Cancer-associated fibroblast exosomes promote prostate cancer metastasis through miR-500a-3p/FBXW7/HSF1 axis under hypoxic microenvironment.

Metastasis is the main cause of deaths in prostate cancer (PCa). However, the exact mechanisms underlying PCa metastasis are not fully understood. In this study, we discovered pronounced hypoxia in primary lesions of metastatic PCa(mPCa). The exosomes secreted by cancer-associated fibroblasts (CAFs) under hypoxic conditions significantly enhance PCa metastasis both in vitro and in vivo. Through miRNA sequencing and reverse transcription quantitative PCR (RT-qPCR), we found that hypoxia elevated miR-500a-3p levels in CAFs exosomes. Subsequent RT-qPCR, western blotting, and dual luciferase reporter assays identified F-box and WD repeat domain-containing 7(FBXW7) as a target of miR-500a-3p. In addition, immunohistochemistry revealed that FBXW7 expression decreased with the progression of PCa, while heat shock transcription factor 1(HSF1) expression increased. Introducing an FBXW7 plasmid into PCa cells reduced their metastatic potential and significantly lowered HSF1 expression. These findings suggest that CAFs exosomes drive PCa metastasis via the miR-500a-3p/FBXW7/HSF1 axis in a hypoxic microenvironment. Targeting either hypoxia or exosomal miR-500a-3p could be a promising strategy for PCa management.

Exploring the Enigma of 5-ARIs Resistance in Benign Prostatic Hyperplasia: Paving the Path for Personalized Medicine.

PURPOSE OF REVIEW: Despite the widespread utilization of 5-alpha reductase inhibitors (5-ARIs) for managing benign prostatic hyperplasia (BPH), certain BPH patients exhibit unresponsiveness to 5-ARIs therapy. This paper provides a comprehensive overview of the current perspectives on the mechanisms of 5-ARIs resistance in BPH patients and integrates potential biomarkers and underlying therapeutic options for 5-ARIs resistance. These findings may facilitate the development of novel or optimize more effective treatment options, and promote personalized medicine for BPH. RECENT FINDINGS: The pathways contributing to resistance against 5-ARIs in certain BPH patients encompass epigenetic modifications, shifts in hormone levels, autophagic processes, and variations in androgen receptor structures, and these pathways may ultimately be attributed to inflammation. Promisingly, novel biomarkers, including intravesical prostatic protrusion, inflammatory factors, and single nucleotide polymorphisms, may offer predictive insights into the responsiveness to 5-ARIs therapy, empowering physicians to fine-tune treatment strategies. Additionally, on the horizon, GV1001 and mTOR inhibitors have emerged as potential alternative therapeutic modalities for addressing BPH in the future. After extensive investigation into BPH's pathological processes and molecular landscape, it is now recognized that diverse pathophysiological mechanisms may contribute to different BPH subtypes among individuals. This insight necessitates the adoption of personalized treatment strategies, moving beyond the prevailing one-size-fits-all paradigm centered around 5-ARIs. The imperative for early identification of individuals prone to treatment resistance will drive physicians to proactively stratify risk and adapt treatment tactics in future practice. This personalized medicine approach marks a progression from the current standard treatment model, emerging as the future trajectory in BPH management.

Upregulation of mir-1199-5p is associated with reduced type 2 5-α reductase expression in benign prostatic hyperplasia.

BACKGROUND: 5-α reductase inhibitors (5-ARIs) are first-line drugs for managing benign prostatic hyperplasia (BPH). Unfortunately, some patients do not respond to 5-ARI therapy and may even show worsening symptoms. The decreased expression of steroid 5-α reductase type 2(SRD5A2) in BPH tissues may explain the failure of 5-ARI therapy, however, the mechanisms underlying SRD5A2 decreased remained unelucidated. OBJECTIVES: To investigate microRNA-mediated regulation of the expression of SRD5A2 resulting in 5-ARI therapy failure. MATERIALS AND METHODS: The expression of SRD5A2 and microRNAs in BPH tissues and prostate cells were detected by immunohistochemistry, western blotting, and quantitative real-time PCR. Dual-luciferase reporter assay was performed to confirm that microRNA directly combine to SRD5A2 mRNA. The apoptosis of prostatic cells was detected by flow cytometry. RESULTS: SRD5A2 expression was variable; it was negative, weak, and strong in 13.6%, 28.8%, and 57.6% of BPH tissues respectively. The normal human prostatic epithelial cell line RWPE-1 strongly expressed SRD5A2, whereas the immortalized human prostatic epithelial cell line BPH-1 weakly expressed SRD5A2. miR-1199-5p expression was remarkably higher in BPH-1 than in RWPE-1 cells(P<0.001), and miR-1199-5p expression was significantly upregulated in BPH tissues with negative SRD5A2 expression than those with positive SRD5A2 expression. Transfection of miR-1199-5p mimics in RWPE-1 cells led to a marked decrease in SRD5A2 expression, whereas miR-1199-5p inhibitor increased SRD5A2 expression in BPH-1 cells. Dual-luciferase reporter assay showed that miR-1199-5p could bind the 3'untranslated region of SRD5A2 mRNA. miR-1199-5p also decreased the RWPE-1 sensibility to finasteride, an inhibitor of SRD5A2. CONCLUSION: Our results show that SRD5A2 expression varies in BPH tissues and miR-1199-5p might be one of the several factors contributing to differential SRD5A2 expression in BPH patients.

