Gene PCR Research Articles

Liver Mitochondrial Morphology and Gene Expression as Markers of Liver Reserve: Prognostic Implications for Native Liver Survival in Biliary Atresia

PurposeHepatocyte mitochondrial morphology and gene expression were compared between biliary atresia (BA), infantile cholestasis (IC), and normal liver (NL) as prognostic indicators. MethodsSpecimens of liver at portoenterostomy (PE) for BA, from intrahepatic bile duct paucity patients for IC, and from choledochal cyst or hepatoblastoma patients for NL were collected prospectively (P) beginning in 2021 (P-BA = 11, P-IC = 9, P-NL = 7) and retrospectively (R) from paraffin-embedded tissue going back to 1981 (R-BA = 25, R-IC = 9, R-NL = 4). The P-cohort had transmission electron microscopy (TEM) to image mitochondria, immunoblotting for heat shock protein 60 (HSP60), and quantitative PCR (qPCR) for HSP60 and mitochondrial functional genes. Both cohorts had immunofluorescence for HSP60 quantified as a ratio to albumin-positive hepatocytes (ALB) with HSP60/ALB<1.0 as a cutoff limit using ImageJ. ResultsHSP60 was significantly lower in BA/IC than NL on qPCR (BA: p < 0.01, IC: p < 0.05) and lower in BA than IC/NL on immunoblotting (p < 0.05). HSP60/ALB was significantly lower in BA than NL/IC (p < 0.001). Despite BA subjects being matched for types of BA and ages at PE, HSP60/ALB did not correlate with jaundice clearance (JC; T-Bil<1.2 mg/dL) but was significantly higher in native liver survivors (NLS) after PE compared with liver transplant (LTx) cases (p < 0.05) and significantly lower in LTx cases achieving JC than NLS achieving JC (p < 0.05). TEM showed BA had significantly more mitochondrial inclusion bodies (p < 0.05) and significantly larger cristae (p < 0.01) than IC/NL. qPCR in BA showed significant repression of mitochondrial functional genes for mRNA stabilization and energy facilitation. ConclusionHSP60/ALB correlates with NLS after PE for BA. Level of evidenceII

A prospective cohort study examining the association between the periconceptual vaginal microbiota and first-trimester miscarriage in Kenyan women.

Studies evaluating the association between the vaginal microbiota and miscarriage have produced variable results. This study evaluated the association between periconceptual and first-trimester vaginal microbiota and women's risk for miscarriage. At monthly preconception visits and at 9-12 weeks gestation, women collected vaginal swabs for molecular characterisation of the vaginal microbiota. Participants who became pregnant were followed to identify miscarriage versus pregnancy continuing to at least 20 weeks gestation. Forty-five women experienced miscarriage and 144 had pregnancies continuing to ≥20 weeks. A principal component analysis of periconceptual and first-trimester vaginal bacteria identified by 16S rRNA gene PCR with next-generation sequencing did not identify distinct bacterial communities with miscarriage versus continuing pregnancy. Using taxon-directed quantitative PCR assays, increasing concentrations of Megasphaera hutchinsoni, Mageeibacillus indolicus, Mobiluncus mulieris and Sneathia sanguinegens/vaginalis were not associated with miscarriage. In exploratory analyses, these data were examined as a binary exposure to allow for multivariable modelling. Detection of Mobiluncus mulieris in first-trimester samples was associated with miscarriage (adjusted relative risk [aRR] 2.14, 95% confidence interval [CI] 1.08, 4.22). Additional analyses compared women with early first-trimester miscarriage (range 4.7-7.3 weeks) to women with continuing pregnancies. Mobiluncus mulieris was detected in all eight (100%) first-trimester samples from women with early first-trimester miscarriage compared to 101/192 (52.6%) samples from women with continuing pregnancy (model did not converge). Detection of Mageeibacillus indolicus in first-trimester samples was also associated with early first-trimester miscarriage (aRR 4.10, 95% CI 1.17, 14.31). The primary analyses in this study demonstrated no association between periconceptual or first-trimester vaginal microbiota and miscarriage. Exploratory analyses showing strong associations between first-trimester detection of Mobiluncus mulieris and Mageeibacillus indolicus and early first-trimester miscarriage suggest the need for future studies to determine if these findings are reproducible.

