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

Based on whole genome data, the identification and expression pattern analysis of the Atropa belladonna WRKY transcription factor family were conducted to provide a theoretical foundation for studying the biological functions and mechanisms of these transcription factors. In this study, bioinformatics methods were employed to identify members of the A. belladonna WRKY gene family and to predict their physicochemical properties, conserved motifs, promoter cis-acting elements, and chromosomal localization. Additionally, the expression patterns of the A. belladonna WRKY gene family under the regulation of environmental factors such as light quality and temperature were analyzed. The results revealed a total of 28 AbWRKY transcription factors, randomly distributed across 16 chromosomes, encoding 324-707 amino acids. Most AbWRKY proteins were acidic, unstable, and hydrophilic. Based on multiple sequence alignment and phylogenetic analysis, the WRKY gene family members were classified into two subfamilies. Conserved motif and domain analysis indicated that WRKY transcription factors in the same subfamily possessed conserved structural features. Promoter analysis predicted that the A. belladonna WRKY family contained light-responsive elements, hormone-responsive elements, and stress-responsive elements. Collinearity analysis showed that AbWRKY24 plays a crucial role in the expansion of the AbWRKY gene family. Then qRT-PCR results indicated that AbWRKY6, AbWRKY8, AbWRKY14, and AbWRKY24 responded to red light stress, while AbWRKY8, AbWRKY14, and AbWRKY24 responded to yellow light/low-temperature combined stress. AbWRKY6 and AbWRKY8 were significantly expressed in leaves and stems, AbWRKY27 and AbWRKY28 were significantly expressed in fibrous roots, and AbWRKY25 was significantly expressed in flowers. This study is the first to identify and analyze the WRKY gene family in A. belladonna and to examine its expression patterns under light and temperature regulation, laying a foundation for in-depth analysis and functional validation of the molecular mechanisms of A. belladonna WRKY transcription factors in responding to light quality and temperature environmental factors.
Transcription Factors/chemistry* ; Plant Proteins/metabolism* ; Phylogeny ; Gene Expression Regulation, Plant ; Light ; Temperature ; Atropa belladonna/metabolism* ; Multigene Family/genetics* ; Promoter Regions, Genetic/genetics* ; Sequence Alignment ; Amino Acid Sequence ; Genome, Plant/genetics*

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment

The amino acid sequence of ancestral enzymes from extinct organisms can be deduced through in silico approach termed ancestral sequence reconstruction (ASR). ASR usually has six steps, which are the collection of nucleic acid/amino acid sequences of modern enzymes, multiple sequence alignment, phylogenetic tree construction, computational deduction of ancestral enzyme sequence, gene cloning, and characterization of enzyme properties. This method is widely used to study the adaptation and evolution mechanism of molecules to the changing environmental conditions on planetary time scale. As enzymes play key roles in biocatalysis, this method has become a powerful method for studying the relationship among the sequence, structure, and function of enzymes. Notably, most of the ancestral enzymes show better temperature stability and mutation stability, making them ideal protein scaffolds for further directed evolution. This article summarizes the computer algorithms, applications, and commonly used computer software of ASR, and discusses the potential application in directed evolution of enzymes.
Amino Acid Sequence ; Evolution, Molecular ; Phylogeny ; Proteins/genetics* ; Sequence Alignment

