Molecular Biology Techniques Research Articles

Identification of Mycoviruses in the Pathogens of Fragrant Pear Valsa Canker from Xinjiang in China.

As a common disease, canker seriously affects the yield and quality of fragrant pear due to the lack of effective control measures. Some fungi have been reported to harbor rich reservoirs of viral resources, and some mycoviruses can be used as biocontrol agents against plant diseases. In this study, 199 isolates were obtained from diseased branches of fragrant pear in the main production areas of Xinjiang. Among them, 134 belonged to Valsa spp., identified using morphological and molecular biological techniques, in which V. mali was the dominant species. The mycoviruses in Valsa spp. were further identified using metatranscriptomic sequencing and RT-PCR. The results revealed that a total of seven mycoviruses were identified, belonging to Botourmiaviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mitoviridae, and Narnaviridae, among which Phomopsis longicolla hypovirus (PlHV) was dominant in all the sample collection regions. The Cryphonectria hypovirus 3-XJ1 (CHV3-XJ1), Botourmiaviridae sp.-XJ1 (BVsp-XJ1), and Fusariviridae sp.-XJ1 (Fvsp-XJ1) were new mycoviruses discovered within the Valsa spp. More importantly, compared with those in the virus-free Valsa spp. strain, the growth rate and virulence of the VN-5 strain co-infected with PlHV and CHV3-XJ1 were reduced by 59% and 75%, respectively, and the growth rate and virulence of the VN-34 strain infected with PlHV were reduced by 42% and 55%, respectively. On the other hand, the horizontal transmission efficiency of PlHV decreased when PlHV was co-infected with CHV3-XJ1, indicating that PlHV and CHV3-XJ1 were antagonistic. In summary, the mycoviruses in Valsa spp. were identified in Xinjiang for the first time, and three of them were newly discovered mycoviruses, with two strains yielding good results. These results will offer potential biocontrol resources for managing pear canker disease and provide a theoretical basis for the control of fruit tree Valsa canker disease.

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

Pan-TRK immunohistochemistry as a tool in the screening for NTRK gene fusions in cancer patients

Therapy with TRK inhibitors is a tumor-agnostic treatment directed against specific molecular changes rather than cancer type. NTRK fusions are rare in most prevalent cancers, accounting for less than 0.5% of cases. However, there is a group of rare cancers in which NTRK fusion is more prevalent. These include secretory carcinoma of the breast and salivary gland, childhood sarcomas, such as infantile fibrosarcoma, and cellular and mixed congenital mesoblastic nephroblastoma. The most common rearrangement pertains to NTRK3 and the most common fusion gene is ETV6 . Identifying patients with NTRK gene fusions who would likely benefit from targeted therapy with TRK inhibitors requires practical diagnostic tools and an appropriate management strategy of diagnostic trajectory. The fusions can be detected by molecular biology techniques or pan-TRK immunohistochemistry. The latter detects NTRK1/2/3 gene fusions independently of the resulting fusion gene but does not determine which of them has been rearranged or what the fusion partner is. The sensitivity and specificity of the method reach 97% and 100%, respectively. Other advantages include the relatively low cost, short duration of examination, and broad accessibility of immunohistochemistry laboratories. These characteristics make this method a useful screening tool for detecting patients with NTRK gene fusions.

