Effective RNA isolation method for gram-positive and acid-fast bacteria: metamorphosed from conventional RNA isolation techniques.

The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.

Development of highly adaptable RT-PCR methods for identifying Delta and BA.1 variants in inactivated COVID-19 vaccines.

Background The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses a significant threat to human health and public safety. While next-generation sequencing (NGS) is capable of detecting and tracking new COVID-19 variants for disease diagnosis and prevention, its high cost and time-consuming nature limit its widespread use. In this study, our aim was to develop a highly adaptable and accurate RT-PCR method for identifying the Delta or BA.1 variants in inactivated COVID-19 vaccine. We devised three two-plex RT-PCR methods targeting specific mutation sites: S: Δ156-157, S: N211-, L212I, and S: Δ142-144, Y145D. The RT-PCR method targeting the S: Δ156-157 mutation site was able to distinguish the Delta variant from other COVID-19 virus strains, while the RT-PCR methods targeting the S: N211-, L212I or S: Δ142-144, Y145D mutation sites were able to distinguish the BA.1 variant from other COVID-19 virus strains. We separately validated these three two-plex RT-PCR methods, and the results demonstrated good linearity, repeatability, reproducibility, and specificity for each method. Moreover, all three methods can be applied in the production of SARS-CoV-2 variant inactivated vaccines, enabling the identification of Delta or BA.1 variants in virus cultures as well as in inactivated vaccine stocks. This study presents a systematic approach to identify COVID-19 variants using multiple RT-PCR methods. We successfully developed three two-plex RT-PCR methods that can identify Delta and BA.1 variants based on specific mutation sites, and we completed the validation of these three methods.

Quantitative method for measuring the proportion of bacterial cells expressing phase 1 or 2 of flagellin of Salmonella enterica serovar Typhimurium.

Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into "Hi" as phase 1 and "H1 or H2" as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.

Mapping the main harmful algal species in the East China Sea (Yangtze River estuary) and their possible response to the Main ecological status and global climate change via a global vision.

The Yangtze River Estuary (YRE) is one of the areas in China most severely affected by harmful algal blooms (HABs). This study explored the distributive patterns of HABs in the YRE and how they are influenced by the El Niño-Southern Oscillation (ENSO) and other environmental factors. Quantitative real-time PCR (qPCR) was employed to detect and quantify the four predominant HAB species in the YRE, Karenia mikimotoi, Margalefidinium polykrikoides, Prorocentrum donghaiense, and Heterosigma akashiwo. Additionally, the study analyzed how turbidity, pH, salinity, and temperature influence these algae. Distribution of the four HAB species in the YRE area shows clear geographical variations: K. mikimotoi is predominantly found in the northwest and central sea areas, M. polykrikoides (East Asian Ribotype, EAR) is mainly distributed in the southeastern part, P. donghaiense is abundant in the northern regions, and H. akashiwo is especially prevalent at stations S26 and S27 in the northeastern part of the study area. HABs dominated by H. akashiwo and P. donghaiense were observed in the northeastern sea area of the YRE on July 22, 2020. Our study reveals that K. mikimotoi, M. polykrikoides (EAR), and P. donghaiense are mainly affected by turbidity, pH, and salinity, while temperature predominantly influences the blooms of H. akashiwo. Moreover, runoff in the YRE has a certain correlation with ENSO events, which may also impact the nutrient content of the region. The findings of this study illustrate the distributive patterns of the four HAB species under various ecological conditions in the YRE and emphasize the importance of establishing practical cases for future warning systems. To better understand how climate change affects HABs, exploring the link between ENSO and HABs is essential.

Development of a biomarker-based platform for comprehensive skin characterization using minimally invasive skin sampling and quantitative real-time PCR.

BACKGROUND: Classifying diverse skin types is crucial for promoting skin health. However, efficiently identifying and analyzing relevant biomarkers from a vast array of available genetic data is challenging. Therefore, this study aimed to develop a precise and efficient platform for analyzing specific skin biomarkers using quantitative real-time PCR (qRT-PCR) with the minimal invasive skin sampling method (MISSM). MATERIALS AND METHODS: MISSM was used for RNA extraction from skin samples, followed by qRT-PCR analysis to quantify the expression of 20 biomarkers associated with skin characteristics (four biomarkers each for five skin characteristics). Noninvasive measurements from 299 Korean participants were utilized to correlate biomarker expression with skin parameters. Statistical analyses were conducted between biomarker expression levels and noninvasive skin measurements to select the relatively best-performing biomarker for each skin characteristic. RESULTS: Collagen type 1 alpha 1 (COL1A1) and moesin (MSN) were identified as skin aging biomarkers. Krüppel-like factor 4 (KLF4) and serine peptidase inhibitor Kazal type 5 (SPINK5) were identified as skin dryness biomarkers, whereas melan-A (MLANA) was selected as a biomarker for understanding pigmentation dynamics. Myelin protein zero like 3 (MPZL3) and high mobility group box 2 (HMGB2) were identified as markers of oily skin and skin sensitivity, respectively. Statistically significant correlations were found between the biomarker expression levels and noninvasive skin characteristic measurements. CONCLUSION: This study successfully developed a platform for the precise evaluation of individual skin characteristics using MISSM and qRT-PCR biomarker analysis. By selecting biomarkers that correlate with noninvasive measurements of skin characteristics, we demonstrated the platform's efficacy in assessing diverse skin conditions.

Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of kifc1 in Hemibarbus labeo (Teleostei, Cypriniformes, Cyprinidae).

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

INTRODUCTION: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. OBJECTIVE: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. METHODOLOGY: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added ß-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. RESULTS: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. CONCLUSION: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

Unraveling the diversity of hyphal explorative traits among Rhizophagus irregularis genotypes.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Cytogenetic and Molecular Effects of Kaolin's Foliar Application in Grapevine (Vitis vinifera L.) under Summer's Stressful Growing Conditions.

Grapevine varieties from "Douro Superior" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer's stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed.

Comprehensive evaluation of genes related to basement membrane in hepatocellular carcinoma.

In all mammals, the basement membrane serves as a pivotal extracellular matrix. Hepatocellular carcinoma (HCC) is a challenge among numerous cancer types shaped by basement membrane-related genes (BMGs). Our research established an innovative prognostic model that is highly accurate in its prediction of HCC prognoses and immunotherapy efficacy to summarize the crucial role of BMGs in HCC. We obtained HCC transcriptome analysis data and corresponding clinical data from The Cancer Genome Atlas (TCGA). To augment our dataset, we incorporated 222 differentially expressed BMGs identified from relevant literature. A weighted gene coexpression network analysis (WGCNA) of 10158 genes demonstrated four modules that were connected to HCC. Additionally, 66 genes that are found at the intersection of BMGs and HCC-related genes were designated as hub HCC-related BMGs. MMP1, ITGA2, P3H1, and CTSA comprise the novel model that was engineered using univariate and multivariate Cox regression analysis. Furthermore, the International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO) datasets encouraged the BMs model's validity. The overall survival (OS) of individuals with HCC may be precisely predicted in the TCGA and ICGC databases utilizing the BMs model. A nomogram based on the model was created in the TCGA database at similar time, and displayed a favorable discriminating ability for HCC. Particularly, when compared to the patients at an elevated risk, the patients with a low-risk profile presented different tumor microenvironment (TME) and hallmark pathways. Moreover, we discovered that a lower risk score of HCC patients would display a greater response to immunotherapy. Finally, quantitative real-time PCR (qRT-PCR) experiments were used to verify the expression patterns of BMs model. In summary, BMs model demonstrated efficacy in prognosticating the survival probability of HCC patients and their immunotherapeutic responsiveness.

The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Detection of prolong excretion of Escherichia albertii in stool specimens of a 7-year-old child by a newly developed Eacdt gene-based quantitative real-time PCR method and molecular characterization of the isolates.

Escherichia albertii is an emerging zoonotic foodborne pathogen. The clinical significance of this bacterium has increasingly been recognized worldwide. However, diagnostic method has not yet been established and its clinical manifestations are not fully understood. Here, we show that an Eacdt gene-based quantitative real-time PCR (qRT-PCR) developed in this study is 100% specific and sensitive when tested with 39 E. albertii and 36 non-E. albertii strains, respectively. Detection limit of the real-time PCR was 10 colony forming unit (CFU) and 1 pg of genomic DNA per PCR tube. When E. albertii was spiked with 4 × 100-106 CFU per mL to stool of healthy person, detection limit was 4.0 × 103 and 4.0 CFU per mL before and after enrichment culture, respectively. Moreover, the qRT-PCR was able to detect E. albertii in five children out of 246 (2%) but none from 142 adults suffering from gastroenteritis. All five E. albertii strains isolated carried eae and paa genes, however, only one strain harbored stx2f genes. Long-term shedding of stx2f gene-positive E. albertii in a child stool could be detected because of the qRT-PCR developed in this study which might have been missed if only conventional PCR and culture methods were employed. Furthermore, E. albertii isolated from siblings with diarrhea showed clonality by PFGE analysis. Taken together, these data suggest that the Eacdt gene-based qRT-PCR developed for the detection of E. albertii is useful and will assist in determining the real burden and clinical manifestation of E. albertii infections.

Establishment of a chip digital PCR detection method for canine circovirus.

Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/µL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

Diagnostic value of combined detection of plasma cfDNA concentration and integrity in NSCLC.

