Genome-wide identification and evolutionary analysis of the NRAMP gene family in the AC genomes of Brassica species.

BACKGROUND: Brassica napus, a hybrid resulting from the crossing of Brassica rapa and Brassica oleracea, is one of the most important oil crops. Despite its significance, B. napus productivity faces substantial challenges due to heavy metal stress, especially in response to cadmium (Cd), which poses a significant threat among heavy metals. Natural resistance-associated macrophage proteins (NRAMPs) play pivotal roles in Cd uptake and transport within plants. However, our understanding of the role of BnNRAMPs in B. napus is limited. Thus, this study aimed to conduct genome-wide identification and bioinformatics analysis of three Brassica species: B. napus, B. rapa, and B. oleracea. RESULTS: A total of 37 NRAMPs were identified across the three Brassica species and classified into two distinct subfamilies based on evolutionary relationships. Conservative motif analysis revealed that motif 6 and motif 8 might significantly contribute to the differentiation between subfamily I and subfamily II within Brassica species. Evolutionary analyses and chromosome mapping revealed a reduction in the NRAMP gene family during B. napus evolutionary history, resulting in the loss of an orthologous gene derived from BoNRAMP3.2. Cis-acting element analysis suggested potential regulation of the NRAMP gene family by specific plant hormones, such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, gene expression pattern analyses under hormonal or stress treatments indicated limited responsiveness of the NRAMP gene family to these treatments, warranting further experimental validation. Under Cd stress in B. napus, expression pattern analysis of the NRAMP gene family revealed a decrease in the expression levels of most BnNRAMP genes with increasing Cd concentrations. Notably, BnNRAMP5.1/5.2 exhibited a unique response pattern, being stimulated at low Cd concentrations and inhibited at high Cd concentrations, suggesting potential response mechanisms distinct from those of other NRAMP genes. CONCLUSIONS: In summary, this study indicates complex molecular dynamics within the NRAMP gene family under Cd stress, suggesting potential applications in enhancing plant resilience, particularly against Cd. The findings also offer valuable insights for further understanding the functionality and regulatory mechanisms of the NRAMP gene family.

Ultrasensitive colorimetric detection of Staphylococcus aureus using wheat germ agglutinin and IgY as a dual-recognition strategy.

A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.

HDAC1 participates in polycystic ovary syndrome through histone modification by regulating H19/miR-29a-3p/NLRP3-mediated granulosa cell pyroptosis.

Histone deacetylase 1 (HDAC1) is known to participate in the molecular etiology of polycystic ovary syndrome (PCOS). However, its role in granulosa cell (GC) pyroptosis remains unclear. This study sought to investigate the mechanism of HDAC1 in PCOS-induced GC pyroptosis through histone modification. Clinical serum samples and the general data of study subjects were collected. PCOS mouse models were established using dehydroepiandrosterone and cell models were established in HGL5 cells using dihydrotestosterone. Expressions of HDAC1, H19, miR-29a-3p, and NLR family pyrin domain containing 3 (NLRP3) and pyroptosis-related proteins and levels of hormones and inflammatory cytokines were determined. Ovarian damage was observed by hematoxylin-eosin staining. Functional rescue experiments were conducted to verify the role of H19/miR-29a-3p/NLRP3 in GC pyroptosis in PCOS. HDAC1 and miR-29a-3p were downregulated whereas H19 and NLRP3 were upregulated in PCOS. HDAC1 upregulation attenuated ovarian damage and hormone disorders in PCOS mice and suppressed pyroptosis in ovarian tissues and HGL5 cells. HDAC1 inhibited H3K9ac on the H19 promoter and H19 competitively bound to miR-29a-3p to improve NLRP3 expression. Overexpressed H19 or NLRP3 or inhibited miR-29a-3p reversed the inhibition of GC pyroptosis by HDAC1 upregulation. Overall, HDAC1 suppressed GC pyroptosis in PCOS through deacetylation to regulate the H19/miR-29a-3p/NLRP3 axis.

Discovery of novel serum metabolic biomarkers in patients with polycystic ovarian syndrome and premature ovarian failure.