Interferon-γ-Induced Myeloid-Derived Suppressor Cells Aggravate Kidney Ischemia-Reperfusion Injury by Regulating Innate Immune Cells.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cells which can suppress T-cell functionality. Herein, we evaluated the functional importance of MDSCs in the context of kidney ischemia-reperfusion injury (IRI) and explored their ability to regulate innate and adaptive immune cell function in this context. METHODS: The differentiation of MDSCs was induced in vitro by treating cells with GM-CSF and interferon (IFN)-γ. In a murine model of renal IRI, serum creatinine and blood urea nitrogen values were measured to monitor kidney function, while histopathological and immunohistochemical approaches were used to assess kidney injury severity. In addition, flow cytometry was employed to assess the phenotypes and apoptosis of kidney cells in these mice. RESULTS: MDSCs induced by treatment with GM-CSF + IFN-γ could suppress T-cell functionality in vitro. The adoptive transfer of these MDSCs into an IRI mouse model system enhanced kidney damage and impaired renal function following the recruitment of these cells to renal tissues in these mice. Following such adoptive transfer, the relative frequency of MDSCs with a CD11b+Ly6G-Ly6Chigh monocytic-MDSC phenotype decreased, whereas cells with a CD11b+Ly6G+Ly6Clow polymorphonuclear-MDSC phenotype become more prevalent within kidney tissues following IRI. Adoptive transfer also coincided with increased frequencies of macrophages and dendritic cells (DCs) in the kidney tissues. This suggested that M-MDSCs contributed to early-stage renal IRI damage by differentiating into these deleterious cell types. However, MDSC-induced suppression of CD4+ and CD8+ T-cell infiltration was not sufficient to prevent the deterioration of renal function in these mice. CONCLUSIONS: Herein, we successfully developed a protocol wherein MDSCs were differentiated in vitro through combination GM-CSF/IFN-γ treatment. When these MDSCs were subsequently adoptively transferred into a murine model of renal IRI, they aggravated kidney damage, likely owing to the differentiation of M-MDSCs into deleterious macrophages and DCs.

Methylated CpG dinucleotides in the 5-α reductase 2 gene may explain finasteride resistance in benign prostatic enlargement patients.

The inhibition of 5-α reductase type 2 (SRD5A2) by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement (BPE). Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy. Our previous work showed that methylated cytosine-phosphate-guanine (CpG) islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression. Here, we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein (scored 0-100) occurred in 10.0% (4/40) of benign adult prostates. We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1 (DNMT1) expression. In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment, and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2'-deoxycytidine (5-Aza-CdR) or N-phthalyl-L-tryptophan (RG108). Furthermore, we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis. Ten methylated CpG dinucleotides, including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon, were found in a CpG island located from -400 bp to +600 bp in SRD5A2, which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression. Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2, which may partially account for the resistance to finasteride observed in certain BPE patients.

Effects of mild hypothermia on serum HMGB1 of brain-dead donors and its impact on kidney transplantation recipients.