Invasive methods for detection of Helicobacter pylori infection in gastric disorders patients undergoing endoscopy from Mosul city, Iraq

Objectives. The identification of H. pylori in Iraq lacks a standardized technique and there is limited information on the efficacy of different diagnostic methods. This research aims to determine the prevalence of H. pylori and evaluate the effectiveness of invasive techniques, namely the Rapid Urease Test (RUT), histopathological examination, and ure A gene by PCR, for detecting H. pylori. Method. Gastric biopsies specimens were collected from 50 patients with gastric disorders who underwent endoscopy after meeting the inclusion criteria. Patients were classified into H. pylori-positive cases and negative cases by testing positive with at least two of the three testing methods, according to the case definition used in the study. Outcomes. Of the 50 gastric biopsies, 58% were H. pylori-positive cases, 26% were H. pylori -negative cases, and the last 16% were indeterminate case according to cases definition. The ure A gene PCR was the most sensitive method, followed by histopathological examination and RUT testing, with sensitivities of 96.6%, 86.2% and 79.3% respectively. Histopathology examination was the most specific with a specificity of 95.2%, followed by ure A gene PCR with 85.7%, while RUT was the least specific with a specificity of 81%. Conclusion. The current study revealed a moderate prevalence of H. pylori infection compared with previous studies. The sensitivity and specificity of the three invasive methods varied, and a combination of two methods may be necessary for a definitive diagnosis of H. pylori infection.

DIVERSITYOF LACTICACID BACTERIAINVOLVEDIN THE FERMENTATION OF THE TAMARIND FRUIT (TAMARINDUS INDICA) COTE DIVOIRE

In Cote dIvoire, tamarind fruits, from trees growing in the northern part of the country, are used for various food purposes (seasoning dishes, producing acidic beverages, etc.), typically after a fermentation process. These fermented fruits could therefore represent a source of beneficial fermentative microorganisms for the development of starters in biotechnology. A study was therefore undertaken to determine the diversity of lactic acid bacteria involved in tamarind fermentation. For this purpose, a total of 400 tamarind samples were collected from various markets in the cities of Korhogo and Abidjan. Lactic acid bacteria from these samples were screened and counted on MRS agar, and then subjected to identification. A total of 664 strains were isolated, of which 430 isolates were retained. Genus characterization was performed by 16S rRNA gene PCR. For species determination of the isolates, MALDI-TOF proteomics was applied. The results obtained showed that the lactic acid bacteria isolated from tamarind belong to the genera Pediococcus, Lactobacillus, Lacococcus, and Weissella, with a dominance of the genus Lactobacillus (63%) compared to the total number of identified strains, followed by the genus Pediococcus (16%) and finally the genera Weissella (12%) and Lactococcus (8%). The genus Lactobacillus was represented by Lactobacillus plantarum (36%), Lactobacillus fermentum (20.5%), Lactobacillus acidophilus (20%), and Lactobacillus rhamnosus (23.5%). The genus Pediococcuswas represented by Pediococcusacidilactici, the Lactococcus strains were solely composed of Lactococcus lactis. As for the genus Weissella, only Weissella sp. was involved in tamarind fermentation.

Time-course transcriptome data of silk glands in day 0–7 last-instar larvae of Bombyx mori (w1 pnd strain)

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

Application of a new designed high resolution melting analysis for mycobacterial species identification

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

Biomass-derived carbon dots as fluorescent quantum probes to visualize and modulate inflammation

Quantum dots, which won the Nobel Prize in Chemistry, have recently gained significant attention in precision medicine due to their unique properties, such as size-tunable emission, high photostability, efficient light absorption, and vibrant luminescence. Consequently, there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors, biomolecular imaging probes, and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs, which often require expensive and toxic precursors, as they offer several merits in eco-friendly synthesis, preparation from renewable sources, and cost-effective production. In this study, we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines, including human dermal fibroblasts, HeLa, cardiomyocytes, induced pluripotent stem cells, and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells, as they were taken up more by cells treated with tumor necrosis factor-α than untreated cells. In addition, our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents, which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases.

Nicotine aggravates liver fibrosis via α7 nicotinic acetylcholine receptor expressed on activated hepatic stellate cells in mice.