Several widely recognized metabolites play a role in regulating the pathophysiological processes of various disorders. Nonetheless, the lack of effective biomarkers for the early diagnosis of polycystic ovarian syndrome (PCOS) and premature ovarian failure (POF) has led to the discovery of serum-based metabolic biomarkers for these disorders. We aimed to identify various differentially expressed metabolites (DEMs) through serum-based metabolic profiling in patients with PCOS and POF and in healthy individuals by using liquid chromatography-mass spectrometry analysis. Furthermore, heatmap clustering, correlation, and Z-score analyses were performed to identify the top DEMs. Kyoto Encyclopedia of Genes and Genomes enriched pathways of DEMs were determined using metabolite-based databases. Moreover, the clinical significance of these DEMs was evaluated on the basis of area under the receiver operating characteristic curve. Significantly dysregulated expressions of several metabolites were observed in the intergroup comparisons of the PCOS, POF, and healthy control groups. Furthermore, 6 DEMs were most frequently observed among the three groups. The expressions of these DEMs were not only directly correlated but also exhibited potential significance in patients with PCOS and POF. Novel metabolites with up/downregulated expressions can be discovered in patients with PCOS and POF using serum-based metabolomics; these metabolites show good diagnostic performance and can act as effective biomarkers for the early detection of PCOS and POF. Furthermore, these metabolites might be involved in the pathophysiological mechanisms of PCOS and POF via interplay with corresponding genes.

Silencing of long non-coding RNA NEAT1 improves Treg/Th17 imbalance in preeclampsia via the miR-485-5p/AIM2 axis.

T-regulatory (Treg)/T-helper 17 (Th17) imbalance is associated with preeclampsia (PE). Herein, we aimed to explore the effect and mechanism of lncRNA NEAT1 on the Treg/Th17 balance. The levels of nuclear enriched abundant transcript 1 (NEAT1), miR-485-5p, and absent in melanoma 2 (AIM2) in CD4+ T cells were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Treg and Th17 cells were examined using flow cytometry. The relationship between miR-485-5p and NEAT1 or AIM2 was assessed using a dual-luciferase reporter assay. Pearson's correlation coefficient was used to analyze the correlation. All the data indicated that NEAT1 was upregulated in PE. The number of Treg cells decreased and was negatively related to NEAT1, whereas the number of Th17 cells increased and was positively related to NEAT1 in PE. Knockdown of NEAT1 increased the Treg cells and Treg/Th17 but decreased Th17 cells. Furthermore, NEAT1 sponges miR-485-5p to suppress the target AIM2 levels. Inhibition of miR-485-5p or upregulation of AIM2 abrogated the effect on Treg/Th17 balance induced by knockdown of NEAT1. In conclusion, silencing of NEAT1 promoted Treg/Th17 balance via the miR-485-5p/AIM2 axis in PE, suggesting that NEAT1 is a potential target for the treatment of PE.

Ultrasensitive and selective detection of Staphylococcus aureus using a novel IgY-based colorimetric platform.

To develop a specific method for the detection of S. aureus, chicken anti-protein A IgY was adopted for specifically capturing S. aureus, depending on the specific recognition of staphylococcal protein A (SPA) by chicken anti-protein A IgY, which can eliminate the interference from protein G-producing Streptococcus. HRP labeled IgG, Fc region of which has a high affinity towards SPA, was paired with IgY for the colorimeter analysis of the system. By optimizing the system, a super-low detection limit of 11 CFU of S. aureus in 100 µL PBS without enrichment, with a linear range from 5.0 × 102 CFU mL-1 to 5.0 × 104 CFU mL-1 was obtained. The entire assay was accomplished in less than 90 min and no cross-reactivity with the other tested bacterial species was observed. Moreover, the developed assay has been applied for the detection of S. aureus in three different types of real samples (sodium chloride injection, apple juice and human urine) with satisfactory results. To the best of our knowledge, it is the first time to report using chicken anti-protein A IgY and any IgG to detect S. aureus based on the dual-recognition mode of SPA. The novel method opened up a way for monitoring S. aureus in food samples with high sensitivity, specificity and simple operation.

