Identification, characterization and expression analysis of rLcn13, an epididymal lipocalin in rats.

As the essential tissue for sperm maturation and storage, the epididymis secretes a number of tissue-specific proteins to exert its functions. Among these proteins, epididymal lipocalins have been intensively studied because of their epididymis-specific expression pattern and clustered genomic organization. In this study, rLcn13, a member of the rat epididymal lipocalin family, is identified and elaborately characterized. The cDNA sequence of rLcn13 consists of 719 nucleotides and encodes a 176 amino-acid protein with a predicted N-terminal signal peptide of 19 amino acids. rLcn13 shares a similar genomic structure and predicted 3D protein structure with other lipocalin family members. A recombinant rLCN13 mature peptide of 157 amino acids is expressed and purified, which is used to raise a polyclonal antibody against rLCN13 with high specificity and sensitivity. Northern blot, western blot, and immunohistochemistry assays reveal that rLcn13 is an epididymis-specific gene which is expressed predominantly in the initial segment and proximal caput epididymis and influenced by androgen. The rLCN13 protein is modified by N-glycosylation and secreted into the epididymal lumen, and then binds to the acrosome region of the sperm. Our data demonstrate that rLcn13 exhibits a specific temporospatial expression pattern and androgen dependence, indicating its potential roles in sperm maturation.

Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs.

Several sialoglycopeptides were isolated from several fish eggs and exerted anti-osteoporosis effects. However, few papers have explored sialoglycopeptide from tuna eggs (T-ES). Here, a novel T-ES was prepared through extraction with KCl solution and subsequent enzymolysis. Pure T-ES was obtained through DEAE-Sepharose ion exchange chromatography and sephacryl S-300 gel filtration chromatography. The T-ES was composed of 14.07% protein, 73.54% hexose, and 8.28% Neu5Ac, with a molecular weight of 9481 Da. The backbone carbohydrate in the T-ES was →4)-ß-D-GlcN-(1→3)-α-D-GalN-(1→3)-ß-D-Glc-(1→2)-α-D-Gal-(1→2)-α-D-Gal-(1→3)-α-D-Man-(1→, with two branches of ß-D-GlcN-(1→ and α-D-GalN-(1→ linking at o-4 in →2,4)-α-D-Gal-(1→. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1→. A peptide chain, Ala-Asp-Asn-Lys-Ser*-Met-Ile that was connected to the carbohydrate chain through O-glycosylation at the -OH of serine. Furthermore, in vitro data revealed that T-ES could remarkably enhance bone density, bone biomechanical properties, and bone microstructure in SAMP mice. The T-ES elevated serum osteogenesis-related markers and reduced bone resorption-related markers in serum and urine. The present study's results demonstrated that T-ES, a novel sialoglycopeptide, showed significant anti-osteoporosis effects, which will accelerate the utilization of T-ES as an alternative marine drug or functional food for anti-osteoporosis.

Structural Characterization and Effects on Insulin Resistance of a Novel Chondroitin Sulfate from Halaelurus burgeri Skin.

Several studies have isolated chondroitin sulphate (CHS) from sharks' jaws or cartilage. However, there has been little research on CHS from shark skin. In the present study, we extracted a novel CHS from Halaelurus burgeri skin, which has a novel chemical structure and bioactivity on improvement in insulin resistance. Results using Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR), and methylation analysis showed that the structure of the CHS was [4)-ß-D-GlcpA-(1→3)-ß-D-GlcpNAc-(1→]n with 17.40% of sulfate group concentration. Its molecular weight was 238.35 kDa, and the yield was 17.81%. Experiments on animals showed that this CHS could dramatically decrease body weight, reduce blood glucose and insulin levels, lower lipid concentrations both in the serum and the liver, improve glucose tolerance and insulin sensitivity, and regulate serum-inflammatory factors. These results demonstrated that the CHS from H. burgeri skin has a positive effect in reducing insulin resistance because of its novel structure, which provides a significant implication for the polysaccharide as a functional food.

Anti-Obesity and Gut Microbiota Regulation Effects of Phospholipids from the Eggs of Crab, Portunus Trituberculatus, in High Fat Diet-Fed Mice.

