Polymerase Chain Reaction Analysis Research Articles

Identification of the Gossypium hirsutum SDG Gene Family and Functional Study of GhSDG59 in Response to Drought Stress.

SET-domain group histone methyltransferases (SDGs) are known to play crucial roles in plant responses to abiotic stress. However, their specific function in cotton's response to drought stress has not been well understood. This study conducted a comprehensive analysis of the SDG gene family in Gossypium hirsutum, identifying a total of 82 SDG genes. An evolutionary analysis revealed that the SDG gene family can be divided into eight subgroups. The expression analysis shows that some GhSDG genes are preferentially expressed in specific tissues, indicating their involvement in cotton growth and development. The transcription level of some GhSDG genes is induced by PEG, with GhSDG59 showing significant upregulation upon polyethylene glycol (PEG) treatment. Quantitative polymerase chain reaction (qPCR) analysis showed that the accumulation of transcripts of the GhSDG59 gene was significantly upregulated under drought stress. Further functional studies using virus-induced gene silencing (VIGS) revealed that silencing GhSDG59 reduced cotton tolerance to drought stress. Under drought conditions, the proline content, superoxide dismutase (SOD) and peroxidase (POD) enzyme activities in the GhSDG59-silenced plants were significantly lower than in the control plants, while the malondialdehyde (MDA) content was significantly higher. Transcriptome sequencing showed that silencing the GhSDG59 gene led to significant changes in the expression levels of 1156 genes. The KEGG enrichment analysis revealed that these differentially expressed genes (DEGs) were mainly enriched in the carbon metabolism and the starch and sucrose metabolism pathways. The functional annotation analysis identified known drought-responsive genes, such as ERF, CIPK, and WRKY, among these DEGs. This indicates that GhSDG59 is involved in the drought-stress response in cotton by affecting the expression of genes related to the carbon metabolism and the starch and sucrose metabolism pathways, as well as known drought-responsive genes. This analysis provides valuable information for the functional genomic study of SDGs and highlights potential beneficial genes for genetic improvement and breeding in cotton.

Identification and characterization of PAL genes involved in the regulation of stem development in Saccharum spontaneumL.

BackgroundSaccharum spontaneumL. is a closely related species of sugarcane and has become an important genetic component of modern sugarcane cultivars. Stem development is one of the important factors for affecting the yield, while the molecular mechanism of stem development remains poorly understanding in S. spontaneum. Phenylalanine ammonia-lyase (PAL) is a vital component of both primary and secondary metabolism, contributing significantly to plant growth, development and stress defense. However, the current knowledge about PAL genes in S. spontaneum is still limited. Thus, identification and characterization of the PAL genes by transcriptome analysis will provide a theoretical basis for further investigation of the function of PAL gene in sugarcane.ResultsIn this study, 42 of PAL genes were identified, including 26 SsPAL genes from S. spontaneum, 8 ShPAL genes from sugarcane cultivar R570, and 8 SbPAL genes from sorghum. Phylogenetic analysis showed that SsPAL genes were divided into three groups, potentially influenced by long-term natural selection. Notably, 20 SsPAL genes were existed on chromosomes 4 and 5, indicating that they are highly conserved in S. spontaneum. This conservation is likely a result of the prevalence of whole-genome replications within this gene family. The upstream sequence of PAL genes were found to contain conserved cis-acting elements such as G-box and SP1, GT1-motif and CAT-box, which collectively regulate the growth and development of S. spontaneum. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that SsPAL genes of stem had a significantly upregulated than that of leaves, suggesting that they may promote the stem growth and development, particularly in the + 6 stem (The sixth cane stalk from the top to down) during the growth stage.ConclusionsThe results of this study revealed the molecular characteristics of SsPAL genes and indicated that they may play a vital role in stem growth and development of S. spontaneum. Altogether, our findings will promote the understanding of the molecular mechanism of S. spontaneum stem development, and also contribute to the sugarcane genetic improving.