Alleviation of allergic asthma by rosmarinic acid via gut-lung axis

BackgroundAsthma affects 3% of the global population, leading to over 0.25 million deaths. Due to its complexity, asthma is difficult to cure or prevent, and current therapies have limitations. This has led to a growing demand for alternative asthma treatments. We found rosmarinic acid (RosA) as a potential new drug candidate from natural medicine. However, RosA has poor bioavailability and remains mainly in the gastrointestinal tract after oral administration, suggesting the involvement of gut microbiota in its bioactivity. PurposeTo investigate the mechanism of RosA in alleviating allergic asthma by gut-lung axis. MethodsWe used 16S rRNA gene sequencing and metabolites analysis to investigate RosA's modulation of gut microbiota. Techniques of molecular biology and metabolomics were employed to study the pharmacological mechanism of RosA. Cohousing was used to confirm the involvement of gut microbiota in RosA-induced improvement of allergic asthma. ResultsRosA decreased cholate levels from spore-forming bacteria, leading to reduced 5-hydroxytryptamine (5-HT) synthesis, bronchoconstriction, vasodilation, and inflammatory cell infiltration. It also increased short-chain fatty acids (SCFAs) levels, facilitating the expression of intestinal tight junction proteins to promote intestinal integrity. SCFAs upregulated intestinal monocarboxylate transporters (MCTs), thereby improving their systemic delivery to reduce Th2/ILC2 mediated inflammatory response and suppress eosinophil influx and mucus production in lung. Additionally, RosA inhibited lipopolysaccharide (LPS) production and translocation, leading to reduced TLR4-NFκB mediated pulmonary inflammation and oxidative stress. ConclusionsThe anti-asthmatic mechanism of oral RosA is primarily driven by modulation of gut microbiota-derived 5-HT, SCFAs, and LPS, achieving a combined synergistic effect. RosA is a safe, effective, and reliable drug candidate that could potentially replace glucocorticoids for asthma treatment.

Current understanding of essential trace elements in intrahepatic cholestasis of pregnancy.

Trace elements are important components in the body and have fundamental roles in maintaining a healthy and balanced pregnancy process.Either deficiency or excess of trace elements, including selenium, iron, zinc, copper, and magnesium can lead to pregnancy complications. As a rare disorder during pregnancy of unknown aetiology, intrahepatic cholestasis of pregnancy (ICP) poses a significant risk to the fetus of perinatal mortality. ICP is a multifactorial complication of which the pathogenesis is still an enigma. Epidemiological studies have demonstrated the association of ICP with some trace elements. Evidence from retrospective studies in humans further revealed the possible contributing roles of trace elements in the pathogenesis of ICP. The published literature on the association of trace elements with ICP was reviewed. Recent advances in molecular biological techniques from animal studies have helped to elucidate the possible mechanisms by how these trace elements function in regulating oxidative reactions, inflammatory reactions and immune balance in the maternal-fetal interface, as well as the influence on hepato-intestinal circulation of bile acid. The scenario regarding the role of trace elements in the pathogenesis of ICP is still developing. The administration or depletion of these trace elements may have promising effects in alleviating the symptoms and improving the pregnancy outcomes of ICP.

Biological parameters for quality evaluation of allografts from the Brazilian National Institute of Traumatology and Orthopedics tissue bank.

Bone allografts are clinically used in a variety of surgical procedures, and tissue banks are responsible for harvesting, processing, quality testing, storing, and delivering these materials for transplantation. In tissue banks, the bone is processed for the removal of all organic content, remaining only the tissue structure (scaffold). However, several studies have shown that even after using different processing methods, viable cells, functional proteins, and DNA may still persist in the tissue, which constitute the main causes of graft rejection. Therefore, the objective of this study was to establish techniques and biological parameters for quality validation of allografts. To this end, we propose the use of 3 combined methods such as microscopy, histology, and molecular biology techniques to evaluate the quality of allografts harvested and processed by the Brazilian National Institute of Traumatology and Orthopedics (INTO) tissue bank according to the donation criteria of the Brazilian National Health Surveillance Agency and the Brazilian National Transplant System. Bone fragments from different processing stages showed no viable cells on histology, an intact extracellular matrix on scanning electron microscopy, and gradual reduction in DNA amount. Different techniques were used to demonstrate the quality of allografts produced by the INTO tissue bank and to establish biological parameters for ensuring the safety and quality of these products. Future studies need to be undertaken to assess and validate the efficacy of the decellularization process in larger bone grafts with diverse architectural configurations.

Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes.

Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the β1-subunit (BK-β1), is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbs2 (Sorbin homology [SoHo] and Src homology 3 [SH3] domains-containing protein 2), is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown. The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and BK-α mRNA, regulating BK channel expression and function in coronary smooth muscle cells. Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α subunits, while both the SH3 and SoHo domains of Sorbs2 interact with BK-β1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-β1 channel current density. Additionally, Sorbs2 is a target gene of the Nrf2 (nuclear factor erythroid-2-related factor 2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary smooth muscle cells. In vivo studies demonstrate that Sorbs2 knockout mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca2+-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice. Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular smooth muscle cells. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.