There are resourceful phospholipids in the eggs of the crab, Portunus trituberculatus (Pt-PL). However, their components and bioactivities regarding obesity were unclear. Here, we investigated the composition of Pt-PL and their fatty acids. Moreover, its effects on obesity and gut microbiota were also evaluated in high fat diet (HFD)-fed mice. The results showed that Pt-PL contained 12 kinds of phospholipids, mainly including phosphatidylcholine (PC, 32.28%), phosphatidylserine (PS, 26.51%), phosphatidic acid (PA, 19.61%), phosphatidylethanolamine (PE, 8.81%), and phosphatidylinositol (PI, 7.96%). Polyunsaturated fatty acids (PUFAs) predominated in the fatty acids components of Pt-PL, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Animal experiments demonstrated that Pt-PL significantly alleviated body weight gain, adipose gain, hepatic gain, fasting blood glucose, serum insulin, lipid levels in serum and the liver, and systematic inflammation in HFD-fed mice. Furthermore, Pt-PL regulated gut microbiota, especially in a dramatic reduction in the ratio of Firmicutes to Bacteroidetes at phylum level, as well as significant amelioration in their subordinate categories. Pt-PL reduced fecal lipopolysaccharide and total bile acids, and elevated fecal short chain fatty acid (SCFA) concentrations, particularly acetate and butyrate. These findings suggest that Pt-PL possesses anti-obesity effects and can alter gut microbiota owing to the abundance of PUFAs. Therefore, Pt-PL may be developed as an effective food supplement for anti-obesity and regulation of human gut health.

A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm.

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

Upregulation of 6-phosphofructo-2-kinase (PFKFB3) by hyperactivated mammalian target of rapamycin complex 1 is critical for tumor growth in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the benign tumor formation in multiple organs. The main etiology of TSC is the loss-of-function mutation of TSC1 or TSC2 gene, which leads to aberrant activation of mammalian target of rapamycin complex 1 (mTORC1). In this research, we found a significant increase of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts (MEFs) compared with the control cells. Inhibition of mTORC1 led to a dramatic decrease of PFKFB3 expression, indicating PFKFB3 regulation by mTORC1. Moreover, suppression of mTORC1 inhibited the expression of PFKFB3 in rat uterine leiomyoma-derived Tsc2-null ELT3 cells and human tumor cells. Furthermore, we identified hypoxia-inducible factor 1α (HIF-1α) as a mediator transmitting the signal from mTORC1 to PFKFB3. Depletion of PFKFB3 inhibited proliferation and tumorigenicity of Tsc1- or Tsc2-deficient cells. In addition, combination of rapamycin with PFK15, a PFKFB3 inhibitor, exerts a stronger inhibitory effect on cell proliferation of Tsc1- or Tsc2-null MEFs than treatment with single drug. We conclude that loss of TSC1 or TSC2 led to upregulated expression of PFKFB3 through activation of mTORC1/HIF-1α signaling pathway and co-administration of rapamycin and PFK15 may be a promising strategy for the treatment of TSC tumors as well as other hyperactivated mTORC1-related tumors.

Impaired sperm maturation in conditional Lcn6 knockout mice.

Human LCN6, a lipocalin protein, exhibits predominant expression in epididymis and location on the sperm surface. However, the biological function of LCN6 in vivo remains unknown. Herein, we found that unlike human LCN6, mouse Lcn6 gene encoded two transcript variants that were both upregulated by androgen. Subsequently, we generated a conditional knockout mouse model to disrupt Lcn6 in the adult and investigate its function. In this model, spermatogenesis was normal and Lcn6 deficiency did not affect the natural birth rate of male mice or in vitro fertilization ability of their cauda epididymal sperm. Nevertheless, sperm from the cauda epididymis of the Lcn6 null mice underwent a sustained increase of acrosome reaction frequency whether capacitated or not (P < 0.01). Consistent with premature acrosome reaction, sperm from knockout mice had significantly increased intracellular calcium content when extracellular calcium was supplied (P < 0.01). These results demonstrate an important function of LCN6 in preventing calcium overload and premature acrosome reaction of sperm and suggest a potential risk factor of LCN6 deficiency for sperm maturation.

