BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

METHODS

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

RESULTS

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

CONCLUSION

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

Sars-cov-2 ; Polymerase Chain Reaction ; Dna Primers ; Oligonucleotide Primers ; Computer Simulation ; Conserved Sequence ; Coronavirus ; Covid-19 ; Disease ; Emergencies ; Evaluation Studies As Topic ; Genes ; Genome ; Global Health ; Health ; Identification (psychology) ; Infection ; Infections ; Membranes ; Methods ; Mutation ; Oligonucleotides ; Organizations ; Population ; Public Health ; Rna ; Rna, Viral ; Sars Virus ; Sequence Alignment ; Severe Acute Respiratory Syndrome ; Syndrome ; Viruses ; Whole Genome Sequencing ; World Health Organization

Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.
Plasmids/genetics* ; Proteins/genetics* ; Bacteria/genetics* ; Recombinases ; Genes, Bacterial ; Anti-Bacterial Agents

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.
Cordyceps/genetics* ; Genes, Mating Type, Fungal/genetics* ; Reproduction/genetics*

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.
Genes, vif ; Phylogeny ; Plant Leaves/genetics* ; Peptides, Cyclic ; Cloning, Molecular ; Caryophyllaceae/genetics*

Aims: The basal stem rot disease in oil palm is caused by the pathogenic Ganoderma boninense, which is infectious after mating and forming dikaryotic hyphae. This study was aimed to generate a mating-type biomarker for the detection of pathogenic Ganoderma species. Methodology and results: Mating-type region of Ganoderma was amplified using polymerase chain reaction (PCR) and primers flanking the mating-type region of other basidiomycetes. Amplified fragments were sequenced and were identified as the Ganoderma pheromone receptor gene of matB locus called the gprb2 gene. Using this biomarker, the pheromone receptor gene was detected in a total of 107 pathogenic Ganoderma spp. while the gene was not detected in the non-pathogenic Ganoderma lucidum. Phylogenetic tree analyses of the gene fragment encoding the partial amino acid sequence of gprb2 showed clades of close evolutionary relationship among the 107 pathogenic Ganoderma spp. Phylogenetic analyses using deduced amino acid sequences of the Ganoderma pheromone receptor b2 gene, gprb2 with homologous pheromone receptors of other basidiomycetous fungi revealed high conservation of this pheromone receptor within their respective taxonomy. Conclusion, significance and impact of study A potential mating-type biomarker was successfully identified that could detect pathogenic Ganoderma spp. The research findings will be helpful in oil palm screening to detect pathogenic Ganoderma spp. and gain further insight into the role of the mating-type loci of Ganoderma towards its pathogenesis in causing the basal stem rot disease of oil palm.
Genes, Mating Type, Fungal ; Ganoderma

Aged ; Animals ; Arcobacter/genetics* ; Chickens ; Diarrhea/microbiology* ; Drug Resistance, Bacterial/genetics* ; Genes, Bacterial ; Gram-Negative Bacterial Infections/veterinary* ; Humans ; Male ; Meat ; Microbial Sensitivity Tests ; Phylogeny ; Poultry Diseases/microbiology* ; Virulence ; Virulence Factors/genetics*

Objective: Our objective was to investigate the occurrence of opportunistic pathogens and characterize the bacterial community structures in the water system of a pulmonary hospital. Methods: The water samples were collected from automatic and manual faucets in the consulting room, treatment room, dressing room, respiratory ward, and other non-medical rooms in three buildings of the hospital. Quantitative polymerase chain reaction was used to quantify the load of several waterborne opportunistic pathogens and related microorganisms, including spp., spp., and . Illumina sequencing targeting 16S rRNA genes was performed to profile bacterial communities. Results: The occurrence rates of spp., spp., and were 100%, 100%, and 76%, respectively in all samples. Higher occurrence rates of were observed in the outpatient service building (building 1, 91.7%) and respiration department and wards (building 2, 80%) than in the office building (building 3), where no was found. were more abundant in automatic faucets (average 2.21 × 10 gene copies/L) than in manual faucets (average 1.03 × 10 gene copies/mL) ( < 0.01). , , , , , and were the dominant bacterial phyla. Disinfectant residuals, nitrate, and temperature were found to be the key environmental factors driving microbial community structure shifts in water systems. Conclusion This study revealed a high level of colonization of water faucets by opportunistic pathogens and provided insight into the characteristics of microbial communities in a hospital water system and approaches to reduce risks of microbial contamination.
China ; Drinking Water ; microbiology ; Genes, Bacterial ; Hospitals ; Legionella ; isolation & purification ; Microbiota ; Mycobacterium ; isolation & purification ; Mycobacterium avium ; isolation & purification ; RNA, Bacterial ; analysis ; RNA, Ribosomal, 16S ; analysis ; Water Quality ; Water Supply