Resistance to preservatives and the viable but non-culturable state formation of Asaia lannensis in flavored syrups.

Food security is a crucial issue that has caused extensive concern, and the use of food flavors has become prevalent over time. we used the molecular biological techniques, preservative susceptibility testing, viable but non-culturable (VBNC) state induction testing, and a transcriptome analysis to examine the bacterial contamination of favored syrup and identify the causes and develop effective control measures. The results showed that Asaia lannensis WLS1-1 is a microorganism that can spoil food and is a member of the acetic acid bacteria families. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests showed that WLS1-1 was susceptible to potassium sorbate (PS), sodium benzoate (SB), and sodium sulffte (SS) at pH 4.0. It revealed a progressive increase in resistance to these preservatives at increasing pH values. WLS1-1 was resistant to PS, SB and SS with an MIC of 4.0, 2.0 and 0.5 g/L at pH 5.0, respectively. The MIC values exceed the maximum permissible concentrations that can be added. The induction test of the VBNC state demonstrated that WLS1-1 lost its ability to grow after 321 days of PS induction, 229 days of SB induction and 52 days of SS induction combined with low temperature at 4°C. Additionally, laser confocal microscopy and a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay showed that WLS1-1 was still alive after VBNC formation. There were 7.192 ± 0.081 (PS), 5.416 ± 0.149 (SB) and 2.837 ± 0.134 (SS) log10(CFU/mL) of viable bacteria. An analysis of the transcriptome data suggests that Asaia lannensis can enter the VBNC state by regulating oxidative stress and decreasing protein synthesis and metabolic activity in response to low temperature and preservatives. The relative resistance of Asaia lannensis to preservatives and the induction of the VBNC state by preservatives are the primary factors that contribute to the contamination of favored syrup by this bacterium. To our knowledge, this study represents the first evidence of the ability of Asaia lannensis to enter the VBNC state and provides a theoretical foundation for the control of organisms with similar types of activity.

Nicotinic Receptors in Human Chromaffin Cells: Characterization, Functional and Physical Interactions between Subtypes and Regulation.

This review summarizes our research on nicotinic acetylcholine receptors in human chromaffin cells. Limited research has been conducted in this field on human tissue, primarily due to the difficulties associated with obtaining human cells. Receptor subtypes were characterized here using molecular biology and electrophysiological patch-clamp techniques. However, the most significant aspect of this study refers to the cross-talk between the two main subtypes identified in these cells, the α7- and α3β4* subtypes, aiming to avoid their desensitization. The article also reviews other aspects, including the regulation of their expression, function or physical interaction by choline, Ca2+, and tyrosine and serine/threonine phosphatases. Additionally, the influence of sex on their expression is also discussed.

Global investigation of the presence of adenovirus in different types of water resources: a systematic review.

Waterborne viruses such as adenoviruses cause major health problems in the world. Human adenoviruses are the second leading cause of childhood gastroenteritis worldwide. In recent years, the presence of the virus in aquatic resources has been shown in several studies. In this paper, the global presence of adenovirus in different types of water resources are reviewed through studying several surveys conducted in different countries worldwide. We designed one search study to collect the maximum number of related articles to this subject in international databases search engine via relevant keywords. After reviewing the articles, the most relevant ones were selected, and after classification and extracting the required information, they were reported in the tables presented in this study. In general, it was found that the highest rate of the presence of adenoviruses has been reported in sewage water, inlet, and outlet of the treatment plant while the lowest rate of the presence of adenovirus in the dam water. These findings demonstrate that treatment plant system has weakness in removing the adenovirus and are strongly recommended for treatment plants to use new and better protocols to remove this virus. In addition, appropriate diagnostic methods that combines molecular biological technique with infectivity assay should be implemented for detection of adenoviruses in water resources.

