Molecular Biological Investigations Research Articles

Identification and Molecular Analysis of Proteins Associated with Non-spinning Disease in the Silkworm Bombyx mori L Induced by BmNPV and BmDNV

Aims: The present study combines morphological and molecular biology investigations to uncover the underlying causes of the non-spinning syndrome in silkworms. To understand the molecular mechanism and immune mechanism after infection of viral pathogen in silkworm Bombyx mori L. Study Design: Infected silkworm samples were collected from the field and isolated the genomic DNA and total proteins. Analyzed the samples by using genomic tools and proteomic tools. Place and Duration of Study: Central Sericultural Research and Training Institute, Mysuru, Karnataka. 01.03.2023 to 31.07.2023 total 04 months. Methodology: Our findings indicate that infections of BmDNV and BmNPV are associated with abnormal growth of the silk gland, leading to the non-spinning phenotype. To confirm pathogen infection in non-spinning silkworms, we collected genomic DNA and conducted PCR analysis using specific primers for DNV and NPV. Results: Our results showed amplification of both viral infections in the isolated samples. SDS-PAGE analysis revealed differential regulation of several proteins involved in physiological and immunological processes in the hemolymph of non-spinning silkworms. MALDI-TOF/MS was used to identify an up-regulated protein of interest, which was found to be a Lepidopteran low molecular weight lipoprotein. This protein is known to play an important role in insect immune mechanisms. Conclusion: Major disease-causing agents such as BmDNV and BmNPV infections and investigate their impact on protein expression patterns in the hemolymph, shedding light on the factors contributing to non-spinning behavior in silkworms.

Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors.

Liquidambar formosana Hance is renowned for its rich leaf color and possesses notable advantages, such as robust adaptability, strong resistance to diseases and pests, and rapid growth, making it a preferred choice for urban greening and carbon sequestration forest initiatives. The completion of whole-genome sequencing of L. formosana has spurred an increased interest in exploring the molecular mechanisms underlying seasonal changes in leaf color, marking a significant focus in L. formosana breeding research. However, there is currently a lack of stable reference genes suitable for analyzing the expression patterns of functional genes in L. formosana exhibiting varying leaf colors. This study selected five L. formosana varieties with significant differences in leaf colors. Through the RT-qPCR analysis, and evaluation using BestKeeper, geNorm, NormFinder, Delta Ct, and RefFinder, the expression stability of 14 candidate reference genes was examined. Consequently, two reference genes (LifEF1-α and LifACT) with stable expression, suitable for RT-qPCR of L. formosana with diverse leaf colors, were identified. The stability of these selected reference genes was further validated by examining the LifbHLH137 gene, which promoted the biosynthesis of anthocyanins. This advancement facilitated molecular biology and genetic breeding investigations of L. formosana, providing essential data for the precise quantification of functional genes associated with leaf color variation.

Pyridazinone-based derivatives as anticancer agents endowed with anti-microbial activity: molecular design, synthesis, and biological investigation.

Cancer patients undergoing chemotherapy are highly susceptible to infections owing to their compromised immune system, which also promotes cancer progression through inflammation. Thus, this study aimed to develop novel chemotherapeutic agents with both anticancer and antimicrobial properties. A series of diarylurea derivatives based on pyridazinone scaffolds were designed, synthesized, and characterized as surrogates for sorafenib. The synthesized compounds were tested for their antimicrobial activity and screened against 60 cancer cell lines at the National Cancer Institute (NCI). Compound 10h exhibited potent antibacterial activity against Staphylococcus aureus (MIC = 16 μg mL-1), whereas compound 8g showed significant antifungal activity against Candida albicans (MIC = 16 μg mL-1). Additionally, ten compounds were further evaluated for VEGFR-2 inhibition, with compound 17a showing the best inhibitory activity. Compounds 8f, 10l, and 17a demonstrated significant anticancer activity against melanoma, NSCLC, prostate cancer, and colon cancer, with growth inhibition percentages (GI%) ranging from 62.21% to 100.14%. Compounds 10l and 17a were selected for five-dose screening, displaying GI50 values of 1.66-100 μM. Compound 10l induced G0-G1 phase cell cycle arrest in the A549/ATCC cell line, increasing the cell population from 85.41% to 90.86%. Gene expression analysis showed that compound 10l upregulated pro-apoptotic genes p53 and Bax and downregulated the anti-apoptotic gene Bcl-2. Molecular docking studies provided insights into the binding modes of the compounds to the VEGFR-2 enzyme. In conclusion, the pyridazinone-based diarylurea derivatives developed in this study show promise as dual-function antimicrobial and anticancer agents, warranting further investigation.

