The structures and activities of enzymes are influenced by pH of the environment. Understanding and distinguishing the adaptation mechanisms of enzymes to extreme pH values is of great significance for elucidating the molecular mechanisms and promoting the industrial applications of enzymes. In this study, the ESM-2 protein language model was used to encode the secreted microbial proteins with the optimal performance above pH 9 and below pH 5, which yielded 47 725 high-pH protein sequences and 66 079 low-pH protein sequences, respectively. A deep learning model was constructed to identify protein acid-base tolerance based on amino acid sequences. The model showcased significantly higher accuracy than other methods, with the overall accuracy of 94.8%, precision of 91.8%, and a recall rate of 93.4% on the test set. Furthermore, we built a website (https://enzymepred.biodesign.ac.cn), which enabled users to predict the acid-base tolerance by submitting the protein sequences of enzymes. This study has accelerated the application of enzymes in various fields, including biotechnology, pharmaceuticals, and chemicals. It provides a powerful tool for the rapid screening and optimization of industrial enzymes.
Deep Learning ; Hydrogen-Ion Concentration ; Amino Acid Sequence ; Enzymes/metabolism* ; Sequence Analysis, Protein ; Proteins/metabolism* ; Bacterial Proteins/metabolism*

In the present study we investigated the changes in miRNA levels inhuman rhinovirus 16 (HRV16)-infected cells. A small RNA deep sequencing experiment was performed through next-generation sequencing. In total, 53 differentially expressed miRNAs were confirmed by RT-qPCR, including 37 known miRNAs and 16 novel miRNAs. Interaction networks between differentially expressed miRNAs and their targets were established by mirDIP and Navigator. The prediction results showed that QKI, NFAT5, BNC2, CELF2, LCOR, MBNL2, MTMR3, NFIB, PPARGC1A, RSBN1, TRPS1, WDR26, and ZNF148, which are associated with cellular differentiation and transcriptional regulation, were recognized by 12, 11, or 9 miRNAs. Many correlations were observed between transcriptional or post-transcriptional regulation of an miRNA and the expression levels of its target genes in HRV16-infected H1-HeLa cells.
CELF Proteins/metabolism* ; DNA-Binding Proteins/genetics* ; Gene Expression Profiling ; Gene Expression Regulation ; HeLa Cells ; High-Throughput Nucleotide Sequencing ; Humans ; MicroRNAs/metabolism* ; Nerve Tissue Proteins/genetics* ; Protein Tyrosine Phosphatases, Non-Receptor ; Repressor Proteins/metabolism* ; Sequence Analysis, RNA ; Transcription Factors/metabolism*

OBJECTIVE: To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T. METHODS: The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp. RESULTS: Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the of voltage-dependence of fast inactivation of I1363T mutant[(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type[(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type. CONCLUSIONS I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.
Gene Expression Profiling ; Humans ; Ion Channel Gating ; genetics ; Muscle, Skeletal ; physiopathology ; Mutation ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Sequence Analysis, Protein

BACKGROUND: Invasive nonfunctioning pituitary adenomas (NFPAs) remain challenging due to their high complication rate and poor prognosis. We aimed to identify the distinctive molecular signatures of invasive NFPAs, compared with noninvasive NFPAs, using gene expression profiling by RNA sequencing. METHODS: We obtained frozen fresh tissue samples from 14 patients with NFPAs who underwent primary transsphenoidal surgery. Three non-invasive and 11 invasive NFPAs were used for RNA sequencing. The bioinformatics analysis included differential gene expression, gene ontology analysis, and pathway analysis. RESULTS: A total of 700 genes were differentially expressed (59 up-regulated and 641 down-regulated genes) between invasive and non-invasive NFPAs (false discovery rate <0.1, and |fold change| ≥2). Using the down-regulated genes in invasive NFPAs, gene ontology enrichment analyses and pathway analyses demonstrated that the local immune response was attenuated and that transforming growth factor-β (TGF-β) RII-initiated TGF-β signaling was down-regulated in invasive NFPAs. The overexpression of claudin-9 (CLDN9) and the down-regulation of insulin-like growth factor-binding protein 5 (IGFBP5), death-associated protein kinase 1 (DAPK1), and tissue inhibitor of metalloproteinase-3 (TIMP3) may be related with invasiveness in NFPAs. CONCLUSION: Invasive NFPAs harbor different gene expression profiles relative to noninvasive NFPAs. In particular, local suppression of the immune response and TGF-β signaling can make PAs prone to invasiveness.
Computational Biology ; Death-Associated Protein Kinases ; Down-Regulation ; Gene Expression ; Gene Expression Profiling ; Gene Ontology ; Humans ; Pituitary Neoplasms ; Prognosis ; Sequence Analysis, RNA ; Tissue Inhibitor of Metalloproteinase-3 ; Transcriptome