Deciphering relationship between depression and microbial molecules based on multi-omics: A case study of Chaigui Granules

ObjectiveTo decipher the antidepression effect of Chaigui Granules (CGKL) from the relationship between depression and microbial molecules based on multi-omics. MethodsMale SD rats were subjected to chronic unpredictable mild stress (CUMS) for seven weeks. The antidepressants CGKL extract and CGKL were administered for the following four weeks. The behavior test and the content of monoamine neurotransmitters were used to evaluate the efficacy of CGKL. The 16S rRNA sequencing, LC-MS technology and molecular biological techniques were used to explore the pharmacological mechanism of CGKL. ResultsCGKL treatment obviously alleviated the depressive behavioral indicators and regulated the content of monoamine neurotransmitters, and presented dose-dependent manner. CGKL could also improve the arginine metabolism disorder of gut microbiota in the jejunum. Meanwhile, the contents of arginine and its metabolites in the serum and hippocampus were regulated to normal levels. Further investigation indicated that the expression of related rate-limiting enzyme genes and proteins in the hippocampus was validated by qRT-PCR and Western blotting. The results showed that the gut microbiota, metabolites, and genes or proteins of rate-limiting enzymes involved in the arginine pathway were significantly regulated by CGKL. ConclusionThe present study demonstrates that CGKL might exert antidepressant effects through regulating arginine metabolism, and its mechanism may be related to modulating the gut microbiota and related metabolic enzyme.

An effective deep learning-based approach for splice site identification in gene expression.

A crucial stage in eukaryote gene expression involves mRNA splicing by a protein assembly known as the spliceosome. This step significantly contributes to generating and properly operating the ultimate gene product. Since non-coding introns disrupt eukaryotic genes, splicing entails the elimination of introns and joining exons to create a functional mRNA molecule. Nevertheless, accurately finding splice sequence sites using various molecular biology techniques and other biological approaches is complex and time-consuming. This paper presents a precise and reliable computer-aided diagnosis (CAD) technique for the rapid and correct identification of splice site sequences. The proposed deep learning-based framework uses long short-term memory (LSTM) to extract distinct patterns from RNA sequences, enabling rapid and accurate point mutation sequence mapping. The proposed network employs one-hot encodings to find sequential patterns that effectively identify splicing sites. A thorough ablation study of traditional machine learning, one-dimensional convolutional neural networks (1D-CNNs), and recurrent neural networks (RNNs) models was conducted. The proposed LSTM network outperformed existing state-of-the-art approaches, improving accuracy by 3% and 2% for the acceptor and donor sites datasets.

Green, yellow, or cyan? Introduction of color change mutations into a green thermostable fluorescent protein and characterization during an introduction to biochemistry lab course.

Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to biochemistry labs utilizing a thermostable green protein (TGP-E) engineered to have unusually high thermostability. This protein allows students to proceed through purification and characterization without the need to keep protein samples on ice. The 5-week lab series begins with an introduction to molecular biology techniques during weeks 1 and 2, where site-directed mutagenesis is used introduce, a single nucleotide change that shifts the fluorescent spectra of TGP-E to either cyan (CTP-E) or yellow (YTP-E). Students identify successful mutagenesis reaction by the color of a small expression sample after induction with IPTG. Next, students purify either the TGP-E (control-typically one group volunteers), YTP-E, or CTP-E protein as a 1-week lab. During the following week's lab, students run SDS-PAGE to verify protein purity, bicinchoninic acid assay to quantify protein yield, and absorbance and fluorescence spectra to characterize their protein's fluorescent character. The final lab in the series investigates the thermostability of YTP-E and CTP-E compared with TGP-E using a fluorescence plate reader. This 5-week series of experiments provide students with experience in several key biochemistry techniques and allows the students to compare properties of mutations. At the end of the course, the students will write a research report and give a short presentation over their results.