Adenosine washing improves the retention rate of fat grafts under conditions of obesity

Background Fat grafting is a commonly employed aesthetic procedure for contour enhancement. However, outcome prediction is challenging due to the complex regeneration and remodeling processes involved. We investigated whether adenosine improves engraftment and fat graft survival under conditions of obesity.Methods Fat was harvested from mice fed a high-fat diet. This fat was washed with either Krebs-Ringer bicarbonate HEPES buffer (the vehicle group) or a buffer containing 500 nM adenosine (the adenosine wash group). Subsequently, the fat was transplanted into normal mice at 0.2 mL per mouse. In both groups, 50% of the mice were sacrificed at 1 week and the remainder at 4 weeks post-transplantation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was conducted during week 1. In week 4, micro-computed tomography, immunofluorescence staining, and RT-qPCR were performed. A sample of the initially harvested fat was set aside for lipolysis assay.Results Adenosine washing improved fat graft retention volumes by up to 50%. One week post-transplantation, the expression of adipogenic and angiogenic genes was found to be upregulated in the adenosine wash group. After 4 weeks, immunofluorescence staining revealed greater adipocyte integrity and an increased number of vessels. Furthermore, adenosine appeared to modulate inflammation by stabilizing the lipolysis rate.Conclusions Adenosine washing increased the fat graft survival rate under conditions of obesity. Clinically, this suggests a simple, cost-effective adjuvant method for improving fat graft survival in individuals with obesity. Further research is warranted to elucidate the underlying mechanisms and explore the applicability of this technique for autologous transplantation.

Predictive value of miR-7110-5p and miR-223-3p as biomarkers for sepsis secondary to pneumonia.

Investigating the secondary sepsis of pneumonia is of great significance for rapid diagnosis and early treatment of sepsis. This study aimed to investigate the predictive value of micro ribonucleic acids (miRNA) 7110-5p and miR-223-3p in sepsis secondary to pneumonia. A miRNA microarray was used to analyze the differences in miRNA expression between patients with pneumonia and those with sepsis secondary to pneumonia. The study included a total of 50 patients with pneumonia and 42 patients with sepsis secondary to pneumonia. Quantitative polymerase chain reaction analysis was conducted to measure the circulating miRNA expression levels in patients and assess their correlations with clinical characteristics and prognosis. In this study, nine miRNAs - hsa-miR-4689-5p, hsa-miR-4621-5p, hsa-miR-6740-5p, hsa-miR-7110-5p, hsa-miR-765, hsa-miR-940, hsa-miR-213-5p, hsa-miR-223-3p, and hsa-miR-122 - met the screening criteria of having a fold change ⩾ 2 or < 0.5; p< 0.01 indicated significant differences in the results. The expression levels of miR-7110-5p and miR-223-3p differed between the two patient groups, being up-regulated in the plasma of patients with sepsis secondary to pneumonia. miR-7110-5p and miR-223-3p showed higher expression levels in both patients with pneumonia and sepsis compared to healthy controls. Moreover, the receiver operating characteristic curve revealed that the areas under the curve for predicting pneumonia using miR-7110-5p were 0.781 while those for predicting sepsis secondary to pneumonia were 0.862. For miR-223-3p, the corresponding values for predicting pneumonia and sepsis secondary to pneumonia were 0.879 and 0.924, respectively. However, there were no significant differences in the levels of miR-7110-5p and miR-223-3p between the plasma of survived and deceased patients with sepsis. MiR-7110-5p and miR-223-3p have the potential to serve as biological indicators for predicting sepsis secondary to pneumonia.