PURPOSE: Dysbiosis of gut microbiota has been reported to participate in the pathogenesis of colorectal cancer, but changes in microbiota due to radiotherapy have not been studied. In this study, we tried to elucidate the changes in the microbiome in rectal cancer after chemoradiotherapy using RNA sequencing analysis.MATERIALS AND METHODS: We included 11 pairs of human rectal cancer tissues before and after irradiation between August 2016 and December 2017 and performed RNA sequencing analysis. Mapped reads to human reference genomes were used for pair-wise transcriptome comparisons, and unmapped (non-human) reads were then mapped to bacterial marker genes using PathSeq.RESULTS: At microbiome level, interindividual variability of mucosal microbiota was greater than the change in microbial composition during radiotherapy. This indicates that rapid homeostatic recovery of the mucosal microbial composition takes place short after radiotherapy. At single microbe level, Prevotella and Fusobacterium, which were identified as important causative microbes of the initiation and progression of rectal cancer were decreased by radiotherapy. Moreover, changes in Prevotella were associated with changes in the human transcriptome of rectal cancer. We also found that there was a gene cluster that increased and decreased in association with changes in microbial composition by chemoradiation.CONCLUSION: This study revealed changes in tumor-associated microbial community by irradiation in rectal cancer. These findings can be used to develop a new treatment strategy of neoadjuvant therapy for locally advanced rectal cancer by overcoming radio-resistance or facilitating radio-sensitivity.
Chemoradiotherapy ; Colorectal Neoplasms ; Dysbiosis ; Fusobacterium ; Gastrointestinal Microbiome ; Genes, vif ; Genome ; Humans ; Microbiota ; Neoadjuvant Therapy ; Prevotella ; Radiotherapy ; Rectal Neoplasms ; Sequence Analysis, RNA ; Transcriptome

Genetically engineered mouse models are used in high-throughput phenotyping screens to understand genotype-phenotype associations and their relevance to human diseases. However, not all mutant mouse lines with detectable phenotypes are associated with human diseases. Here, we propose the “Target gene selection system for Genetically engineered mouse models” (TarGo). Using a combination of human disease descriptions, network topology, and genotype-phenotype correlations, novel genes that are potentially related to human diseases are suggested. We constructed a gene interaction network using protein-protein interactions, molecular pathways, and co-expression data. Several repositories for human disease signatures were used to obtain information on human disease-related genes. We calculated disease- or phenotype-specific gene ranks using network topology and disease signatures. In conclusion, TarGo provides many novel features for gene function prediction.
Animals ; Computational Biology ; Genes, vif ; Genetic Association Studies ; Humans ; Mice ; Phenotype ; Systems Biology

Serine protease inhibitor Kazal-type 1 (SPINK1) is a gene expressed from pancreatic acinar cell which its mutation is known to be associated with chronic pancreatitis (CP) and pancreatic cancer. We report a case of a 47-years-old female with nausea and weight loss with yellow discoloration of skin. Initial imaging and endoscopic study led us to an impression of chronic pancreatitis with pancreatic cancer with common bile-duct dilation. Biopsy result was confirmed with pancreatic adenocarcinoma and additional imaging revealed lymph node and bone metastasis. Our genetic analysis revealed 194+2T>C mutation of SPINK1. Biliary obstruction was successfully decompressed by stent insertion and underwent chemotherapy and radiotherapy. Although there is accumulating evidence of association between SPINK1 mutation and CP, the relationship between SPINK1 mutation and pancreatic cancer in CP patient is an emerging concept. Genetic analysis should be considered in patients with young age especially when diagnosed with both CP and pancreatic cancer.
Acinar Cells ; Adenocarcinoma ; Biopsy ; Drug Therapy ; Female ; Genes, vif ; Humans ; Jaundice, Obstructive ; Lymph Nodes ; Nausea ; Neoplasm Metastasis ; Pancreatic Neoplasms ; Pancreatitis, Chronic ; Radiotherapy ; Serine Proteases ; Skin ; Stents ; Weight Loss