Renal Injury During Viral Infections

Renal pathologies following viral infections have become an emerging public health problem in both developed and developing countries. Diagnostic criteria are complex. In most cases, they involve correlation between clinical, biological and histological data, with occasional recourse to molecular biology techniques. Several mechanisms are involved in the pathogenesis of virus-related nephropathy, including virus tropism in the kidney, formation of immune complexes in situ or in the bloodstream, direct cytopathogenic effects, and multiple organ failure. The hepatitis C virus is responsible for three main types of kidney disease: membranoproliferative glomerulnephritis, cryoglobulinemia and membranous nephropathy. Hepatitis B virus is associated with membranous nephropathy, membranoproliferative glomerulnethritis, and IgA nephropathy. HIV (human immunodeficiency virus) infection is associated with several glomerular and tubular kidney damage. HIVAN (HIV-associated nephropathy), a specific entity mainly affecting Africans and African-Americans, presents as a rapidly progressive glomerulonephritis rapidly progressing to the terminal stage. Infections secondary to adenovirus, cytomegalovirus, epstein-barr virus, poliomavirus and coronavirus are often responsible for acute or chronic tubulointerstitial nephritis. Treatment is mainly symptomatic, based on nephroprotection measures, rarely combined with antiviral therapy. Prophylaxis with vacci-nation, when available, remains the best means of preventing viral nephropathy.

Breeding approaches for improvement of temperate fruit crops and nuts - a review

Fruit breeding is an ancient technology with dynamic current technology and an exciting future (Janick and Moore, 1975, 1996). In its broadest sense, fruit breeding refers to the purposeful genetic improvement of fruit crops through various techniques including selection, hybridization, mutation induction, and molecular techniques. Its origins trace to the domestication process in prehistory and antiquity, where useful species were chosen and cultivated, and improved by continuous selection (Janick, 2005, 2011). Much of the world fruit industry is still based on grower selection from chance seedlings as well as mutations (sports) and as a result many fruit species are characterized by a narrow germplasm base. Spontaneous hybridization between wild plants and cultivated clones was critical to the early domestication of fruits. The lengthy juvenile period and the large field space required to grow temperate fruits have imposed major limitations on conventional breeding and various molecular biological techniques have been employed to make fruit breeding easier. (Igarashi et al.2016). Marker-assisted selection (MAS) uses DNA markers to provide an early DNA-based evaluation of genetic performance potential of seedlings, with the aim of improving cost and/or genetic efficiency of seedling selection (Ru, et al., 2015). Transgenic technology has facilitated the development of fruit trees with resistance to fungal or bacterial diseases, improved fruit quality, or root stocks with better rooting or dwarfing ability. In recent years, new technologies for genetic improvement, such as transgrafting, and genome-editing, have emerged. (Mudge, et al. 2009; Jia and Wang 2014). Using these techniques, no foreign genes are present in the final product, and some of them show considerable promise for application to apple breeding. Furthermore, genome editing, by which only the target gene can be accurately modified, is emerging as a novel breeding technology. These new technologies will undoubtedly facilitate fruit breeding, and yield novel and attractive fruit cultivars.

One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far.IMPORTANCEKlebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

BackgroundAs a dual-function metabolite, succinate has emerged in cell function and plays a key signaling role in linking mitochondrial function to other cellular functions. Succinate accumulation in the cytoplasm is commonly associated with hypoxia in the microenvironment and immune cell activation. Extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, which boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate plays an important role in the development of inflammatory diseases. Whether succinate facilitates the progression of endometriosis (EMs), characterized by chronic inflammation and peritoneal adhesion, is worth exploring.ObjectiveWe mimicked the ectopic milieu in vitro and in vivo to evaluate the main source and potential role of succinate in endometriosis. We assessed the molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity. The effect of succinate/SUCNR1 signaling on ectopic endometrial stromal cells (ESCs) was further explored in this study.MethodsIn this study, we used targeted organic acid metabolomics analysis and in vitro assays to assess the potential accumulation of succinate in the peritoneal fluid of EMs patients. We examined its correlation with disease severity, Visual Analogue Scale, and the Endometriosis Fertility Index. Flow cytometry, enzyme linked immunosorbent assay, western blot assay, quantitative real-time PCR, and other molecular biology techniques were used to explore the potential mechanisms.ResultsBy mimicking the ectopic milieu, we constructed an in vitro co-culture system and found that M1 polarized macrophages and that the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarized the macrophages and significantly enhanced the invasive survival of ESCs, and the adhesion to the peritoneum. We further investigated the pathological effects of extracellular succinate in vivo using a xenograft mouse models of endometriosis.ConclusionsSuccinate-SUCNR1 signaling facilitates the creation of inflammatory cells and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanisms underlying succinate accumulation and function will help elucidate the phenotypic mysteries of pain and infertility in EMs.BkiB34NQBDvkGHWZYD1GB2Video