SYVN1 modulates papillary thyroid carcinoma progression by destabilizing HMGB1

E3 ubiquitin ligase synoviolin (SYVN1) has been reported to participate in many human cancers. This study aimed to investigate SYVN1’s roles and molecular pathways in papillary thyroid cancer (PTC). The functions of SYVN1 in PTC were further analyzed using gain- and loss-of-function methods and numerous investigations in cellular function and molecular biology. The findings demonstrated that the overexpression of SYVN1 markedly suppressed the proliferation, migration, and invasion of PTC cell lines (NPA87 and TPC-1). We found that SYVN1 interacted with HMGB1 and promoted its ubiquitination and degradation. In addition, SYVN1 effectively impairs cell proliferation, migration, invasion, and the formation of tumor xenografts in mice models. However, this effect may be partly reversed by overexpressing HMGB1. Thus, SYVN1 may inhibit the proliferation, migration, and invasion of PTC cells by disrupting HMGB1. Consequently, SYVN1 might be considered a promising therapeutic target for PTC.

Molecular modelling, cytotoxicity & biological investigation of novel fluorinated diphenylamine chalcone derivatives

A unique series of 1-(3-(3,4-difluorophenyl)amino)phenyl)ethan-1-one linked fluorinated diphenylamine chalcones (A1-A7) were synthesized and characterized by NMR, IR, and MS. The compounds were studied in vitro for antibacterial and antifungal activities. In vitro studies show that the compound had significant antibacterial (MICs 25–250 µg/ml) and antifungal (MICs 250–1000 µg/ml) properties. Chemicals A1, A2, and A4 significantly inhibited all bacterial strains, whereas compounds A1, A4, and A7 inhibited fungal very marginally. Compounds A1 and A2 had a high anti-Plasmodium falciparum activity. A molecular docking investigation discovered that compounds A1 and A2 significantly bind to the active site of falcilysin. Compound A1 maintains its stability through numerous van der Waals interactions with falcilysin protein residues. Compound A2, on the other hand, shows a hydrogen bond with Asn421, the secondary amine linked to the phenyl ring. The Molecular Dynamic simulation at 100 ns to investigate the dynamic stability of protein-ligand complexes. Compounds A1 and A2 exhibited the highest binding affinity and interaction. According to the MD simulation results, the proposed drug maintains the molecular interaction and structural integrity with P. falciparum. In HeLa cells, the cytotoxicity of compounds A1 and A4 was examined. A1 and A4 had computed LC50 values of 19.14 µg and 21.01 µg, respectively. Both compounds are cytotoxic to HeLa cells, according to the observations above. Compounds A1 and A2 are potential lead compounds for novel drug-like molecules having antibacterial, antifungal, and antimalarial properties.

A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Characteristics of dermal vascularity in melasma and solar lentigo.