PURPOSE: Molecular testing in non-small cell lung cancer (NSCLC) aids in identifying oncogenic alterations. The aim of this study was to compare the rates of detection of oncogenic alterations and responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) according to EGFR mutation status as determined by peptide nucleic acid (PNA) clamping or direct sequencing (DS). MATERIALS AND METHODS: We performed a systematic literature search using MEDLINE, EMBASE, and the Cochrane Central Register. Data from included studies were pooled to yield summary sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and receiver operating characteristic curves. A meta-regression analysis was conducted to identify potential sources of heterogeneity between selected studies. RESULTS: We identified 10 studies comprising 924 patients. Oncogenic alterations were detected in 340 of 924 cases (36.8%) with PNA clamping and in 250 of 924 (27.1%) with DS. The pooled sensitivities of PNA clamping and DS were 0.93 [95% confidence interval (CI): 0.90−0.95] and 0.69 (95% CI: 0.64−0.73), respectively. According to meta-regression analysis, none of the covariates were found to be significant sources of heterogeneity. With respect to treatment responses to EGFR-TKIs, there was no significant difference therein between EGFR mutations detected by PNA clamping and DS (53.4% vs. 50.8%; risk ratio, 0.99; 95% CI 0.83−1.19; p=0.874). CONCLUSION: We demonstrated that PNA clamping has a higher sensitivity than DS for detecting oncogenic alterations in NSCLC. Our findings suggest that PNA clamping is a more useful method for clinical practice.
Antineoplastic Agents/therapeutic use ; Carcinoma, Non-Small-Cell Lung/drug therapy/*genetics ; Constriction ; Humans ; Lung Neoplasms/*genetics ; Molecular Diagnostic Techniques ; Mutation ; Peptide Nucleic Acids/*genetics ; Protein Kinase Inhibitors/*therapeutic use ; Receptor Protein-Tyrosine Kinases/*genetics ; Receptor, Epidermal Growth Factor/*genetics ; Sensitivity and Specificity ; Sequence Analysis ; Sequence Analysis, DNA ; Translocation, Genetic

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.
Animals ; Axons ; physiology ; Brain ; anatomy & histology ; Brain Mapping ; Brain Stem ; cytology ; GABAergic Neurons ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Pathways ; physiology ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Transduction, Genetic ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

The NAC family is an important transcription factor which regulate plant growth and development， signal transduction， and stress response.In this study， the protein identification， subfamily classification， the determination of physical and chemical properties， protein structure， and expression pattern of NAC family were performed using bioinformatic methods based on the RNA-seq data of ginger. The results showed that a total of 72 NAC transcription factors were identified in 271.1 Mb total nucleotides， and they could be clustered into 13 subfamilies according to the phylogenetic tree.The physical and chemical properties， structure analysis revealed that the amino acid number and isoelectric point were different among 13 NAC subfamilies; the secondary structure of NACs transcription factors mainly consist of random coil， and the tertiary structure is similar.In addition，the expression patterns of genes under different soil moisture and Ralstonia solanacearum infection showed that 23 NACs were differentially expressed， which were mainly distributed in Ⅷ，Ⅶ， and ⅩⅤ subfamilies related to plant senescence， hormone metabolism and cell wall metabolism.The results provide some valuable information for the research and development of NAC transcription factors in ginger.
Gene Expression Regulation, Plant ; Ginger ; genetics ; Multigene Family ; Phylogeny ; Plant Proteins ; genetics ; Protein Structure, Tertiary ; RNA, Plant ; genetics ; Sequence Analysis, RNA ; Transcription Factors ; genetics

Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Mutation ; Sequence Analysis, DNA ; methods ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.
Animals ; Classification ; Clone Cells ; Enterotoxins ; Epitopes ; Genetic Variation ; Genotype ; Humans ; Protein Folding ; Protein Processing, Post-Translational ; Rotavirus ; Sequence Analysis ; Viral Nonstructural Proteins ; Virulence