Smoking is a risk factor for liver cirrhosis; however, the underlying mechanisms remain largely unexplored. The α7 nicotinic acetylcholine receptor (α7nAChR) has recently been detected in nonimmune cells possessing immunoregulatory functions. We aimed to verify whether nicotine promotes liver fibrosis via α7nAChR. We used osmotic pumps to administer nicotine and carbon tetrachloride to induce liver fibrosis in wild-type and α7nAChR-deficient mice. The severity of fibrosis was evaluated using Masson trichrome staining, hydroxyproline assays, and real-time PCR for profibrotic genes. Furthermore, we evaluated the cell proliferative capacity and COL1A1 mRNA expression in human HSCs line LX-2 and primary rat HSCs treated with nicotine and an α7nAChR antagonist, methyllycaconitine citrate. Nicotine exacerbated carbon tetrachloride-induced liver fibrosis in mice (+42.4% in hydroxyproline assay). This effect of nicotine was abolished in α7nAChR-deficient mice, indicating nicotine promotes liver fibrosis via α7nAChR. To confirm the direct involvement of α7nAChRs in liver fibrosis, we investigated the effects of genetic suppression of α7nAChR expression on carbon tetrachloride-induced liver fibrosis without nicotine treatment. Profibrotic gene expression at 1.5 weeks was significantly suppressed in α7nAChR-deficient mice (-83.8% in Acta2, -80.6% in Col1a1, -66.8% in Tgfb1), and collagen content was decreased at 4 weeks (-22.3% in hydroxyproline assay). The in vitro analysis showed α7nAChR expression in activated but not in quiescent HSCs. Treatment of LX-2 cells with nicotine increased COL1A1 expression (+116%) and cell proliferation (+10.9%). These effects were attenuated by methyllycaconitine citrate, indicating the profibrotic effects of nicotine via α7nAChR. Nicotine aggravates liver fibrosis induced by other factors by activating α7nAChR on HSCs, thereby increasing their collagen-producing capacity. We suggest the profibrotic effect of nicotine is mediated through α7nAChRs.

Factors underlying the long-term efficacy of faecal microbiota transplantation for patients with irritable bowel syndrome

The long-term effects of the transplant dose, its administration route and repeated faecal microbiota transplantation (FMT) on the outcomes of FMT for patients with irritable bowel syndrome (IBS) are unknown. This study included 171 patients (125 females and 46 males): 90 g of donor feces was administered into the large intestine (LI) in 58, into the small intestine (SI) in 57, and into the SI twice (repeated SI) in 56. The patients provided a fecal sample and completed five questionnaires at the baseline and at 2 years after FMT. Fecal bacteria and the dysbiosis index were analyzed using 16S rRNA gene PCR DNA amplification/probe. The response rates at 2 years after FMT were 47.2%, 80.9%, and 76.6% in the LI, SI, and repeated-SI groups, respectively. The response rate was significantly higher in the SI and repeated SI groups than in the LI group. IBS symptoms at 2 years after FMT were less severe in the SI and repeated-SI groups than in the LI group. Fluorescent signals of several bacteria were significantly correlated with IBS symptoms and fatigue after FMT. No long-term adverse events were observed. In conclusion, administering the transplant to the SI increased the long-term response rate and reduced IBS symptom severity compared with administering it to the LI, and led to the long-term colonization of beneficial bacteria. There was no long-term difference between one and two FMT procedures (www.clinicaltrials.gov: NCT04236843).

Molecular Identification of Morus ssp. in Duhok Using Nuclear ITS Region and Chloroplast Matk Gene

Since Mulberries (Morus)is a tree species with a considerable plant variety. Molecular techniques are methods used to distinguish between species accurately, easily and quickly. This study examines a Molecular method for distinguishing different Morus species in the Duhok - Kurdistan region/ Iraq. The method is based on the use of four techniques: matK gene, the ITS region, PCR-RFLP, and SRAP markers. Twelve Morus species have been selected for this study from different region of Duhok. The ITS region's PCR result was 700 bp, but the matK gene's PCR produce was 900 bp. The same restriction site was found for all utilized cultivars when the 700bp of ITS fragment was used for PCR-RFLP with two restriction enzymes, RsaI GT/AC and HaeIII GG/CC. This study also used six combinations of SRAP markers to aid in grouping and identifying genetic similarities. The results of PCR-RFLP demonstrated an insufficient link between Morus physical appearance and genetic traits, but differences across studied cultivars could be identified using SRAP markers. Furthermore, this study demonstrated the possibility of DNA barcoding Morus cultivars, as well as additional sequence analysis and the identification of probable SNP between cultivars.