Melasma and solar lentigo (SL) are major benign hyperpigmented lesions, and both have been shown to involve the dermal vasculature. This review discusses current knowledge regarding the clinical characteristics of dermal vascularity in melasma and SL, as well as the results of relevant molecular biological investigations. PubMed and Google Scholar were searched in December 2023 to identify articles related to melasma, SL, and the dermal vasculature in these lesions. Vascular morphologies in melasma and SL have been detected by histological and non-invasive methods, including modalities such as optical coherence tomography. Biological studies have indicated that factors secreted from vascular endothelial cells, such as stem cell factor and endothelin-1, can promote melanogenesis. With respect to phototherapy, blood vessel-targeting laser treatments are expected to provide long-term suppression of pigmentation, but this regimen is only effective when dilated capillaries are visible. In both melasma and SL, clinical and experimental investigations are revealing the contributions of dermal vascularity to hyperpigmentation. More effective treatment may require identification of hyperpigmentation subtypes. In the future, knowledge of treatment (including phototherapy) is expected to accumulate through reliable and validated non-invasive measurements.

Identification and expression analysis of ATP-binding cassette (ABC) transporters revealed its role in regulating stress response in pear (Pyrus bretchneideri)

BackgroundATP-binding cassette (ABC) transporter proteins constitute a plant gene superfamily crucial for growth, development, and responses to environmental stresses. Despite their identification in various plants like maize, rice, and Arabidopsis, little is known about the information on ABC transporters in pear. To investigate the functions of ABC transporters in pear development and abiotic stress response, we conducted an extensive analysis of ABC gene family in the pear genome.ResultsIn this study, 177 ABC transporter genes were successfully identified in the pear genome, classified into seven subfamilies: 8 ABCAs, 40 ABCBs, 24 ABCCs, 8 ABCDs, 9 ABCEs, 8 ABCFs, and 80 ABCGs. Ten motifs were common among all ABC transporter proteins, while distinct motif structures were observed for each subfamily. Distribution analysis revealed 85 PbrABC transporter genes across 17 chromosomes, driven primarily by WGD and dispersed duplication. Cis-regulatory element analysis of PbrABC promoters indicated associations with phytohormones and stress responses. Tissue-specific expression profiles demonstrated varied expression levels across tissues, suggesting diverse functions in development. Furthermore, several PbrABC genes responded to abiotic stresses, with 82 genes sensitive to salt stress, including 40 upregulated and 23 downregulated genes. Additionally, 91 genes were responsive to drought stress, with 22 upregulated and 36 downregulated genes. These findings highlight the pivotal role of PbrABC genes in abiotic stress responses.ConclusionThis study provides evolutionary insights into PbrABC transporter genes, establishing a foundation for future research on their functions in pear. The identified motifs, distribution patterns, and stress-responsive expressions contribute to understanding the regulatory mechanisms of ABC transporters in pear. The observed tissue-specific expression profiles suggest diverse roles in developmental processes. Notably, the significant responses to salt and drought stress emphasize the importance of PbrABC genes in mediating adaptive responses. Overall, our study advances the understanding of PbrABC transporter genes in pear, opening avenues for further investigations in plant molecular biology and stress physiology.

Dermorphin [D-Arg2, Lys4] (1-4) Amide Alleviates Frostbite-Induced Pain by Regulating TRP Channel-Mediated Microglial Activation and Neuroinflammation.

Cold injury or frostbite is a common medical condition that causes serious clinical complications including sensory abnormalities and chronic pain ultimately affecting overall well-being. Opioids are the first-choice drug for the treatment of frostbite-induced chronic pain; however, their notable side effects, including sedation, motor incoordination, respiratory depression, and drug addiction, present substantial obstacle to their clinical utility. To address this challenge, we have exploited peripheral mu-opioid receptors as potential target for the treatment of frostbite-induced chronic pain. In this study, we investigated the effect of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripheral mu-opioid receptor agonist, on frostbite injury and hypersensitivity induced by deep freeze magnet exposure in rats. Animals with frostbite injury displayed significant hypersensitivity to mechanical, thermal, and cold stimuli which was significant ameliorated on treatment with different doses of DALDA (1, 3, and 10 mg/kg) and ibuprofen (100 mg/kg). Further, molecular biology investigations unveiled heightened oxido-nitrosative stress, coupled with a notable upregulation in the expression of TRP channels (TRPA1, TRPV1, and TRPM8), glial cell activation, and neuroinflammation (TNF-α, IL-1β) in the sciatic nerve, dorsal root ganglion (DRG), and spinal cord of frostbite-injured rats. Treatment with DALDA leads to substantial reduction in TRP channels, microglial activation, and suppression of the inflammatory cascade in the ipsilateral L4-L5 DRG and spinal cord of rats. Overall, findings from the present study suggest that activation of peripheral mu-opioid receptors mitigates chronic pain in rats by modulating the expression of TRP channels and suppressing glial cell activation and neuroinflammation.