Magnetostriction enhanced self-powered nanofiber sheet as cardiac patch with magnetoelectric synergistic effect on actuating Na+ k+-ATPase

Cardiac tissue engineering has great promise in the treatment of heart disease. Aiming to develop the novel magnetoelectric cardiac patch for improving the non-conduction of injured myocardium after myocardial infarction, the self-powered nanofiber sheet was electro-spun by doping magnetostrictive CoFe2O4 in piezoelectric Poly (vinylidene fluoride) (PVDF). The piezoelectric ratios increased several folds with the doping of CoFe2O4 due to the increased β phase and crystallinity of CoFe2O4-PVDF confirmed by experimental data. Molecular Dynamics and Finite Element Simulation demonstrated that non-β phase transformed to β phase of PVDF with doping CoFe2O4 and the deformation of CoFe2O4 enhanced the piezoelectric effect of PVDF, respectively. The proliferation and spread area of Doxorubicin-induced oxidative stress injured cardiomyocytes, H9c2 cells, were significantly improved under magnetoelectric cue compared with those without magnetoelectric cue when co-cultured with CoFe2O4-PVDF. Importantly, a new mechanism pathway of magnetoelectric cue on cardiomyocytes has been found, including up-regulating of steroid hormone biosynthesis, enhancing of sodium–potassium enzyme, and increasing of intracellular Ca2+, which would trigger the cardiomyocyte contraction to overcome the non-conduction of injured cardiomyocytes by RNA-seq transcriptome sequencing, Polymerase Chain Reaction and downstream expression analysis. Hence, this CoFe2O4-PVDF composite nanofiber sheet would be potentially used as the magnetoelectric cardiac patch to treat injured myocardium after myocardial infarction clinical.

Exploring the effects of ursodeoxycholic acid-probiotic combination on Lactobacillus and liver enzyme levels for cholestasis management: An experimental study

Background: Cholestasis arises from compromised bile secretion due to hepatocyte dysfunction, leading to liver impairment. Available treatments show limited efficacy, with ursodeoxycholic acid (UDCA) being a primary option. Cholestatic conditions influence gut microbiota; therefore, probiotic therapy emerges as a potential approach. Objective: This investigation aimed to evaluate the impact of combined administration of probiotics and UDCA on Lactobacillus levels, as well as the levels of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in rats with restricted common bile ducts .Methods: A total of 35 male Sprague Dawley rats were randomly assigned to 7 groups, each comprising 5 members: K1 (healthy control), K2 (negative control with cholestasis), K3 (positive control with cholestasis given UDCA), K4 (cholestasis given 36 mg probiotics), K5 (cholestasis given 18 mg probiotics and UDCA), K6 (cholestasis given 36 mg probiotics and UDCA), and K7 (cholestasis given 54 mg probiotics and UDCA). The treatment duration was 21 days, during which blood samples were collected for AST and ALT analysis. Lactobacillus count was determined by quantitative polymerase chain reaction (PCR) analysis of fecal samples.Results: When UDCA and probiotics were given together in three different dosages, the Lactobacillus count significantly increased (p&lt;0.05) compared to the other groups. Furthermore, compared to the other treatment groups, the UDCA-probiotic combination group exhibited noticeably lower AST and ALT values.Conclusion: Combining UDCA and probiotics elevated Lactobacillus count and decreased AST and ALT levels in cases of cholestasis more effectively than single therapy.

Identification of Periostin Positive Cell Population during the Long-Term Culture of Mouse Tendon-Derived Cells in Late Passage.

Tendon-derived cells exhibit phenotypic changes and gradually lose their proliferative capacity during serial passages in vitro. This study aimed to characterize the changes in the growth and stem cell characteristics of tendon-derived cells over a long-term culture. Mouse flexor digitorum profundus tendon-derived cells were obtained by enzymatic digestion and seeded at an initial density of 5,000/cm2. Cells were characterized by morphology, growth, senescence staining, trilineage differentiation assays, real-time polymerase chain reaction, immunocytochemistry, flow cytometry, and RNA sequencing analysis. Tendon-derived cells underwent a proliferative stage in the first three passages, followed by a gradual senescence. However, a novel cell population expressing periostin (Postn+) emerged during the long-term culture from passages 5-8, which possessed a high rate of proliferation without significant senescence over successive passages. Compared to early passage cells, Postn+ cells exhibited enhanced osteogenic differentiation potential and attenuated chondrogenic differentiation potential, decreased expression of SSEA-1, Oct3/4, tenomodulin, scleraxis, CD90.2, CD73, CD105, Sca-1, and CD44, and increased expression of collagen III and CD34. RNA-sequencing analysis of Postn+ and early passage cells identified 908 differentially expressed genes, with extracellular matrix-receptor interaction and focal adhesion as the top pathways, and integrins as hub genes. This study highlights the dynamics of tendon-derived cells during serial passages. We identify a Postn+ cell population during long-term culture in late passages, with high proliferative ability and prominent osteogenic differentiation potential. Further investigations are needed to elucidate the origin and potential applications of Postn+ tendon-derived cells.