Molecular surveillance of Helicobacter species with high prevalence from two streams with various wastewater pollution in Taiwan

Helicobacter species are potential zoonotic pathogens classified as either enterohepatic or gastric. Helicobacter infection can be transmitted through wastewater from households and livestock and through water from irrigation and streams. In this study, the distribution and source of Helicobacter species in the Donggang and Yenshui rivers, two natural water bodies with different characteristics, were analyzed. A total of 44 water samples were collected over the four seasons. The samples were subjected to Helicobacter 16 s rRNA gene PCR, followed by sequencing and comparison for identification and analysis. The detection rate of Helicobacter species in both rivers was 79.55%, with H. kayseriensis (10/35, 28.57%) being the most common species. Analysis of the environment around the sampling sites showed a high detection rate in the livestock-rich area, and the results of BLAST for species identification and comparison indicated feces as the contamination source. The area around the Donggang River was developed for animal husbandry, led to a high detection rate of Helicobacter species. Many Helicobacter species were identified to have a risk of zoonotic transmission, especially if the stream is used as a source of drinking, agricultural, or even aquacultural water. The high presence of Helicobacter species in natural water bodies suggests that wastewater treatment is an effective strategy to control pathogen spread. Therefore, investigation and monitoring of pathogens in wastewater are highly important. However, methods for the isolation and culture of Helicobacter species in natural waters have yet to be developed. Hence, future research should focus on developing such methods.

Conversion of boreal forests to agricultural systems: soil microbial responses along a land-conversion chronosequence

BackgroundBoreal regions are warming at more than double the global average, creating opportunities for the northward expansion of agriculture. Expanding agricultural production in these regions will involve the conversion of boreal forests to agricultural fields, with cumulative impacts on soil microbial communities and associated biogeochemical cycling processes. Understanding the magnitude or rate of change that will occur with these biological processes will provide information that will enable these regions to be developed in a more sustainable manner, including managing carbon and nitrogen losses. This study, based in the southern boreal region of Canada where agricultural expansion has been occurring for decades, used a paired forest-adjacent agricultural field approach to quantify how soil microbial communities and functions were altered at three different stages post-conversion (< 10, > 10 and < 50, and > 50 years). Soil microbial functional capacity was assessed by quantitative PCR of genes associated with carbon (C), nitrogen, and phosphorous (P) cycling; microbial taxonomic diversity and community structure was assessed by amplicon sequencing.ResultsFungal alpha diversity did not change, but communities shifted from Basidiomycota to Ascomycota dominant within the first decade. Bacterial alpha diversity increased, with Gemmatimonadota groups generally increasing and Actinomycetota groups generally decreasing in agricultural soils. These altered communities led to altered functional capacity. Functional genes associated with nitrification and low molecular weight C cycling potential increased after conversion, while those associated with organic P mineralization potential decreased. Stable increases in most N cycling functions occurred within the first decade, but C cycling functions were still changing 50 years post conversion.ConclusionsMicrobial communities underwent a rapid shift in the first decade, followed by several decades of slower transition until stabilizing 50 years post conversion. Understanding how the microbial communities respond at different stages post-conversion improves our ability to predict C and N losses from emerging boreal agricultural systems, and provides insight into how best to manage these soils in a way that is sustainable at the local level and within a global context.

Methyl-CpG-binding protein 2 regulates CYP27A1-induced myometrial contraction during preterm labor.

Persistent and intense uterine contraction is a risk factor for preterm labor. We previously found that methyl-CpG-binding protein 2 (MeCP2), as a target of infection-related microRNA miR-212-3p, may play an inhibitory role in regulating myometrium contraction. However, the molecular mechanisms by which MeCP2 regulates myometrial contraction are still unknown. In this study, we found that MeCP2 protein expression was lower in myometrial specimens obtained from preterm labor cases, compared to those obtained from term labor cases. Herein, using RNA sequence analysis of global gene expression in human uterine smooth muscle cells (HUSMCs) following siMeCP2, we show that MeCP2 silencing caused dysregulation of the cholesterol metabolism pathway. Notably, MeCP2 silencing resulted in the upregulation of CYP27A1, the key enzyme involved in regulating cholesterol homeostasis, in HUSMCs. Methylation-specific PCR, chromatin immunoprecipitation, and dual luciferase reporter gene technology indicated that MeCP2 could bind to the methylated CYP27A1 promoter region and repress its transcription. Administration of siCYP27A1 in a lipopolysaccharide (LPS)-induced preterm labor mouse model delayed the onset of preterm labor. Human preterm myometrium and the LPS-induced preterm labor mouse model both showed lower expression of MeCP2 and increased expression of CYP27A1. These results demonstrated that aberrant upregulation of CYP27A1 induced by MeCP2 silencing is one of the mechanisms facilitating inappropriate myometrial contraction. CYP27A1 could be exploited as a novel therapeutic target for preterm birth.