Investigate the effect of mild hypothermia on serum inflammatory factor HMGB1 of brain-dead donors, and its significance for renal transplantation recipients.In our hospital between January 2018 and January 2019 up to the standard of brain death donor (aged 18 to 65 years old) prospective cohort study, brain death donor were randomly divided into mild hypothermia group and the non-mild hypothermia group. Serum were collected from donor at different periods, and enzyme-linked immunoassay (ELISA) was used to determine the serum HMGB1 concentration to compare the difference between the 2 donor groups. The early recovery of renal function after renal transplantation was followed up, and the incidence of delayed graft function (DGF) and early recovery of renal function were compared between the 2 groups. The correlation between donor HMGB1 and recipient DGF was analyzed.Between 17 donors in the mild hypothermia group and 17 in the non-mild hypothermia group, there were no statistically significant differences in the age, perioperative urine volume and ICU stay between the 2 groups. After mild hypothermia treatment, serum HMGB1 levels of brain death donors were significantly decreased. While in non-mild hypothermia brain death donor group without treatment, serum HMGB1 was significantly increased. There were no statistically significant differences in age and preoperative creatinine between the 2 recipient groups, including 33 patients in the mild hypothermia group and 34 patients in the non-mild hypothermia group. DGF incidence was lower in mild hypothermia group comparing with non-mild hypothermia group with statistical significance. The levels of HMGB1 from donor before procurement is correlated with the occurrence of DGF of the recipient.Mild hypothermia therapy can reduce the levels of serum HMGB1, improve the function of donor organs. The levels of HMGB1 before donor procurement can be used to predict the occurrence of DGF in kidney transplant recipients. Our study shows that HMGB1 can be potentially used as therapeutic target of early intervention for brain death donors. Furthermore, mild hypothermia therapy can be applied in the maintenance of brain death donors for kidney transplant recipient to improve the successful rate of transplantation.

Genetic characteristics of Jiaji Duck by whole genome re-sequencing.

Jiaji Duck (JJ) is a Muscovy duck species that possesses many superior characteristics, and it has become an important genetic resource in China. However, to date, its genetic characteristics and genetic relationship with other duck breeds have not been explored yet, which greatly limits the utilization of JJ. In the present study, we investigated the genome sequences of 15 individual ducks representing five different duck populations, including JJ, French Muscovy duck (FF), mallard (YD), hong duck (HD) and Beijing duck (BD). Moreover, we investigated the characteristics of JJ-specific single nucleotide polymorphisms (SNPs) and compared the genome sequences of JJ vs. YD and JJ vs. BD using integrated strategies, including mutation detection, selective screening, and Gene Ontology (GO) analysis. More than 40 Gb of clean data were obtained for each population (mean coverage of 13.46 Gb per individual). A total number of 22,481,367 SNPs and 4,156,829 small insertion-deletions (Indels) were identified for the five duck populations, which could be used as molecular markers in breeding and utilization of JJ. Moreover, we identified 1,447,932 JJ-specific SNPs, and found that genes covering at least one JJ-specific SNP mainly involved in protein phosphorylation and dephosphorylation, as well as DNA modification. Phylogenetic tree and principal components analysis (PCA) revealed that the genetic relationship of JJ was closest to FF, while it was farthest to BD. A total of 120 and 111 genes were identified as positive selection genes for JJ vs. BD and JJ vs. YD, respectively. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the positive selection genes for JJ vs. BD ducks mainly involved in pigmentation, muscle contraction and stretch, gland secretion, and immunology, while the positive selection genes obtained from JJ vs. YD ducks mainly involved in embryo development, muscle contraction and stretch, and gland secretion. Taken together, our findings enabled us to better understand the characteristics of JJ and provided a molecular basis for the breeding and hybrid utilization of JJ in the future.

Landscape of alternative splicing in Capra_hircus.

Alternative splicing (AS) is a fundamental regulatory process in all higher eukaryotes. However, AS landscapes for a number of animals, including goats, have not been explored to date. Here, we sequenced 60 samples representing 5 tissues from 4 developmental stages in triplicate using RNA-seq to elucidate the goat AS landscape. In total, 14,521 genes underwent AS (AS genes), accounting for 85.53% of intron-containing genes (16,697). Among these AS genes, 6,342 were differentially expressed in different tissues. Of the AS events identified, retained introns were most prevalent (37.04% of total AS events). Functional enrichment analysis of differential and specific AS genes indicated goat AS mainly involved in organ function and development. Particularly, AS genes identified in leg muscle were associated with the "regulation of skeletal muscle tissue development" GO term. Given genes were associated with this term, four of which (NRG4, IP6K3, AMPD1, and DYSF) might play crucial roles in skeletal muscle development. Further investigation indicated these five genes, harbored 13 ASs, spliced exclusively in leg muscle, likely played a role in goat leg muscle development. These results provide novel insights into goat AS landscapes and a valuable resource for investigation of goat transcriptome complexity and gene regulation.

Persistent accumulation of circulating monocytic myeloid-derived suppressor cells contributes to post-infectious immunosuppression in renal transplant recipients with bacterial infection: A pilot study.

