific miRNAs in the grass carp. Table S9. Expression correlation between neighbouring miRNAs of grass carp. Table S10. Expression level of grass carp miRNAs with different genomic context. Table S11. The predicted common miRNA/target pairs.