Association of Foxp3 and TGF-ß1 Polymorphisms with Pre-Eclampsia Risk in Chinese Women.

PURPOSE: The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes that encode forkhead box p3 (Foxp3) (rs3761549 C>T, rs2280883T>C, rs2232365 A>G and rs3761548 C>A) and transforming growth factor (TGF)-ß1 (rs11466359 C>T, rs11466345 A>G and rs1800469 T>C) are associated with pre-eclampsia (PE) risk in Chinese women. MATERIALS AND METHODS: SNPs were identified by polymerase chain reaction and ligase detection reaction. Allelic variant and genotype frequencies for Foxp3 and TGF-ß1 were compared between PE women (n = 203) and healthy pregnant (HP) controls (n = 243). RESULTS: The TGF-ß1 rs1800469 TT genotype was found more frequently in PE patients than in HP controls [CC vs. CT+TT: odds ratio (OR) = 1.71; 95% confidence interval (CI): 1.04-2.81; p = 0.033], indicating that the T allele of rs1800469 confers a risk for PE [OR = 1.46; 95% CI: 1.12-1.92; p = 0.006]. The Foxp3 rs2232365 A allele was associated with severe PE specifically [OR = 1.70; 95% CI: 1.12-2.58; p = 0.01], compared with mild PE. There were no haplotype associations with PE. CONCLUSIONS: These findings indicate that allelic variants of TGF-ß1 rs1800469 T influence PE risk in Chinese women. Pregnant Han Chinese women carrying the rs1800469 TT genotype were at increased risk of PE.

A novel colorimetric sensing platform for the detection of S. aureus with high sensitivity and specificity.

In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 103 CFU mL-1 and a wide linear range of 3.1 × 103 to 2.0 × 105 CFU mL-1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples.

Brittle Culm 1 Encodes a COBRA-Like Protein Involved in Secondary Cell Wall Cellulose Biosynthesis in Sorghum.

Plant mechanical strength contributes to lodging resistance and grain yield, making it an agronomically important trait in sorghum (Sorghum bicolor). In this study, we isolated the brittle culm 1 (bc1) mutant and identified SbBC1 through map-based cloning. SbBC1, a homolog of rice OsBC1 and Arabidopsis thaliana AtCOBL4, encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchored protein. A single-nucleotide mutation in SbBC1 led to reduced mechanical strength, decreased cellulose content, and increased lignin content without obviously altering plant morphology. Transmission electron microscopy revealed reduced cell wall thickness in sclerenchyma cells of the bc1 mutant. SbBC1 is primarily expressed in developing sclerenchyma cells and vascular bundles in sorghum. RNA-seq analysis further suggested a possible mechanism by which SbBC1 mediates cellulose biosynthesis and cell wall remodeling. Our results demonstrate that SbBC1 participates in the biosynthesis of cellulose in the secondary cell wall and affects the mechanical strength of sorghum plants, providing additional genetic evidence for the roles of COBRA-like genes in cellulose biosynthesis in grasses.

Contribution of regulatory T cells to immune tolerance and association of microRNA­210 and Foxp3 in preeclampsia.

Increasing evidence suggests that an exaggerated maternal systemic inflammatofrery response may play a central role in the pathogenesis of preeclampsia (PE). Considering the growing evidence on microRNAs (miRNAs) and tissue­specific regulators of gene expression, we investigated the potential association of miR­210 and forkhead box p3 (Foxp3) in preeclamptic patients. Serum levels of the cytokines interleukin (IL)­6, IL­10, IL­17, and transforming grown factor­ß1 were detected with ELISA. Reverse­transcription­quantitative polymerase chain reaction was performed to detect mRNA expression for maternal placenta retinoic acid­related orphan receptor C, Foxp3 and miRNA (miR)­210. Foxp3 protein expression was evaluated by western blot analysis. Serum levels of cytokines IL­10 were significantly lower in preeclamptic patients than in normal pregnant women. mRNA expression of Foxp3 was significantly lower in placenta of PE. mRNA expression of miR­210 was significantly increased in PE. Results of western blot analysis indicated that Foxp3 protein expression was lower in PE than in normal pregnant women. Our data suggest that PE manifests as a decreased number of regulatory T cells (Tregs), which regulate maternal tolerance of the fetus. In placenta from women with PE, compared with normal pregnant women, mRNA expression of Foxp3 was significantly decreased, and expression of miR­210 was significantly increased.

[Effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction].

OBJECTIVE: To investigate the effect of fluorofenidone on renal interstitial fibrosis in rats with unilateral ureteral obstruction (UUO) and to observe the effect of fluorofenidone on the expressions of collagen type I (Col I), collagen type III (Col III), α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), platelet derived growth factor (PDGF) in the renal tissues of UUO rats.
 Methods: Male Sprague-Dawley (SD) rats were randomly divided into a sham-operated group, a UUO group, and a flurofenidone group (n=5). UUO model was induced by ligating the left ureter in rats. The rats were treated with 125 mg/(kg.d) fluorofenidone by gastric gavage in the fluorofenidone group at 24 h before the operation, and the rats were treated with the identical dose of 0.5% sodium carboxyl methyl cellulose (CMC-Na) in the other 2 groups. The rats were sacrificed at 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expressions of Col I, Col III, α-SMA, PDGF and CTGF were detected by real-time PCR, and the protein expressions of Col I, Col III, PDGF and CTGF were detected by immunohistochemical staining.
 Results: The renal interstitial damage index, relative collagen area and mRNA and protein expressions of Col I and Col III in the renal tissues of the rats in the UUO group significantly increased (P<0.05), and fluorofenidone could reduce these indexes (P<0.05). Compared with the sham-operated group, the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF in the renal tissues of the rats in the UUO group were increased, but fluorofenidone could decrease the protein expressions of α-SMA, PDGF, CTGF and the mRNA expressions of PDGF and CTGF (P<0.05).
 Conclusion: Fluorofenidone (125 mg/kg.d) could attenuate renal interstitial fibrosis through inhibition of fibroblast proliferation, myofibroblastic activation, PDGF and CTGF expression.

Loss of the Protein Cystathionine ß-Synthase During Kidney Injury Promotes Renal Tubulointerstitial Fibrosis.

BACKGROUND/AIMS: Renal tubulointerstitial fibrosis (TIF) is the common pathway of progressive chronic kidney disease. Inflammation has been widely accepted as the major driving force of TIF. Cystathionine ß-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway. CBS is considered to play protective role in liver and pulmonary fibrosis, but its role in TIF remains unknown. The purpose of this study was to investigate the potential role and mechanism of CBS in renal inflammation and TIF. METHODS: Renal function, tubulointerstitium damage index score, extracellular matrix (ECM) deposition, and the expressions of collagen I, collagen III, fibronectin, CD3, CD68, IL-1ß, TNF-α were measured in sham operation and unilateral ureteral obstruction (UUO) rats. Proteomics and gene array analysis were performed to screen differentially expressed molecules in the development of renal inflammation and TIF in UUO rats. The expression of CBS was detected in patients with obstructive nephropathy and UUO rats. We confirmed the expression of CBS using western blot and real-time PCR in HK-2 cells. Overexpression plasmid and siRNA were transfected specifically to study the possible function of CBS in HK-2 cells. RESULTS: Abundant expression of CBS, localized in renal tubular epithelial cells, was revealed in human and rat renal tissue, which correlated negatively with the progression of fibrotic disease. Expression of CBS was dramatically decreased in the obstructed kidney from UUO rats as compared with the sham group (SHM). In addition, knocking down CBS exacerbated extracellular matrix (ECM) deposition, whereas CBS overexpression attenuated TGF-ß1-induced ECM deposition in vitro. Inflammatory and chemotactic factors were also increased in CBS knockdown HK-2 cells stimulated by IL-1ß. CONCLUSIONS: These findings establish CBS as a novel inhibitor in renal fibrosis and as a new therapeutic target in patients with chronic kidney disease.