Effects of High-Dose Cyclophosphamide on Ultrastructural Changes and Gene Expression Profiles in the Cardiomyocytes of C57BL/6J Mice.

The pathogenesis of cyclophosphamide (CY)-induced cardiotoxicity remains unknown, and methods for its prevention have not been established. To elucidate the acute structural changes that take place in myocardial cells and the pathways leading to myocardial damage under high-dose CY treatments, we performed detailed pathological analyses of myocardial tissue obtained from C57BL/6J mice subjected to a high-dose CY treatment. Additionally, we analysed the genome-wide cardiomyocyte expression profiles of mice subjected to the high-dose CY treatment. Treatment with CY (400 mg/kg/day intraperitoneally for two days) caused marked ultrastructural aberrations, as observed using electron microscopy, although these aberrations could not be observed using optical microscopy. The expansion of the transverse tubule and sarcoplasmic reticulum, turbulence in myocardial fibre travel, and a low contractile protein density were observed in cardiomyocytes. The high-dose CY treatment altered the cardiomyocyte expression of 1210 genes (with 675 genes upregulated and 535 genes downregulated) associated with cell-cell junctions, inflammatory responses, cardiomyopathy, and cardiac muscle function, as determined using microarray analysis (|Z-score| > 2.0). The expression of functionally important genes related to myocardial contraction and the regulation of calcium ion levels was validated using real-time polymerase chain reaction analysis. The results of the gene expression profiling, functional annotation clustering, and Kyoto Encyclopedia of Genes and Genomes pathway functional-classification analysis suggest that CY-induced cardiotoxicity is associated with the disruption of the Ca2+ signalling pathway.

Echinococcus granulosus Sensu Stricto and Echinococcus multilocularis in a Gray Wolf (Canis lupus) in Turkey: Further Evidence for Increased Risk of Alveolar Echinococcosis in Urban Areas.

The aim of this study was to identify Echinococcus species by morphological and molecular means. A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted. The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively. This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted.

PD-L1 Expression in Nonbacterial Chronic Cystitis and Bladder Cancer.

The objective was to assess PD-L1 expression in nonbacterial chronic cystitis (NCC) and bladder cancer (BC). The present study included 20 NCC and 20 BC patients. The degree of inflammation of the bladder wall was assessed on slides stained with H&E. Viral pathogens (herpes simplex virus, Epstein-Barr virus, cytomegalovirus, and high-risk HPVs) were detected using real-time polymerase chain reaction analyses of the bladder specimens. Immunohistochemistry was performed to assess the PD-L1 expression in bladder tissue. Expression of PD-L1 was detected in 40% of NCC patients and 85% of BC patients. Viral pathogens were found in 50% of NCC patients and 60% of BC patients, with EBV being the most common. In NCC patients the immune cell score correlated strongly with the degree of inflammatory infiltration of the bladder wall (r = 0.867, p < 0.001), the presence of lymphoid aggregates in the submucosa (r = 0.804, p < 0.001), koilocytosis (r = 0.620, p = 0.004), and the presence of viral pathogens (r = 0.784, p < 0.001). In BC patients the immune cell score correlated with the degree of inflammatory infiltration of the bladder wall (r = 0.534, p = 0.015) and the presence of viral pathogens (r = 0.626, p = 0.003), but not with the presence of lymphoid aggregates in the submucosa (r = 0.083, p = 0.729), and koilocytosis (r = 0.366, p = 0.112). Expression of PD-L1 was detected in a cohort of NCC patients, although the PD-L1 positivity rate was lower than that in BC. Our results demonstrate that the degree of PD-L1 expression in bladder tissue is associated with the presence of viral infections and with the degree of inflammatory infiltration of the bladder wall in both NCC and BC.