INTRODUCTION: Post-infectious immunosuppression is disadvantageous to patients' long-term outcomes, especially in transplant recipients receiving large doses of immunosuppressants. A growing body of evidence indicates the immunoregulatory ability of myeloid-derived suppressor cells (MDSCs). We herein investigate the characteristics of monocytic-MDSCs (M-MDSCs) in a cohort of renal transplant recipients with/without infection to clarify the potential involvement in post-infectious immunosuppression. METHODS: The study prospectively included 28 adult recipients who underwent allogeneic ABO-compatible renal transplant. Blood samples were drawn at day 0, 7, 14, 28, 60 and 90 postoperation. The incidence of infection and treatment strategies were recorded. The frequency and absolute number of peripheral blood M-MDSCs as well as other immune cells were determined by flow cytometry. Immnosuppressive functions of M-MDSCs were analyzed by inhibition of T cells proliferation. mRNA levels of immunosuppressive molecules in sorted M-MDSCs were also examined. RESULTS: 7 recipients were diagnosed with bacterial (n = 5) or viral (n = 2) infection and 3/5 of bacterial-infected recipients suffered from secondary infection during further follow-up. In the non-infected group, M-MDSCs numbers increased transiently during the early postoperative period, however, bacterial but not viral infection led to significant and persistent accumulation of M-MDSCs that remained at high levels after anti-infective treatments. M-MDSCs from infected recipients demonstrated potent ability to suppress T cells proliferation in vitro and negatively correlated with lymphocytes in vivo, yet not in the non-infected group. Inducible nitric oxide synthase (iNOS) mRNA levels were higher in sorted M-MDSCs when compared with monocytes, and suppressive activity was reversed by addition of a NOS inhibitor. CONCLUSIONS: Circulating M-MDSCs underwent significant and persistent increases after bacterial infection in renal transplant recipients, contributing to post-infectious immunodeficiency. Therefore, special attention should be given to M-MDSCs during the monitoring of immune status and infection management.

Lower Expression of SLC27A1 Enhances Intramuscular Fat Deposition in Chicken via Down-Regulated Fatty Acid Oxidation Mediated by CPT1A.

Intramuscular fat (IMF) is recognized as the predominant factor affecting meat quality due to its positive correlation with tenderness, juiciness, and flavor. Chicken IMF deposition depends on the balance among lipid synthesis, transport, uptake, and subsequent metabolism, involving a lot of genes and pathways, however, its precise molecular mechanisms remain poorly understood. In the present study, the breast muscle tissue of female Wenchang chickens (WC) (higher IMF content, 1.24 in D120 and 1.62 in D180) and female White Recessive Rock chickens (WRR; lower IMF content, 0.53 in D120 and 0.90 in D180) were subjected to RNA-sequencing (RNA-seq) analysis. Results showed that many genes related to lipid catabolism, such as SLC27A1, LPL, ABCA1, and CPT1A were down-regulated in WC chickens, and these genes were involved in the PPAR signaling pathway and formed an IPA® network related to lipid metabolism. Furthermore, SLC27A1 was more down-regulated in WRR.D180.B than in WRR.D120.B. Decreased cellular triglyceride (TG) and up-regulated CPT1A were observed in the SLC27A1 overexpression QM-7 cells, and increased cellular triglyceride (TG) and down-regulated CPT1A were observed in the SLC27A1 knockdown QM-7 cells. These results suggest that lower lipid catabolism exists in WC chickens but not in WRR chickens, and lower expression of SLC27A1 facilitate IMF deposition in chicken via down-regulated fatty acid oxidation mediated by CPT1A. These findings indicate that reduced lipid catabolism, rather than increased lipid anabolism, contributes to chicken IMF deposition.

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases.

Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Tms increased by 0.6-3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.

Improvement of the thermostability and catalytic efficiency of a highly active ß-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.

BACKGROUND: ß-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported ß-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes. RESULTS: A novel endo-ß-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyces leycettanus JCM12802 was produced in Pichia pastoris and characterized. For potential industrial applications, recombinant TlGlu16A exhibits favorable enzymatic properties over most reported glucanases, i.e., remarkable stability over a wide pH range from 1.0 to 10.0 and superior activity on glucan substrates (up to 15,197 U/mg). The only weakness of TlGlu16A is the thermolability at 65 °C and higher. To improve the thermostability, the enzyme thermal stability system was then used to engineer TlGlu16A through optimization of residual charge-charge interactions. Eleven mutants were constructed and compared to the wild-type TlGlu16A. Four mutants, H58D, E134R, D235G and D296K, showed longer half-life time at 80 °C (31, 7, 25, 22 vs. 0.5 min), and two mutants, D235G and D296K, had greater specific activities (158.2 and 122.2 %, respectively) and catalytic efficiencies (k cat/K m, 170 and 114 %, respectively). CONCLUSIONS: The engineered TlGlu16A has great application potentials from the perspectives of enzyme yield and properties. Its thermostability and activity were apparently improved in the engineered enzymes through charge optimization. This study spans the genetic, functional and structural fields, and provides a combination of gene mining and protein engineering approaches for the systematic improvement of enzyme performance.