Population genetic analyses inferred a limited genetic diversity across the pvama-1 DI domain among Plasmodium vivax isolates from Khyber Pakhtunkhwa regions of Pakistan.

BACKGROUND: Plasmodium vivax apical membrane antigen-1 (pvama-1) is an important vaccine candidate against Malaria. The genetic composition assessment of pvama-1 from wide-range geography is vital to plan the antigen based vaccine designing against Malaria. METHODS: The blood samples were collected from 84 P. vivax positive malaria patients from different districts of Khyber Pakhtunkhwa (KP) province of Pakistan. The highly polymorphic and immunogenic domain-I (DI) region of pvama-1 was PCR amplified and DNA sequenced. The QC based sequences raw data filtration was done using DNASTAR package. The downstream population genetic analyses were performed using MEGA4, DnaSP, Arlequin v3.5 and Network.5 resources. RESULTS: The analyses unveiled total 57 haplotypes of pvama-1 (DI) in KP samples with majorly prevalent H-14 and H-5 haplotypes. Pairwise comparative population genetics analyses identified limited to moderate genetic distinctions among the samples collected from different districts of KP, Pakistan. In context of worldwide available data, the KP samples depicted major genetic differentiation against the Korean samples with Fst = 0.40915 (P-value = 0.0001), while least distinction was observed against Indian and Iranian samples. The statistically significant negative values of Fu and Li's D* and F* tests indicate the evidence of population expansion and directional positive selection signature. The slow LD decay across the nucleotide distance in KP isolates indicates low nucleotide diversity. In context of reference pvama-1 sequence, the KP samples were identified to have 09 novel non-synonymous single nucleotide polymorphisms (nsSNPs), including several trimorphic and tetramorphic substitutions. Few of these nsSNPs are mapped within the B-cell predicted epitopic motifs of the pvama-1, and possibly modulate the immune response mechanism. CONCLUSION: Low genetic differentiation was observed across the pvama-1 DI among the P. vivax isolates acquired from widespread regions of KP province of Pakistan. The information may implicate in future vaccine designing strategies based on antigenic features of pvama-1.

Effects of sea salt intake on metabolites, steroid hormones, and gut microbiota in rats.

High salt intake is positively linked to many health problems, but the effect of mineral-rich sea salt (SS) has rarely been studied. To better understand the physiological effects of SS intake, the changes in general characteristics, metabolites, steroid hormones, and gut microbiota of SS-fed rats were investigated. Male rats were fed either a normal diet (ND, control) or ND containing 1% SS or 4% SS for 5 weeks. SS intake decreased fat, spleen, liver, and body weight, and increased blood urea nitrogen (BUN), water intake, and gut salt content. Accumulated gut salt content led to a decrease in beneficial bacteria, such as Lachnospiraceae and Lactobacillus, but an increase in potentially harmful bacteria, resulting in a change in lipid metabolites associated with gut health. Interestingly, most renal lysophosphatidylcholines (LPCs) associated with many renal functions were dramatically decreased and female hormones, such as estrogens, were significantly more altered than the male hormones by high SS intake. Although further investigation is needed, these data suggest that high SS intake could be positively linked to kidney dysfunction and gut health problems, and salt-related physiological changes may be sex-specific. Additionally, these data will be useful to better under-stand the physiological effects of SS intake.

Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood.

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27 kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84 h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27 kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Effects of Salt Treatment Time on the Metabolites, Microbial Composition, and Quality Characteristics of the Soy Sauce Moromi Extract.

Salt is one of the most important factors for fermented foods, but the effect of salt treatment time on the quality of fermented foods has rarely been studied. In this study, the effect of different salt treatment times (0, 48, and 96 h) after the start of fermentation on the quality of the soy sauce moromi extract (SSME) was investigated. As the salt treatment time was delayed, the population of Aspergillus oryzae, Lactobacillaceae, and Enterococcaecea in SSME increased, whereas the population of Staphylococcaceae and Bacillaceae decreased, leading to changes in the enzymatic activity and metabolite profiles. In particular, the contents of amino acids, peptides, volatile compounds, acidic compounds, sugars, and secondary metabolites were significantly affected by the salt treatment time, resulting in changes in the sensory quality and appearance of SSME. The correlation data showed that metabolites, bacterial population, and sensory parameters had strong positive or negative correlations with each other. Moreover, based on metabolomics analysis, the salt treatment-time-related SSME metabolomic pathway was proposed. Although further studies are needed to elucidate the salt treatment mechanism in fermented foods, our data can be useful to better understand the effect of salt treatment time on the quality of fermented foods.

Bile Ductal Transcriptome Identifies Key Pathways and Hub Genes in Clonorchis sinensis-Infected Sprague-Dawley Rats.

Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes cholangiocarcinoma. The underlying molecular pathogenesis occurring in the bile duct by the infection is little known. In this study, transcriptome profile in the bile ducts infected with C. sinensis were analyzed using microarray methods. Differentially expressed genes (DEGs) were 1,563 and 1,457 at 2 and 4 weeks after infection. Majority of the DEGs were temporally dysregulated at 2 weeks, but 519 DEGs showed monotonically changing expression patterns that formed seven distinct expression profiles. Protein-protein interaction (PPI) analysis of the DEG products revealed 5 sub-networks and 10 key hub proteins while weighted co-expression network analysis (WGCNA)-derived gene-gene interaction exhibited 16 co-expression modules and 13 key hub genes. The DEGs were significantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes pathways, which were related to original systems, cellular process, environmental information processing, and human diseases. This study uncovered a global picture of gene expression profiles in the bile ducts infected with C. sinensis, and provided a set of potent predictive biomarkers for early diagnosis of clonorchiasis.

Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr.

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40°C, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.