CASE REPORT: POSSIBLE EMERGING OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR HAVANA INFECTION IN NEWBORN KID GOATS

Although salmonellosis has been considered an important problem in the ruminant farm from 10 weeks to 14 months old, there is no information about the disease in goats, especially in 2 to 3-day-old kids, in recent years. The goat farmers complained that the 2 to 3-day-old kids showed clinical signs of depression, weakness, lameness, and diarrhea. The kids might die within one week after the symptoms appeared. The mortality reached 60% (48/80). After many antimicrobial agents were dosed, no better outcome was expected. The farmer sent one kid to the official deputy for further investigation. By helping with bacterial and molecular biological techniques, autopsy, serotyping, and histopathological diagnosis, the kids were infected by Salmonella enterica subsp. enterica serovar Havana strain. After a farm visit, a moldy floor contaminated by 2 to 3[Formula: see text]cm thick feces was identified on the floor. After removing the mold and implementing disinfection, the outbreak was stopped. In conclusion, this report proves that salmonellosis has recently been identified in goat farms, especially in kids, within one week. Moreover, good hygiene and keeping feces out of barns are essential to preventing salmonellosis.

Ghrelin/GHSR signaling in the lateral septum ameliorates chronic stress-induced depressive-like behaviors

Ghrelin is a gastrointestinal hormone on feeding and metabolism regulation, and acts through its receptor-growth hormone secretagogue receptor (GHSR), which is widely distributed throughout the central nervous system. Recent studies have suggested that ghrelin plays an important role in the regulation of depression, but the underlying mechanisms remain uncertain. Lateral septum (LS) is a critical brain region in modulating depression. Therefore, we investigated the role of ghrelin/GHSR signaling in the LS on the depressive-like behaviors of mice under conditions of chronic stress by using behavioral tests, neuropharmacology, and molecular biology techniques. We found that infusion of ghrelin into the LS produced antidepressant-like responses in mice. Activation of LS GABAergic neurons was involved in the antidepressant effect of ghrelin. Importantly, GHSR was highly expressed and distributed in the LS neurons. Blockade of GHSR in the LS reversed the ghrelin-induced antidepressant-like effects. Molecular knockdown of GHSR in the LS induced depressive-like symptoms in mice. Furthermore, administration of ghrelin into the LS alleviated depressive-like behaviors induced by chronic social defeat stress (CSDS). Consistent with the neuropharmacological results, overexpression of GHSR in the LS reversed CSDS-induced depressive-like behaviors. Our findings clarify a key role for ghrelin/GHSR signaling in the regulation of chronic stress-induced depressive-like behaviors, which could provide new strategies for the treatment of depression.

Rivaroxaban down-regulates pyroptosis and the TLR4/NF-κB/NLRP3 signaling pathway to promote flap survival

BackgroundFlap placement remains the primary method for wound repair, but postoperative ischemic flap necrosis is of major concern. This study explored whether rivaroxaban, a factor Xa inhibitor, enhanced flap survival. MethodsThirty-six rats were randomly divided into control, low-dose rivaroxaban (3 mg/kg/day), and high-dose rivaroxaban (7 mg/kg/day) groups. On postoperative day 7, the flap survival rate was analyzed and the average survival area calculated. After the rats were euthanized, immunological and molecular biological techniques were employed to assess vascular regeneration, pyroptosis, and inflammation. ResultsRivaroxaban upregulated VEGF expression, in turn enhancing angiogenesis, and it downregulated IL-1β, IL-6, and TNF-α expression, thereby mitigating inflammation. The drug also suppressed TLR4, NF-κB p65, NLRP3, caspase-1, and IL-18 syntheses, thus inhibiting pyroptosis. ConclusionsRivaroxaban enhanced random flap survival by down-regulating the TLR4/NF-κB/NLRP3 signaling pathway to suppress pyroptosis, promoting vascular regeneration and inhibiting inflammation.