Glycine max acyl-acyl carrier protein thioesterase B gene overexpression alters lipid content and fatty acid profile of Arabidopsis seeds.

The fatty acyl-acyl carrier protein thioesterase B (FATB ) gene, involved in the synthesis of saturated fatty acids, plays an important role in the content of fatty acid and composition of seed storage lipids. However, the role of FATB in soybeans (Glycine max ) has been poorly characterised. This paper presents a preliminary bioinformatics and molecular biological investigation of 10 hypothetical FATB members. The results revealed that GmFATB1B , GmFATB2A and GmFATB2B contain many response elements involved in defense and stress responses and meristem tissue expression. Moreover, the coding sequences of GmFATB1A and GmFATB1B were significantly longer than those of the other genes. Their expression varied in different organs of soybean plants during growth, with GmFATB2A and GmFATB2B showing higher relative expression. In addition, subcellular localisation analysis revealed that they were mainly present in chloroplasts. Overexpression of GmFATB1A , GmFATB1B , GmFATB2A and GmFATB2B in transgenic Arabidopsis thaliana plants increased the seed oil content by 10.3%, 12.5%, 7.5% and 8.4%, respectively, compared to that in the wild-type and led to significant increases in palmitic and stearic acid content. Thus, this research has increased our understanding of the FATB family in soybeans and provides a theoretical basis for subsequent improvements in soybean quality.

Innovative microwave-assisted biosynthesis of copper oxide nanoparticles loaded with platinum(ii) based complex for halting colon cancer: cellular, molecular, and computational investigations.

In the current study, we biosynthesized copper oxide NPs (CuO NPs) utilizing the essential oils extracted from Boswellia carterii oleogum resin, which served as a bioreductant and capping agent with the help of microwave energy. Afterwards, the platinum(ii) based anticancer drug, carboplatin (Cr), was loaded onto the CuO NPs, exploiting the electrostatic interactions forming Cr@CuO NPs. The produced biogenic NPs were then characterized using zeta potential (ZP), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction spectroscopy (XRD), and Fourier transform infrared spectroscopy (FTIR) techniques. In addition, the entrapment efficiency and release profile of the loaded Cr were evaluated. Thereafter, SRB assay was performed, where Cr@CuO NPs demonstrated the highest cytotoxic activity against human colon cancer cells (HCT-116) with an IC50 of 5.17 μg mL-1, which was about 1.6 and 2.2 folds more than that of Cr and CuO NPs. Moreover, the greenly synthesized nanoparticles (Cr@CuO NPs) displayed a satisfactory selectivity index (SI = 6.82), which was far better than the free Cr treatment (SI = 2.23). Regarding the apoptosis assay, the advent of Cr@CuO NPs resulted in an immense increase in the cellular population percentage of HCT-116 cells undergoing both early (16.02%) and late apoptosis (35.66%), significantly surpassing free Cr and CuO NPs. A study of HCT-116 cell cycle kinetics revealed the powerful ability of Cr@CuO NPs to trap cells in the Sub-G1 and G2 phases and impede the G2/M transition. RT-qPCR was utilized for molecular investigations of the pro-apoptotic (Bax and p53) and antiapoptotic genes (Bcl-2). The novel Cr@CuO NPs treatment rose above single Cr or CuO NPs therapy in stimulating the p53-Bax mediated mitochondrial apoptosis. The cellular and molecular biology investigations presented substantial proof of the potentiated anticancer activity of Cr@CuO NPs and the extra benefits that could be obtained from their use.