Antibacterial potential of lactic acid bacteria isolated from raw cow milk in Sylhet district, Bangladesh: A molecular approach.

The most prevalent probiotic bacterium employed in the food industry is Lactobacillus because it can produce metabolites with antibacterial capabilities and exhibits hostility towards infections and microorganisms that cause spoilage. This study set out to identify naturally occurring Lactobacillus and plantaricin (pln EF) coding genes in raw cow milk and to assess the antibacterial potency of isolated Lactobacillus isolates. Following enrichment in De Man, Rogosa and Sharpe (MRS) broth, single colonies were isolated, and pure colonies were obtained by streaking on MRS agar. The 16S rRNA gene was amplified using polymerase chain reaction (PCR) to confirm the cultural positivity of all isolates. Additionally, the presence of plantaricin was verified by targeting the pln EF gene through PCR. Out of the 166 raw milk specimens acquired from cows, 153 (91.17%; CI: 86.98-95.76) were identified as positive for Lactobacillus through both culture and biochemical screening. Subsequently, 121 (72.89%; CI: 65.46-79.49) of the isolates were affirmed to harbour Lactobacillus through PCR analysis. Within this subset, 6 isolates (4.96%; CI: 1.84-10.48) were found to possess the plnEF gene. When exposed to Lactobacillus isolates, Salmonella Typhimurium and Salmonella enterica displayed an average maximum zone of inhibition with a diameter measuring 24mm. In contrast, Escherichia coli exhibited an average minimum zone of inhibition, featuring a diameter of 11mm. Additionally, the Lactobacillus isolates demonstrated inhibitory zones against Staphylococcus aureus, Klebsiella pneumoniae and Klebsiella oxytoca, measuring 14, 22 and 19mm, respectively. Lactic acid bacteria, particularly Lactobacilli, are plentiful in cow milk and possess broad-spectrum antibacterial properties.

Genome-wide identification and analysis of DEAD-box RNA helicases in Gossypium hirsutum

DEAD-box RNA helicases, a prominent subfamily within the RNA helicase superfamily 2 (SF2), play crucial roles in the growth, development, and abiotic stress responses of plants. This study identifies 146 DEAD-box RNA helicase genes (GhDEADs) and categorizes them into four Clades (Clade A-D) through phylogenetic analysis. Promoter analysis reveals cis-acting elements linked to plant responses to light, methyl jasmonate (MeJA), abscisic acid (ABA), low temperature, and drought. RNA-seq data demonstrate that Clade C GhDEADs exhibit elevated and ubiquitous expression across different tissues, validating their connection to leaf development through real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Notably, over half of GhDEADs display up-regulation in the leaves of virus-induced gene silencing (VIGS) plants of GhVIR-A/D (members of m6A methyltransferase complex, which regulate leaf morphogenesis). In conclusion, this study offers a comprehensive insight into GhDEADs, emphasizing their potential involvement in leaf development.

Biological significance of METTL5 in atherosclerosis: comprehensive analysis of single-cell and bulk RNA sequencing data.