Correcting motion in multiplanar cardiac magnetic resonance images.

BACKGROUND: Misalignment in cardiac magnetic resonance (CMR) images can adversely affect three-dimensional left ventricle modelling and downstream quantitative analysis. Currently, there are two types of approaches for dealing with realignment and motion distortion problems, one image based and the other geometry based. Image-based approaches are limited by the inherent non-homogeneity and anisotropy of CMR images. Geometry-based approaches rely on idealized models and over-simplified assumptions. This study was motivated by the need for a robust and effective approach for correcting motion related distortions due to misalignment in CMR images. METHODS: A cine cardiac magnetic resonance image sequence was acquired using our routine clinical imaging protocol. The left ventricular endocardium was delineated manually with software assistance on all long and short-axis images. Long and short-axis contours were projected onto a patient-based coordinate system and then realigned using iterative registration. The realigned contour points were used to reconstruct the shape of the left ventricle for quantitative validation. RESULTS: The method was tested on five myocardial infarction patients whose images showed substantial misalignment. Realignment time was about 16 seconds per case, using a 2.5 GHz CPU desktop with obvious elimination of the distortion in the reconstructed model. Using the long-axis contour as a reference in evaluating the reconstructed models, it was apparent that the models with realigned contours had better accuracy than the non-realigned ones. CONCLUSION: This study presents a novel, geometry-based method for correcting motion distortions in CMR images. The method incorporates (1) manual delineation, (2) registration based on a generalized, iterative closest point algorithm, and (3) reconstruction of the shape of the left ventricle for quantitative validation. The effectiveness of our approach is corroborated both visually and by quantitative assessment. We envision the use of our method in current clinical practice as a means of improving accuracy in the evaluation of cardiac function.

Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility.

Sulforaphane inhibits the growth of prostate cancer by regulating the microRNA-3919/DJ-1 axis.

Background: Prostate cancer (PCa) is the second most common solid cancer among men worldwide and the fifth leading cause of cancer-related deaths in men. Sulforaphane (SFN), an isothiocyanate compound, has been shown to exert inhibitory effects on a variety of cancers. However, the biological function of SFN in PCa has not been fully elucidated. The objective of this study was conducted to further investigate the possible underlying mechanism of SFN in PCa using in vitro cell culture and in vivo tumor model experiments. Methods: Cell viability, migration, invasion, and apoptosis were analyzed by Cell Counting Kit-8 (CCK-8), wound healing assay, transwell assay, or flow cytometry. Expression of microRNA (miR)-3919 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) or in situ hybridization assay. Xenograft assay was conducted to validated the antitumor effect of miR-3919. The targeting relationship between miR-3919 and DJ-1 was verified by dual-luciferase reporter assay. The level of DJ-1was measured by qRT-PCR or western blotting (WB). Results: In the present study, SFN downregulated mRNA and protein expression of DJ-1, an oncogenic gene. Small RNA sequencing analysis and dual-luciferase reporter assay confirmed that microRNA (miR)-3919 directly targeted DJ-1 to inhibition its expression. Furthermore, miR-3919 overexpression impeded viability, migration, and invasion and promoted apoptosis of PCa cells. Tumor growth in nude mice was also inhibited by miR-3919 overexpression, and miR-3919 expression in PCa tissues was lower than that in peritumoral tissues in an in situ hybridization assay. Transfection with miR-3919 inhibitors partially reversed the effects of SFN on cell viability, migration, invasion, and apoptosis. Conclusion: Overall, the miR-3919/DJ-1 axis may be involved in the effects of SFN on the malignant biological behavior of PCa cells, which might be a new therapeutic target in PCa.

WEE Family Kinase Inhibitors Combined with Sorafenib Can Selectively Inhibit HCC Cell Proliferation.