Dual-Layer Spectral CT as Innovative Imaging Guidance in Lung Biopsies: Could Color-Coded Z-Effective Images Allow More Diagnostic Samplings and Biomarkers Information?

The aim of the study was to try to obtain more information on diagnostic samplings and biomarkers using dual-layer spectral CT in lung biopsies. Lung biopsies were performed by merging images obtained with CBCT with those from spectral CT to use them as functional guidance, experimenting with double sampling to determine the difference between the area with a higher Z-effective number and that with a lower Z-effective number. Ten patients with large lung lesions on spectral CT were selected and underwent percutaneous transthoracic lung mass biopsy. Technical success was calculated. The percentage of neoplastic, inflammatory, fibrotic, necrotic cells, or non-neoplastic lung parenchyma was reported. The possibility of carrying out immunohistochemical or molecular biology investigations was analyzed. All lesions were results malignant in 10/10 samples in the Zmax areas; in the Zmin areas, malignant cells were found in 7/10 samples. Technical success was achieved in 100% of cases for Zmax sampling and in 70% for Zmin sampling (p-value: 0.2105). The biomolecular profile was detected in 9/10 (90%) cases in Zmax areas, while in 4/10 (40%) cases in Zmin areas (p-value: 0.0573). The advantage of Z-effective imaging would be to identify a region of the lesion that is highly vascularized and probably richer in neoplastic cells, thus decreasing the risk of obtaining a non-diagnostic biopsy sample.

In situ photo-crosslinked hydrogel promotes oral mucosal wound healing through sustained delivery of ginsenoside Rg1.

Oral mucosal wounds exhibit an increased susceptibility to inflammation as a consequence of their direct exposure to a diverse range of microorganisms. This causes pain, slow healing, and other complications that interfere with patients' daily activities like eating and speaking. Consequently, patients experience a significant decline in their overall quality of life. Therefore, the pursuit of novel treatment approaches is of great importance. In this study, ginsenoside Rg1, a natural active substance extracted from ginseng root, was chosen as a therapeutic agent. It was encapsulated in a screened photo-crosslinked hydrogel scaffold for the treatment of mucosal defects in the rat palate. The results demonstrated that Rg1-hydrogel possessed excellent physical and chemical properties, and that oral mucosa wounds treated with Rg1-hydrogel exhibited the greatest healing performance, as evidenced by more pronounced wound re-epithelialization, increased collagen deposition, and decreased inflammatory infiltration. Subsequent investigations in molecular biology confirmed that Rg1-hydrogel stimulated the secretion of repair-related factors and inhibited the secretion of inflammatory factors. This study demonstrated that the hydrogel containing ginsenoside Rg1 significantly promotes oral mucosal tissue healing in vivo. Based on the findings, it can be inferred that the Rg1-hydrogel has promising prospects for the therapeutic management of oral mucosal wounds.

Sida cordifolia L. attenuates behavioral hypersensitivity by interfering with KIF17-NR2B signaling in rat model of neuropathic pain