N6-methyladenosine (m6A) methylation is involved in the pathogenesis of atherosclerosis (AS). Limited studies have examined the role of the m6A methyltransferase METTL5 in AS pathogenesis. This study subjected the AS dataset to differential analysis and weighted gene co-expression network analysis to identify m6A methylation-associated differentially expressed genes (DEGs). Next, the m6A methylation-related DEGs were subjected to consensus clustering to categorize AS samples into distinct m6A subtypes. Single-cell RNA sequencing (scRNA-seq) analysis was performed to investigate the proportions of each cell type in AS and adjacent healthy tissues and the expression levels of key m6A regulators. The mRNA expression levels of METTL5 in AS and healthy tissues were determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. AS samples were classified into two subtypes based on a five-m6A regulator-based model. scRNA-seq analysis revealed that the proportions of T cells, monocytes, and macrophages in AS tissues were significantly higher than those in healthy tissues. Additionally, the levels of m6A methylation were significantly different between AS and healthy tissues. METTL5 expression was upregulated in macrophages, smooth muscle cells (SMCs), and endothelial cells (ECs). qRT-PCR analysis demonstrated that the METTL5 mRNA level in AS tissues was downregulated when compared with that in healthy tissues. METTL5 is a potential diagnostic marker for AS subtypes. Macrophages, SMCs, and ECs, which exhibit METTL5 upregulation, may modulate AS progression by regulating m6A methylation levels.

Impact of donor &lt;i&gt;CYP3A5&lt;/i&gt; genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients

Background. In the paediatric liver transplant programme in Johannesburg, South Africa (SA), tacrolimus is the calcineurin inhibitor of choice, comprising an essential component of the immunosuppression regimen. It is characterised by a narrow therapeutic index and wide interpatient variability, necessitating therapeutic drug monitoring of whole-blood concentrations. Pharmacogenetic research, although not representative of SA population groups, suggests that single-nucleotide polymorphisms within the cytochrome P450 3A5 (CYP3A5) gene contribute to the variability in tacrolimus dosing requirements. The rs776746 polymorphism, CYP3A5*3, results in a splice defect and a non-functional enzyme. Clinically, to reach the same tacrolimus concentration-to-dose ratio (CDR), expressors (CYP3A5*1/*1 and *1/*3) require a higher tacrolimus dose than non-expressors (*3/*3). Objectives. To compare the pharmacokinetics of tacrolimus in paediatric liver transplant recipients with their donors’ CYP3A5 genotypes, considering both donor and recipient characteristics. Methods. Blood samples from 46 living liver donors were collected, their genomic DNA was extracted, and their CYP3A5 genotype was established (polymerase chain reaction and restriction fragment length polymorphism analysis, validated by Sanger sequencing). The relationship of donor and recipient characteristics with the mean tacrolimus CDR was analysed using a general linear model. Non- confounding significant variables were included in a multiple regression model. Results. The study showed that all expressor donors genotyped as CYP3A5*1/*1 were of black African self-reported race and ethnicity. During the first 15 days post-transplant, we found that children who received grafts from donor CYP3A5 expressors (CYP3A5*1/*1 and *1/*3) had significantly lower mean tacrolimus CDRs compared with those who received grafts from donor CYP3A5 non-expressors (*3/*3); the recipients of CYP3A5 expressor grafts therefore require higher doses of oral tacrolimus to achieve the same therapeutic target range. In addition, graft-to-recipient weight ratio and the CYP3A5 donor genotypes were independent factors that significantly (p&lt;0.05) affected mean tacrolimus CDRs in recipients. Conclusion. In this study, we showed that all CYP3A5*1 homozygote donors were of black African self-reported race and ethnicity, and tacrolimus CDRs in paediatric living-donor liver transplant recipients were significantly affected by donor graft size and donor CYP3A5 genotypes. Information from this study may inform the development of an Afrocentric tacrolimus precision-medicine algorithm to optimise recipient safety and graft outcomes.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&amp;D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p &amp;lt; 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p &amp;lt; 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p &amp;lt; 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Detection of Macrolide-Resistant Streptococcus pneumoniae Genes and Its Clinical Outcomes in a Tertiary Teaching Hospital in Malaysia.