Air trap and removal on a pressure driven PDMS-based microfluidic device.

With the development of microfluidic technology, microfluidic chips have played a positive role in applications such as cell culture, microfluidic PCR, and nanopore gene sequencing. However, the presence of bubbles interferes with fluid flow and has a significant impact on experimental results. There are many reasons for the generation of bubbles in microfluidic chips, such as pressure changes inside the chip, air vibration inside the chip, and the open chip guiding air into the chip when driving fluid. This study designed and prepared a microfluidic device based on polydimethylsiloxane. First, air was actively introduced into the microfluidic chip, and bubbles were captured through the microfluidic device to simulate the presence of bubbles inside the chip in biological experiments. To remove bubbles trapped in the microfluidic chip, distilled water, distilled water containing surfactants, and mineral oil were pumped into the microfluidic chip. We compared and discussed the bubble removal efficiency under different driving fluids, driving pressures, and open/closed channel configurations. This study helps to understand the mechanism of bubble formation and removal in microfluidic devices, optimize chip structure design and experimental reagent selection, prevent or eliminate bubbles, and reduce the impact of bubbles on experiments.

Gut-muscle axis: establishing the connection between microbial signaling and skeletal muscle function in a mouse model (mdx) of Duchenne muscular dystrophy

The metabolic and endocrine nature of skeletal muscle allows for systemic signaling and influence over the enteric environment influencing gut microbial populations. Concomitantly, commensal microbial signaling can alter the composition and development of skeletal muscle forming a bidirectional gut-muscle axis. Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle atrophy, fibrosis, and ultimately cardiorespiratory failure. Previous studies have shown that gut microbial signaling can alter the pathways involved in muscle atrophy, enhancing atrophy when dysbiotic, or blunting the atrophic effect by restoring gut microbial populations to a healthy status; thus, we sought to characterize the metabolic signaling between the gut and the diaphragm in a mouse model of DMD ( mdx) to identify potential therapeutic targets. The study was divided into two arms and conducted following institutional ethical approval. The first arm investigated metabolomic signalling. 6-month-old male BL10 wildtype (WT; n = 12) and mdx (n = 12) mice were anaesthetized (induction by 5% isoflurane) and decapitated. Following collection of trunk blood, the diaphragm was excised, and colonic/cecal contents were collected. The samples were then processed by mass-spectrometry to isolate the metabolomic profiles of semi-polar, secondary bile acid, and short chain fatty acid (SCFA) targets in the gut, plasma and diaphragm of WT and mdx mice. The second arm of the study explored the gut microbial profile and diaphragm gene expression, structure and function. 6-month-old male WT (n = 15) and mdx (n = 15) were anesthetized (induction by 5% isoflurane) followed by cervical dislocation. The diaphragm was excised, and longitudinal strips were isolated for structural analysis (i.e., histological staining and immunohistochemistry for fibre type and size), PCR gene expression, and isometric force assessment ex vivo. Additionally, faecal pellets were collected and analysed via shotgun metagenomic sequencing to assess microbial populations. Diaphragm twitch and tetanic force in mdx were profoundly decreased compared to WT. Metabolic analysis of faecal metabolites indicated impairment in the three main tryptophan metabolic pathways in mdx mice: kynurenine, indole and serotonin. Metagenomic analysis indicated a profound genotype effect between WT and mdx gut microbial populations. Two main producers of the short chain fatty acid acetate (Lactobacillus and Bifidobacterium) were significantly decreased within the mdx group. Acetate serves as a substate in the TCA cycle contributing to ATP production and positively affects skeletal muscle via numerous pathways including the down regulation of key atrogenes associated with atrophy. Pathway enrichment analysis showed a global disruption to both purine and pyrimidine metabolism in all three tissue types. Purine/pyrimidine metabolism have been tightly linked with microbial metabolism and affect skeletal muscle health and function. Further understanding of the gut-muscle axis in dystrophic models may pave the way for novel microbiota-directed therapeutic strategies to ameliorate muscle dysfunction in DMD. Funded by Science Foundation Ireland SFI/19/6628 INSPIRE DMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Production of Recombinant Human Interferon alpha 2b (rhIFN?-2b) in genetically engineered strain of Bacillus subtilis

