Computational design of experimentally validated multi-epitopes vaccine against hepatitis E virus: An immunological approach.

Hepatitis E virus (HEV) is one of the leading acute liver infections triggered by viral hepatitis. Patients infected with HEV usually recover and the annual death rate is negligible. Currently, there is no HEV licensed vaccine available globally. This study was carried out to design a multi-epitope HEV peptide-based vaccine by retrieving already experimentally validated epitopes from ViPR database leading to epitope prioritization. Epitopes selected as potential vaccine candidates were non-allergen, immunogenic, soluble, non-toxic and IFN gamma positive. The epitopes were linked together by AAY linkers and the linker EAAAK was used to join adjuvant with epitopes. Toll-like receptor (TLR)-4 agonist was used as an adjuvant to boost efficacy of the vaccine. Furthermore, codon optimization followed by disulfide engineering was performed to analyse the designed vaccine's structural stability. Computational modeling of the immune simulation was done to examine the immune response against the vaccine. The designed vaccine construct was docked with TLR-3 receptor for their interactions and then subjected to molecular dynamic simulations. The vaccine model was examined computationally towards the capability of inducing immune responses which showed the induction of both humoral and cell mediated immunity. Taken together, our study suggests an In-silico designed HEV based multi-epitope peptide-based vaccine (MEPV) that needs to be examined in the wet lab-based data that can help to develop a potential vaccine against HEV.

Biochemical Recurrence in Prostate Cancer Is Associated with the Composition of Lactobacillus: Microbiome Analysis of Prostatic Tissue.

Many human pathologies, such as malignancy, are linked with specific bacteria and changes in the constituents of the microbiome. In order to examine the association between an imbalance of bacteria and prostate carcinoma, a comparison of the microbiomes present in patients with biochemical recurrence (BCR) or NO BCR (NBCR) was performed. Additionally, 16S rRNA-based next-generation sequencing was applied to identify the bacterial profiles within these tumors in terms of the bacteria and operational genes present. The percentage average taxonomic composition between the taxa indicated no difference between BCR and NBCR. In addition, alpha and beta diversity indices presented no distinction between the cohorts in any statistical method. However, taxonomic biomarker discovery indicated a relatively higher population of Lactobacillus in the NBCR group, and this finding was supported by PCR data. Along with that, differences in the operational activity of the bacterial genes were also determined. It is proposed that the biochemical recurrence was linked to the quantity of Lactobacillus present. The aim of this study was to investigate the microbiome involved in prostate carcinoma and the potential association between them.

Oxidative Stress and Dopaminergic Metabolism: A Major PD Pathogenic Mechanism and Basis of Potential Antioxidant Therapies

Reactive oxygen species (ROS)-induced oxidative stress triggers the vicious cycle leading to the degeneration of dopaminergic neurons in the nigra pars compacta. ROS produced during the metabolism of dopamine is immediately neutralized by the endogenous antioxidant defense system (EADS) under physiological conditions. Aging decreases the vigilance of EADS and makes the dopaminergic neurons more vulnerable to oxidative stress. As a result, ROS left over by EADS oxidize the dopamine-derived catechols and produces a number of reactive dopamine quinones, which are precursors to endogenous neurotoxins. In addition, ROS causes lipid peroxidation, uncoupling of the electron transport chain, and DNA damage, which lead to mitochondrial dysfunction, lysosomal dysfunction, and synaptic dysfunction. The mutations in genes such as DNAJC6, SYNJ1, SH3GL2, LRRK2, PRKN, and VPS35 caused by ROS have been associated with synaptic dysfunction and the pathogenesis of Parkinson's disease (PD). The available drugs that are used against PD can only delay the progression of the disease, but they produce various side effects. Through their antioxidant activity, flavonoids can substantiate the EADS of dopaminergic neurons and disrupt the vicious cycle incepted by oxidative stress. In this review, we show how the oxidative metabolism of dopamine generates ROS and dopamine-quinones, which then exert unrestrained OS, causing mutations in several genes involved in the proper functioning of mitochondrion, synapse, and lysosome. Besides, we also present some examples of approved drugs used for the treatment of PD, therapies in the clinical trial phase, and an update on the flavonoids that have been tested to boost the EADS of dopaminergic neurons.

The Effects of Iron Deficiency on the Gut Microbiota in Women of Childbearing Age.

Iron deficiency anemia (IDA) is the most prevalent and common nutritional deficiency worldwide and is a global health problem with significant risk, particularly among women of reproductive age. Oral iron supplementation is the most widely used and cost-effective treatment for iron deficiency and IDA. However, there are limitations regarding side effects such as enteritis, treatment compliance, and bioavailability. Intestinal microbiome characteristic research has been recently conducted to overcome these issues, but more is needed. Against this background, a metagenomics study on the 16S gene in the feces of young women vulnerable to IDA was conducted. As a result of analyzing 16 normal subjects and 15 IDA patients, significant differences in bacterial community distribution were identified. In particular, a significant decrease in Faecalibacterium was characteristic in IDA patients compared with normal subjects. Furthermore, in the case of patients who recovered from IDA following iron supplementation treatment, it was confirmed that Faecalibacterium significantly recovered to normal levels. However, no significance in beta diversity was seen compared with before treatment. There were also no differences in the beta diversity results between the recovered and normal subjects. Therefore, intestinal dysbiosis during the disease state was considered to be restored as IDA improved. Although the results were derived from a limited number of subjects and additional research is needed, the results of this study are expected to be the basis for developing treatment and prevention strategies based on host-microbiome crosstalk in IDA.