Streptococcus pneumoniae is one of the leading causes of mortality and morbidity worldwide. The dramatic increase in in-vitro resistance of antimicrobial agents, particularly beta-lactams and macrolides, makes pneumococcal infections difficult to treat. The aim of this study was to describe the drug resistance rate, assess the prevalence of macrolide-resistant genes and review the clinical complications of pneumococcal infections among patients presented to Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia. This is a descriptive cross-sectional study. All S. pneumoniae isolates collected from clinical specimens within a 1-year period were subjected to selected antimicrobial susceptibility testing using E-test strips. Polymerase chain reaction (PCR) analysis was conducted to detect macrolide-resistant determinants. The patient's clinical data were obtained from clinical notes. A total of 113 patients with a positive growth of S. pneumoniae were included in the study. The most common predisposing factors among them were bronchopulmonary diseases (15.9%). The penicillin-resistant rate was 7.1%, with minimal inhibitory concentration (MIC) ranging between 0.012 μg/mL and >32 μg/mL, and the erythromycin-resistant rate was 26.5%, with a MIC range of 0.03 μg/mL-> 256 μg/mL. Most of the erythromycin-resistant isolates were found to have the mef(A) gene (50.4%) and the erm(B) gene (20%); 16.7% had a combination of genes mef(A) and erm(B), and 13.3% had none of the two genes. Community-acquired pneumonia is the predominant type of pneumococcal infection. There was no significant association between the presence of macrolide resistance determinants and mortality (P = 0.837) or complications (P > 0.999 for empyema and cardiac complication; P = 0.135 for subdural abscess). The majority of erythromycin-resistant isolates were found to have the mef(A) gene, followed by the erm(B) gene and a combination of genes mef(A) and erm(B).

Targeting genomic receptors in voided urine for confirmation of benign prostatic hyperplasia

AbstractObjectivesThe objective of this study is to validate a hypothesis that a non‐invasive optical imaging assay targeting genomic VPAC receptors on malignant cells shed in voided urine will represent either benign prostatic hyperplasia (BPH) or prostatic cancer (PCa). Risk for BPH in men 50–70 years old is 50–70% and PCa is 17%. BPH and PCa can coexist in 20% of men with BPH. Most commonly practiced methods to diagnose BPH do not distinguish BPH from PCa.Patients (or Materials) and MethodsMales with BPH (N = 97, 60.8 ± 6.3 years, prostate‐specific antigen 0.7 ± 0.4 ng/mL) and without oncologic disease (N = 35, 63.4 ± 5.8 years, prostate‐specific antigen &lt; 1.5 ng/mL) signed informed consent form and provided voided urine. Urine was cytocentrifuged, cells collected on glass slide, fixed, treated with VPAC specific fluorophore TP4303 (Kd 3.1 × 10−8M), washed, incubated with DAPI and observed using a fluorescence microscope. Cells with no VPAC did not fluoresce (BPH) and those with VPAC had red‐orange fluorescence (PCa). Real‐time polymerase chain reaction analyses for VPAC and NKX3.1 assay for cell origin were performed.ResultsEighty‐seven subjects were negative for VPAC expression. Positive VPAC expression was noted in 10 subjects. Patient chart review for clinical data on these 10 VPAC positive subjects showed five had nephrolithiasis, three had renal cysts, one had prostatitis and one was being treated with finasteride. Real‐time polymerase chain reaction analysis‐VPAC expressions for 7 normal and 12 BPH subjects were 1.31 ± 1.26 and 0.94 ± 0.89, respectively (P = 0.46). NKX3.1 showed cells of prostate origin for finasteride‐treated patient. Specificity for VPAC urine assay for excluding prostate cancer in this BPH cohort was 88.5%, positive predictive value 0.00% and negative predictive value 100%.ConclusionVPAC assay may contribute extensively for BPH diagnosis and warrant continued investigation.

Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis.

Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury, and finally leads to liver cirrhosis or even hepatocellular carcinoma. The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells (HSCs), which can transdifferentiate into myofibroblasts to produce an excess of the extracellular matrix (ECM). Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis. Therefore, activated hepatic stellate cells (aHSCs), the principal ECM producing cells in the injured liver, are a promising therapeutic target for the treatment of hepatic fibrosis. To explore the effect of taurine on aHSC proliferation and the mechanisms involved. Human HSCs (LX-2) were randomly divided into five groups: Normal control group, platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) treated group, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 mmol/L, respectively) with PDGF-BB (20 ng/mL) treated group. Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs. Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species (ROS), malondialdehyde, glutathione, and iron concentration. Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62. Taurine promoted the death of aHSCs and reduced the deposition of the ECM. Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation, by decreasing autophagosome formation, downregulating LC3B and Beclin 1 protein expression, and upregulating p62 protein expression. Meanwhile, treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Furthermore, bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4, exhibiting the best average binding affinity of -20.99 kcal/mol. Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

Genome-Wide Analysis of the HSF Gene Family Reveals Its Role in Astragalus mongholicus under Different Light Conditions.

Astragalus mongholicus is a traditional Chinese medicine (TCM) with important medicinal value and is widely used worldwide. Heat shock (HSF) transcription factors are among the most important transcription factors in plants and are involved in the transcriptional regulation of various stress responses, including drought, salinity, oxidation, osmotic stress, and high light, thereby regulating growth and developmental processes. However, the HFS gene family has not yet been identified in A. mongholicus, and little is known regarding the role of HSF genes in A. mongholicus. This study is based on whole genome analysis of A. mongholicus, identifying a total of 22 AmHSF genes and analyzing their physicochemical properties. Divided into three subgroups based on phylogenetic and gene structural characteristics, including subgroup A (12), subgroup B (9), and subgroup C (1), they are randomly distributed in 8 out of 9 chromosomes of A. mongholicus. In addition, transcriptome data and quantitative real time polymerase chain reaction (qRT-PCR) analyses revealed that AmHSF was differentially transcribed in different tissues, suggesting that AmHSF gene functions may differ. Red and blue light treatment significantly affected the expression of 20 HSF genes in soilless cultivation of A. mongholicus seedlings. AmHSF3, AmHSF3, AmHSF11, AmHSF12, and AmHSF14 were upregulated after red light and blue light treatment, and these genes all had light-corresponding cis-elements, suggesting that AmHSF genes play an important role in the light response of A. mongholicus. Although the responses of soilless-cultivated A. mongholicus seedlings to red and blue light may not represent the mature stage, our results provide fundamental research for future elucidation of the regulatory mechanisms of HSF in the growth and development of A. mongholicus and its response to different light conditions.

Identification of metabolism-related key genes as potential biomarkers for pathogenesis of immune thrombocytopenia

Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction. A biomarker is a biological entity that contributes to disease pathogenesis and reflects disease activity. Metabolic alterations are reported to be associated with the occurrence of various diseases. As metabolic biomarkers for ITP have not been identified. This study aimed to identify metabolism-related differentially expressed genes as potential biomarkers for pathogenesis of ITP using bioinformatic analyses.The microarray expression data of the peripheral blood mononuclear cells were downloaded from the Gene Expression Omnibus database (GSE112278 download link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112278). Key module genes were intersected with metabolism-related genes to obtain the metabolism-related key candidate genes. The hub genes were screened based on the degree function in the coytoscape sofware. The key ITP-related genes were subjected to functional enrichment analysis. Immune infiltration analysis was performed using a single-sample gene set enrichment analysis algorithm to evaluate the differential infiltration levels of immune cell types between ITP patient and control. Molecular subtypes were identified based on the expression of hub genes. The expression of hub genes in the ITP patients was validated using quantitative real-time polymerase chain reaction analysis. This study identified five hub genes (ADH4, CYP7A1, CYP1A2, CYP8B1, and NR1H4), which were be associated with the pathogenesis of ITP, and two molecular subtypes of ITP. Among these hub genes, CYP7A1 and CYP8B1 involved in cholesterol metabolism,were further verified in clinical samples.