Dirigent(DIR) proteins are involved in the biosynthesis of lignin, lignans, and gossypol in plants and respond to biotic and abiotic stresses. Based on the full-length transcriptome of Schisandra chinensis, bioinformatics methods were used to preliminarily identify the DIR gene family and analyze the physico-chemical properties, subcellular localization, conserved motifs, phylogeny, and expression patterns of the proteins. The results showed that a total of 34 DIR genes were screened and the encoded proteins were 156-387 aa. The physico-chemical properties of the proteins were different and the secondary structure was mainly random coil. Half of the DIR proteins were located in chloroplast, while the others in extracellular region, endoplasmic reticulum, cytoplasm, etc. Phylogenetic analysis of DIR proteins from S. chinensis and the other 8 species such as Arabidopsis thaliana, Oryza sativa, and Glycine max demonstrated that all DIR proteins were clustered into 5 subfamilies and that DIR proteins from S. chinensis were in 4 subfamilies. DIR-a subfamily has the unique structure of 8 β-sheets, as verified by multiple sequence alignment. Finally, through the analysis of the transcriptome of S. chinensis fruit at different development stages, the expression pattern of DIR was clarified. Combined with the accumulation of lignans in fruits at different stages, DIR might be related to the synthesis of lignans in S. chinensis. This study lays a theoretical basis for exploring the biological functions of DIR genes and elucidating the biosynthesis pathway of lignans in S. chinensis.
Fruit/genetics* ; Lignans/analysis* ; Phylogeny ; Schisandra ; Sequence Alignment

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
Cinnamomum camphora/genetics* ; Cloning, Molecular ; Genes, Plant ; Phosphotransferases (Phosphate Group Acceptor)/genetics* ; Phylogeny ; Sequence Alignment

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.
DNA, Chloroplast/genetics* ; Genetic Markers ; Genetics, Population ; INDEL Mutation ; Panax notoginseng/genetics* ; Polymorphism, Single Nucleotide ; Sequence Alignment

In this study, we investigated the genetics, clinical features, and therapeutic approach of 14 patients with 5α-reductase deficiency in China. Genotyping analysis was performed by direct sequencing of PCR products of the steroid 5α-reductase type 2 gene (SRD5A2). The 5α-reductase activities of three novel mutations were investigated by mutagenesis and an in vitro transfection assay. Most patients presented with a microphallus, variable degrees of hypospadias, and cryptorchidism. Eight of 14 patients (57.1%) were initially reared as females and changed their social gender from female to male after puberty. Nine mutations were identified in the 14 patients. p.G203S, p.Q6X, and p.R227Q were the most prevalent mutations. Three mutations (p.K35N, p.H162P, and p.Y136X) have not been reported previously. The nonsense mutation p.Y136X abolished enzymatic activity, whereas p.K35N and p.H162P retained partial enzymatic activity. Topical administration of dihydrotestosterone during infancy or early childhood combined with hypospadia repair surgery had good therapeutic results. In conclusion, we expand the mutation profile of SRD5A2 in the Chinese population. A rational clinical approach to this disorder requires early and accurate diagnosis, especially genetic diagnosis.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Adolescent ; Adult ; Asian People/genetics* ; Child ; Child, Preschool ; China ; Disorder of Sex Development, 46,XY/genetics* ; Follicle Stimulating Hormone/blood* ; Genitalia, Male/abnormalities* ; Humans ; Hypospadias/genetics* ; Luteinizing Hormone/blood* ; Male ; Membrane Proteins/genetics* ; Mutation/genetics* ; Sequence Alignment ; Steroid Metabolism, Inborn Errors/genetics* ; Testosterone/blood* ; Young Adult

OBJECTIVE: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Computational Biology ; DNA Methylation ; DNA, Circular/genetics* ; DNA, Viral/genetics* ; Genome, Human ; Genomics ; Genotype ; Hepacivirus/genetics* ; Hepatitis C/virology* ; Humans ; Leukocytes, Mononuclear/virology* ; Polymerase Chain Reaction ; RNA, Viral/genetics* ; Sequence Alignment

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA, MeJA and Ag, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.
Amino Acid Sequence ; Benzofurans ; Biosynthetic Pathways ; genetics ; Cinnamates ; Depsides ; Gene Expression Regulation, Plant ; genetics ; Oxidoreductases ; genetics ; Phenylpropionates ; metabolism ; Phenylpyruvic Acids ; metabolism ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; chemistry ; enzymology ; genetics ; metabolism ; Plants, Genetically Modified ; Recombinant Proteins ; analysis ; biosynthesis ; Salvia miltiorrhiza ; chemistry ; enzymology ; genetics ; metabolism ; Sequence Alignment