Malignant Prostate Tissue Is Associated with Different Microbiome Gene Functions.

Specific microorganisms and changes in the constituents of the microbiome are linked with pathologies in humans, such as malignancy. Within the prostate, certain bacterial communities may locate advantageous conditions and establish themselves, thus outperforming alternative species. In this study, a comparison of malignant (MT) and benign prostate tissues (BT) or benign prostate hyperplasia (BPH) was performed in order to delineate the respective microbiomes in each sample type and to determine their pertinence to prostatic tumourigenesis. Specimens of MT (n = 26) and PT (n = 13)/BPH (n = 10) were acquired from patients. No variations in the make-up of the microbiome were seen when MT and PT specimens were compared. Changes in the bacterial constituents and functional genes were seen in the specimens obtained from patients with MT when contrasted against samples from those with BPH. Pelomonas was the genus with the highest abundance in MT specimens. It is proposed that dissimilar microbiome gene functions are present in the contexts of MT and PT samples.

Characterization of Fecal Microbiomes of Osteoporotic Patients in Korea.

An imbalanced gut microbiome has been linked to a higher risk of many bone-related diseases. The objective of this study was to discover biomarkers of osteoporosis (OP). So, we collected 76 stool samples (60 human controls and 16 OP patients), extracted DNA, and performed 16S ribosomal ribonucleic acid (rRNA) gene-based amplicon sequencing. Among the taxa with an average taxonomic composition greater than 1%, only the Lachnospira genus showed a significant difference between the two groups. The Linear Discriminant Effect Size analysis and qPCR experiments indicated the Lachnospira genus as a potential biomarker of OP. Moreover, a total of 11 metabolic pathways varied between the two groups. Our study concludes that the genus Lachnospira is potentially crucial for diagnosing and treating osteoporosis. The findings of this study might help researchers better understand OP from a microbiome perspective. This research might develop more effective diagnostic and treatment methods for OP in the future.

Characteristic alterations of gut microbiota in uncontrolled gout.

Microbiome research has been on the rise recently for a more in-depth understanding of gout. Meanwhile, there is a need to understand the gut microbiome related to uric acid-lowering drug resistance. In this study, 16S rRNA gene-based microbiota analysis was performed for a total of 65 stool samples from 17 healthy controls and 48 febuxostat-treated gout patients (including 28 controlled subjects with decreased uric acid levels and 20 uncontrolled subjects with non-reduced uric acid levels). Alpha diversity of bacterial community decreased in the healthy control, controlled, and uncontrolled groups. In the case of beta diversity, the bacterial community was significantly different among groups (healthy control, controlled, and uncontrolled groups). Taxonomic biomarker analysis revealed the increased population of g-Bifidobacterium in healthy controls and g-Prevotella in uncontrolled patients. PCR further confirmed this result at the species level. Additionally, functional metagenomics predictions led to the exploration of various functional biomarkers, including purine metabolism. The results of this study can serve as a basis for developing potential new strategies for diagnosing and treating gout from microbiome prospects.

Characteristics and Microbiome Profiling of Korean Gochang Bokbunja Vinegar by the Fermentation Process.

As NGS (next-generation sequencing) technology develops, metagenomics-based microbial ecology, that is, microbiome research, has recently led to the science of fermented food. Based on the above technology, a study was conducted to understand the characteristics of vinegar made from bokbunja, a local crop in Gochang-gun, Korea. Physicochemical characteristics of vinegar, organic acid analysis, microbial community analysis, and electronic tongue analysis were explored while fermenting the vinegar for 70 days under eight fermentation conditions according to the concentration of bokbunja liquid (100% or 50%), type of fermenter (porcelain jar or stainless container), and fermentation environment (natural outdoor conditions or temperature/oxygen controlled). As a result, distinct microbial community patterns were found in the stage of acetic acid fermentation and, accordingly, this fermentation of Gochang vinegar is classified into three categories. Vinegar prepared by the traditional method of outdoor fermentation using jars showed characteristics of "Acetobacter (42.1%)/Lactobacillus (56.9%) fusion fermentation". Under conditions where oxygen and temperature were controlled indoors using jars, characteristics of "Komagataeibacter (90.2%) fermentation" were found. "Lactobacillus (92.2%) fermentation" characteristics were discovered under natural outdoor conditions using stainless steel containers. These fermentation pattern differences were related to taxonomic phylogenetic diversity, which was also considered involved in determining organic acid production and taste. These results will be helpful as a scientific basis for understanding the fermentation characteristics of Gochang vinegar and developing high-value-added traditional vinegar products.

Human Organic Anion Transporting Polypeptide 1B3 Applied as an MRI-Based Reporter Gene.

OBJECTIVE: Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. MATERIALS AND METHODS: CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 µL/g). RESULTS: Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. CONCLUSION: The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

Isolation of MLL1 Inhibitory RNA Aptamers.

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers-APT1 and APT2-represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5΄-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3΄. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

Anti-adipogenic effects of KD025 (SLx-2119), a ROCK2-specific inhibitor, in 3T3-L1 cells.

Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK's activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.