Interferon alpha 2b (IFN?-2b) is a critical therapeutic protein with immunomodulatory properties, widely used in the treatment of various diseases, including viral infections and cancer. This study presents a comprehensive methodology for the cloning and characterization of the IFN?-2b gene, including the identification of point mutations, followed by expression in Bacillus subtilis. The research begins with gene isolation, primer design, RNA extraction, cDNA synthesis, and PCR amplification. Cloning into a T/A vector and subsequent sequencing reveal point mutations within the gene. The transformed construct is expressed in Bacillus subtilis, and the resulting protein is analyzed using SDS-PAGE. The identification of point mutations and successful protein expression in Bacillus subtilis. This study provides valuable insights into the IFN?-2b gene and its potential applications.

Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

Ticks are arachnid ectoparasites that rank second only to mosquitoes in the transmission of human diseases including bacteria responsible for anaplasmosis, ehrlichiosis, spotted fevers, and Lyme disease among other febrile illnesses. Due to the paucity of data on bacteria transmitted by ticks in Kenya, this study undertook a bacterial metagenomic-based characterization of ticks collected from Isiolo, a semi-arid pastoralist County in Eastern Kenya, and Kwale, a coastal County with a monsoon climate in the southern Kenyan border with Tanzania. A total of 2,918 ticks belonging to 3 genera and 10 species were pooled and screened in this study. Tick identification was confirmed through the sequencing of the Cytochrome C Oxidase Subunit 1 (COI) gene. Bacterial 16S rRNA gene PCR amplicons obtained from the above samples were sequenced using the MinION (Oxford Nanopore Technologies) platform. The resulting reads were demultiplexed in Porechop, followed by trimming and filtering in Trimmomatic before clustering using Qiime2-VSearch. A SILVA database pretrained naïve Bayes classifier was used to classify the Operational Taxonomic Units (OTUs) taxonomically. The bacteria of clinical interest detected in pooled tick assays were as follows: Rickettsia spp. 59.43% of pools, Coxiella burnetii 37.88%, Proteus mirabilis 5.08%, Cutibacterium acnes 6.08%, and Corynebacterium ulcerans 2.43%. These bacteria are responsible for spotted fevers, query fever (Q-fever), urinary tract infections, skin and soft tissue infections, eye infections, and diphtheria-like infections in humans, respectively. P. mirabilis, C. acnes, and C. ulcerans were detected only in Isiolo. Additionally, COI sequences allowed for the identification of Rickettsia and Coxiella species to strain levels in some of the pools. Diversity analysis revealed that the tick genera had high levels of Alpha diversity but the differences between the microbiomes of the three tick genera studied were not significant. The detection of C. acnes, commonly associated with human skin flora suggests that the ticks may have contact with humans potentially exposing them to bacterial infections. The findings in this study highlight the need for further investigation into the viability of these bacteria and the competency of ticks to transmit them. Clinicians in these high-risk areas also need to be appraised for them to include Rickettsial diseases and Q-fever as part of their differential diagnosis.