Ethnopharmacological relevanceSida cordifolia L., a perennial subshrub belonging to the Malvaceae family, holds noteworthy significance in the Indian Ayurvedic System and global texts. Roots of this plant are reported to be useful in neurodegenerative disorders, facial paralysis, and treating several neuropathic pain conditions such as neuralgia, and sciatica. However, despite these claims, there remains a dearth of experimental evidence showcasing the effectiveness of Sida cordifolia L. roots in mitigating neuropathic pain. Aim of the studyThe primary objective of this study was to assess the analgesic properties of the whole extract (SCE) obtained from the roots of Sida cordifolia L., as well as its aqueous fraction (SAF) in rat model of chronic constriction injury (CCI)-induced neuropathic pain. Furthermore, in-depth phytochemical and molecular biology studies were conducted to identify the potential phytoconstituents and unveil the underlying mechanisms of action. Material and methodsDCM: Methanol (1:1) was used to extract the roots of Sida cordifolia L. to get whole extract (SCE) and was subjected to phytochemical investigations including LC-MS analysis. Analgesic potential of SCE was evaluated in chronic constriction injury (CCI) model of neuropathic pain in rats followed by its bioactivity guided fractionation using in-vitro anti-inflammatory assay and assessment of most potent fraction (SAF) in in-vivo pain model. We have also performed the detailed phytochemical and molecular biology investigations to delineate the mechanism of action of Sida cordifolia root extract. ResultsChronic constriction injury leads to significant decrease in paw withdrawal threshold and paw withdrawal latency indicating development of hypersensitivity in rodents. Treatment with SCE and its most potent aqueous fraction (SAF) leads to significant and dose-dependent reduction in pain-like behavior of nerve injured rats. Pro-inflammatory cytokines (TNF-α, IL-1β), glia cell markers (Iba1, ICAM1), neuropeptides (CGRP and Substance P), KIF-17 and NR2B expressions were found to be significantly upregulated in DRG and spinal cord of nerve injured rats. Treatment with SCE and SAF suppressed oxido-inflammatory cascade along with attenuation of KIF-17 mediated NR2B trafficking and neuroinflammation in DRG and spinal tissues of neuropathic rats. HPTLC and HR-MS analysis suggest betaine as major constituent in SAF which along with other phytoconstituents. ConclusionsBoth the whole extract (SCE) and the aqueous fraction (SAF) demonstrate a significant reduction in mechanical and thermal hypersensitivity by inhibiting KIF-17 mediated NR2B signaling in nerve injured rats and may be used as a potential alternative for the treatment of chronic pain. Our findings support the use of roots of Sida cordifolia L. in neuropathic pain conditions as acclaimed by its traditional use.

Hepatitis A virus subgenotypes in Latvia, 2008–2021

BackgroundIn Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017–2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. MethodsTo determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. ResultsFrom the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. ConclusionsAll of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

Engineered Human Tissue as A New Platform for Mosquito Bite-Site Biology Investigations.

Vector-borne diseases transmitted through the bites of hematophagous arthropods, such as mosquitoes, continue to be a significant threat to human health globally. Transmission of disease by biting arthropod vectors includes interactions between (1) saliva expectorated by a vector during blood meal acquisition from a human host, (2) the transmitted vector-borne pathogens, and (3) host cells present at the skin bite site. Currently, the investigation of bite-site biology is challenged by the lack of model 3D human skin tissues for in vitro analyses. To help fill this gap, we have used a tissue engineering approach to develop new stylized human dermal microvascular bed tissue approximates-complete with warm blood-built with 3D capillary alginate gel (Capgel) biomaterial scaffolds. These engineered tissues, termed a Biologic Interfacial Tissue-Engineered System (BITES), were cellularized with either human dermal fibroblasts (HDFs) or human umbilical vein endothelial cells (HUVECs). Both cell types formed tubular microvessel-like tissue structures of oriented cells (82% and 54% for HDFs and HUVECs, respectively) lining the unique Capgel parallel capillary microstructures. Female Aedes (Ae.) aegypti mosquitoes, a prototypic hematophagous biting vector arthropod, swarmed, bit, and probed blood-loaded HDF BITES microvessel bed tissues that were warmed (34-37 °C), acquiring blood meals in 151 ± 46 s on average, with some ingesting ≳4 µL or more of blood. Further, these tissue-engineered constructs could be cultured for at least three (3) days following blood meal acquisitions. Altogether, these studies serve as a powerful proof-of-concept demonstration of the innovative BITES platform and indicate its potential for the future investigation of arthropod bite-site cellular and molecular biology.