Aim: To evaluate the value of combined detection of plasma cfDNA concentration and integrity in the early diagnosis of NSCLC. Methods: Real-time fluorescence quantitative PCR was used to determine the concentration and integrity of plasma cfDNA in 71 NSCLC patients and 53 healthy people. Results: Combined detection of plasma cfDNA concentration and integrity had higher diagnostic power in differentiating NSCLC patients with stage I/II from healthy people than detection of plasma cfDNA concentration alone or integrity alone. The AUC, sensitivity and specificity of the combined detection of plasma cfDNA concentration and integrity were 0.781, 0.62 and 0.85. Conclusion: Combined detection of plasma cfDNA concentration and integrity could improve the diagnostic value in NSCLC detection.

The discovery of cfDNA has opened up a wide range of new possibilities for the diagnosis of cancer. CfDNA provides a noninvasive diagnostic approach for early screening, early detection and monitoring of patients with cancer. Currently, the application of cfDNA in clinical practice for NSCLC patients has been widely reported, which mainly focused on DNA methylation detection, oncogenic driver gene mutation detection. However, few studies have evaluated the diagnostic value of combined detection of plasma cfDNA concentration and integrity for NSCLC patients. Our study suggests that the combination of plasma cfDNA concentration and integrity has higher AUC value in differentiating NSCLC patients from healthy individuals than plasma cfDNA concentration alone or integrity alone.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

Relationship of TNFα-238 G/A (rs 361525) genotypes with TNFα gene expression in liver and pancreas disorders in sample of beta thalassemia major adult Iraqi patients.

BACKGROUND: Tumor necrosis factor-α (TNFα) is a crucial physiologic regulator of immune responses, and several disorders have been associated with its dysregulation. OBJECTIVE: This study aimed to understand TNFα gene expression in adult patients with liver and pancreas disorders and examine the impact of TNFα-238 genotypes on this population. METHODS: At the Ibn Al-Baladi Hospital in Baghdad, blood samples were collected from forty patients who were diagnosed with beta thalassemia together with pancreatic disease, forty patients who were diagnosed with thalassemia together with liver disorder, and forty patients who were diagnosed with thalassemia without pancreas or liver disorder. For the purpose of establishing a control group, forty samples were collected from persons who were of the same age and gender and seemed to be in good health. All of these individuals were deemed to be older than 18 years old. Through the utilization of real-time polymerase chain reaction (PCR), the level of TNF-α gene expression was investigated and assessed. The T-ARMS-PCR method was performed for detection and genotyping of TNFα-238 in thalassemia patients and healthy control samples. RESULTS: The result showed that TNF α gene expression assessment showed that group B (thalassemia patients with liver disorder) had higher folding than other groups while the lowest gene expression was in group D (as control group). Furthermore, the relationship between TNFα gene expressions folding with TNFα-238 genotypes in beta thalassemia major patients, discovered a considerable increase at GA genotype patients in TNFα gene expression level, followed by AA genotype compared to the GG genotype. Furthermore, the results of the current study showed an association between the presence of the mutant (A) allele whether heterozygous (GA) and homozygous (AA) with the TNF-α gene expression in thalassemia patients with liver and pancreatic disorders. CONCLUSION: Based on the results, it can be concluded that there is a relationship between the presence of the mutant (A) allele, whether heterozygous (GA) or homozygous (AA) of TNF-α 238, and TNF-α gene expression in liver and pancreatic diseases as well as in patients with thalassemia.

Yak IGFBP3 promotes hepatocyte proliferation through PI3K-Akt signaling pathway.

IGFBP3 (Insulin-like growth factor binding protein 3) constitutes a crucial constituent of the insulin-like growth factor (IGF), which are intimately associated with the organism's growth and development processes. Despite its significance, the precise function of IGFBP3 in yak liver development remains largely unexplored. In the present study, we systematically examined the expression profile of IGFBP3 in the liver tissues of yaks across various growth stages, elucidated its influence on the activity of yak hepatocytes, and probed its effects on murine liver development. A comparative analysis revealed that the expression of IGFBP3 was significantly higher in the liver tissue of 5-year-old yaks compared to their 15-month-old and 1-day-old counterparts (P < 0.01). To further validate its biological function, pET-28a-BgIGFBP3 prokaryotic expression vector was constructed. Upon exposing yak hepatocytes to varying concentrations of Bos grunniens (Bg) IGFBP3 protein, we observed augmented cellular activities and elevated colony formation rates. Moreover, our investigation revealed the upregulation of key genes within the PI3K-Akt signaling pathway, including ERBB2, IRS1, PIK3R1, AKT1, RAF1, MAP2K2, and MAPK3, in both yak hepatocyte cultures and murine models. These findings collectively indicate that BgIGFBP3 promotes the proliferation of yak hepatocytes and enhances murine liver development by modulating the PI3K-Akt signaling pathway. The functional relevance of BgIGFBP3 was substantiated through in vivo and in vitro experiments, thereby underscoring its potential as a regulatory factor in liver development processes.

Development of real-time PCR assay for quantitative detection of Clostridium septicum.

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.