Occurrence of Coxiellosis in ruminants and its associated risk factors

Coxiellosis in animals is caused by the zoonotic pathogen, Coxiella burnetii. Although the disease is of public health importance it remains underdiagnosed and underreported. The cross- sectional study was aimed to estimate the occurrence of the disease in livestock of study area and also to identify the risk factors associated with the disease in animals. Blood, serum, and vaginal swabs samples were collected from 200 ruminants (cattle, sheep, and goats), across various farms in Karnataka, India. These samples were then screened using ELISA and PCR (com1 and IS1111). A questionnaire was administered to the farm owners to collect the risk factor-related information. About 5.26 % cattle, 12.3 % sheep, and 12.5 % goats were positive by ELISA. By PCR, 9.47 % cattle, 9.3 % sheep, and 10 % goats were positive. Overall, the occurrence of 14.73 %, 18.46 % and 17.5 % was estimated in cattle, sheep and goat, respectively. PCR targeting the IS1111 gene detected higher number of samples as positive as compared to the com1 gene PCR. Higher number of vaginal swab samples were detected as positive as compared to blood. History of reproductive disorders (OR: 4.30; 95 %CI:1.95- 9.46), abortion (OR: 30.94; 95 %CI:6.30- 151.84) and repeat breeding (OR:11.36; 95 %CI:4.16- 30.99) were significantly associated with coxiellosis (p < 0.005). Multivariable analysis by logistic regression model analysis suggested retained abortion, repeat breeding and rearing of animal in semi-intensive system as factors significantly associated with the infection. Cultural identification of the PCR positive samples were cultured using embryonated egg propagation and cell culture techniques and positivity was confirmed in six samples. Phylogenetic analysis of the com1 and IS1111 gene revealed clustering based on similar geographic locations. The study estimated the occurrence of the disease in the study area and identified the potential risk factors.

Cumulative mortalities in white leg shrimp, Litopenaeus vannamei Boone 1931, cultured in biofloc system in Egypt reflected new record of Fusarium verticillioides infection

The Egyptian aquaculture sector is still struggling to establish L. vannamei culture. In an effort to stimulate shrimp production with minimal environmental impact and less water loss, biofloc technology (BFT) has been employed. Chronic mortality was recently observed in L. vannamei which was raised in the BFT system. Examining samples of moribund shrimp revealed sporadic melanization on the musculature and gills, suggesting the potential existence of black gill disease. The present work was carried out to isolate and identify potentially implicated fungus species based on mycological properties of interest. To isolate and serve as initial identification tools, wet mount preparations and colonial features, along with slide culture techniques, were used. PCR assays, gene sequencing, and phylogenetic analyses were used in the molecular identification process to clarify the confirmation of the fungal isolates that were acquired. The pathogenicity tests were designed to fulfil Koch’s postulates in addition to clarifying the infectivity of isolated fungus. Moreover, histopathological examination was employed to notice tissues’ alterations associated with experimentally infected shrimp. The results revealed the isolation of three fungal isolates, namely, BNS 1117, BNS 2117, and BNS 3117, that were confirmatively identified as Fusarium verticillioides, and their selected sequences were submitted to Genbank. Moreover, experimental infection using F. verticillioides selected isolate (BNS 31,117) at both high (5 × 105 CFU/mL, group A) and low (3 × 105 CFU/mL, group B) doses of its conidial suspensions experienced cumulative mortality of 72.2% and 21.1%, respectively. Besides, the selected isolate was re-isolated from the experimentally infected shrimp in a pure form, which satisfied Koch’s postulates. Histopathological alterations demonstrated several degrees of either degenerative, oedema, or proliferative changes in the hepatopancreas, musculatures, and gills, respectively. This study sets a new record for F. verticillioides to be included in Fusarium species linked to shrimp mortality.

Temperature and immune challenges modulate the transcription of genes of the ubiquitin and apoptosis pathways in two high-latitude Notothenioid fish across the Antarctic Polar Front.

Thermal variations due to global climate change are expected to modify the distributions of marine ectotherms, with potential pathogen translocations. This is of particular concern at high latitudes where cold-adapted stenothermal fish such as the Notothenioids occur. However, little is known about the combined effects of thermal fluctuations and immune challenges on the balance between cell damage and repair processes in these fish. The aim of this study was to determine the effect of thermal variation on specific genes involved in the ubiquitination and apoptosis pathways in two congeneric Notothenioid species, subjected to simulated bacterial and viral infections. Adult fish of Harpagifer bispinis and Harpagifer antarcticus were collected from Punta Arenas (Chile) and King George Island (Antarctica), respectively, and distributed as follows: injected with PBS (control), LPS (2.5mg/kg) or Poly I:C (2mg/kg) and then submitted to 2, 5 and 8°C. After 1week, samples of gills, liver and spleen were taken to evaluate the expression by real-time PCR of specific genes involved in ubiquitination (E3-ligase enzyme) and apoptosis (BAX and SMAC/DIABLO). Gene expression was tissue-dependent and increased with increasing temperature in the gills and liver while showing an opposite pattern in the spleen. Studying a pair of sister species that occur across the Antarctic Polar Front can help us understand the particular pressures of intertidal lifestyles and the effect of temperature in combination with biological stressors on cell damage and repair capacity in a changing environment.