Molecular modeling and biological investigation of novel s-triazine linked benzothiazole and coumarin hybrids as antimicrobial and antimycobacterial agents

A novel series of s-triazine linked benzothiazole and coumarin hybrids (6a-6d, 7a-7d, and 8a-8d) were synthesized and characterized by IR, NMR, and mass spectrometry. The compound’s in vitro antibacterial and antimycobacterial activities were also evaluated. Remarkable antibacterial activity with MIC in the range of 12.5–62.5 μM and antifungal activity of 100–200 μM were demonstrated by in vitro antimicrobial analysis. Compounds 6b, 6d, 7b, 7d, and 8a strongly inhibited all bacterial strains, while 6b, 6c, and 7d had good to moderate efficacy against M. tuberculosis H37Rv. Synthesized hybrids are observed in the active pocket of the S. aureus dihydropteroate synthetase enzyme, according to a molecular docking investigations. Among the docked compounds, 6d had a strong interaction and a greater binding affinity, and the dynamic stability of protein-ligand complexes was examined using molecular dynamic simulation with various settings at 100 ns. The proposed compounds successfully maintained their molecular interaction and structural integrity inside the S. aureus dihydropteroate synthase, according to the MD simulation analysis. These in silico analyses supported the in vitro antibacterial results of compound 6d, which demonstrated outstanding in vitro antibacterial efficacy against all bacterial strains. In the quest for new antibacterial drug-like molecules, compounds 6d, 7b, and 8a have been identified as promising lead compounds. Communicated by Ramaswamy H. Sarma

Identification and the Related Molecular Mechanism of B(A)04 Allele

To investigate the identification and molecular biological mechanism of a case of B(A)04 allele. The ABO blood groups of the proband and his nine family members were analyzed serologically and DNA sequencing was used to accurately determine the genotypes of these ten specimens. The cartoon models of local active center of enzymes of the GTA,GTB and the GTB mutant were constructed to explore the possible molecular mechanism leading to abnormal enzyme-catalyzed A antigen synthesis. The serological results suggested that the ABO blood groups of the proband, his elder brother and his maternal grandmother were AweakB or B(A); the ABO blood group of his mother was type AB, his uncle and elder aunt were type B, and his father was type O. ABO blood group gene sequencing results showed that 6 out of 10 members of the family carried the B(A)04 allele. Molecular structure models suggested that the spatial distance of critical amino acid residues in the catalytic center of the GTB mutant enzyme was greater than that of GTB, which might cause the enzyme to abnormally catalyze the synthesis of A antigen. The characteristics of serological reactions of B(A) blood subgroup are complicated, and its identification needs to be combined with molecular biology and pedigree investigation. It is speculated that the B(A) phenotype may be associated with a larger volume of the catalytic center in the GTB mutant.

Role of G-Proteins and GPCR-Mediated Signalling in Neuropathophysiology.

G-protein-coupled receptors (GPCRs) are activated by manifold neurotransmitters, and their activation, in turn, evokes slow synaptic transmission. They are profoundly related to numerous psychiatric and neurological disorders such as schizophrenia and Parkinson's disease. The significant malady indications for GPCR modulators demonstrate a change towards obesity, diabetes, and Alzheimer's disease, while other central nervous system disorders persist highly represented. GPR52, GPR6, and GPR8 are recognised as orphan GPCRs, co-exist either with both the dopamine D2 and D1 receptors in neurons of the basal ganglia or with the dopamine D2 receptor alone, and recommend that between these orphan receptors, GPR52 has the maximum potential of being a therapeutic psychiatric receptor. Genetically modified creature models and molecular biological investigations have suggested that these improved GPCRs could be potential therapeutic psychiatric receptors. In this perspective, the role of molecular targets in GPCR-mediated signalling has been discussed that would be novel drug design and discovery options for a scientist to elaborate previous knowledge with modern techniques.