BACKGROUND: Sorafenib is currently the first choice for the treatment of patients with advanced hepatocellular carcinoma, but its therapeutic effect is still limited. OBJECTIVES: This study aims to examine whether WEE family kinase inhibitors can enhance the anticancer effect of sorafenib. METHODS: We analyzed the expression levels of PKMYT1 kinase and WEE1 kinase in HCC, studied the inhibitory effect of PKMYT1 kinase inhibitor RP-6306, WEE1 kinase inhibitor adavosertib combined with sorafenib on the proliferation of HCC cells, and detected the effect of drug combination on CDK1 phosphorylation. RESULTS: We found that PKMYT1 and WEE1 were upregulated in HCC and were detrimental to patient survival. Cell experiments showed that both RP-6306 and adavosertib (1-100 µM) inhibited the proliferation of HCC cell lines in a dose-dependent manner alone, and the combination of the two drugs had a synergistic effect. In HCC cell lines, sorafenib combined with RP-6306 or adavosertib showed a synergistic antiproliferation effect and less toxicity to normal cells. Sorafenib combined with RP-6306 and adavosertib further inhibited the proliferation of HCC cells and caused complete dephosphorylation of CDK1. CONCLUSION: Taken together, our findings provide experimental evidence for the future use of sorafenib in combination with RP-6306 or adavosertib for the treatment of HCC.

The chemokine CCL20 can assist AFP in serological diagnosis of hepatocellular carcinoma.

The chemokine 20 (CCL20) is a member of the CC chemokine family and plays a role in tumor immunity and autoimmune disease. This work investigated the value of CCL20 as a serum diagnostic marker for primary hepatocellular carcinoma (HCC). Based on the data of hepatocellular carcinoma patients in the TCGA database, the up-regulated genes encoding secretory proteins were analyzed in each pathological stage, and the candidate marker CCL20 gene was selected. Serum concentrations of CCL20 in patients with primary HCC, benign liver disease, and healthy subjects were analyzed by enzyme-linked immunosorbent assay (ELISA). The ROC curve evaluated the efficacy of CCL20 alone or in combination with AFP in the diagnosis of HCC. It was found the expression of CCL20 in HCC patients was significantly higher than that in the benign liver disease group and healthy controls (P < 0.05); The AUC of ROC curve to distinguish HCC patients from healthy controls was 0.859, the sensitivity was 73.42%, and the specificity was 86.84%. After combination with AFP, the AUC increased to 0.968, the sensitivity was 88.16%, and the specificity was 97.37%. Although CCL20 was increased in the serum of patients with benign liver diseases, combined with AFP, the AUC to distinguish HCC patients from non-HCC cohorts (benign liver disease group and healthy control group) was 0.902, with a sensitivity of 91.67% and a specificity of 75.26%. Collectively, serum CCL20 is closely related to the occurrence of HCC, and detection of serum CCL20 can assist AFP in improving the diagnostic sensitivity of HCC.

Augmented ERO1α upon mTORC1 activation induces ferroptosis resistance and tumor progression via upregulation of SLC7A11.

BACKGROUND: The dysregulated mechanistic target of rapamycin complex 1 (mTORC1) signaling plays a critical role in ferroptosis resistance and tumorigenesis. However, the precise underlying mechanisms still need to be fully understood. METHODS: Endoplasmic reticulum oxidoreductase 1 alpha (ERO1α) expression in mTORC1-activated mouse embryonic fibroblasts, cancer cells, and laryngeal squamous cell carcinoma (LSCC) clinical samples was examined by quantitative real-time PCR (qRT-PCR), western blotting, immunofluorescence (IF), and immunohistochemistry. Extensive in vitro and in vivo experiments were carried out to determine the role of ERO1α and its downstream target, member 11 of the solute carrier family 7 (SLC7A11), in mTORC1-mediated cell proliferation, angiogenesis, ferroptosis resistance, and tumor growth. The regulatory mechanism of ERO1α on SLC7A11 was investigated via RNA-sequencing, a cytokine array, an enzyme-linked immunosorbent assay, qRT-PCR, western blotting, IF, a luciferase reporter assay, and a chromatin immunoprecipitation assay. The combined therapeutic effect of ERO1α inhibition and the ferroptosis inducer imidazole ketone erastin (IKE) on mTORC1-activated cells was evaluated using cell line-derived xenografts, LSCC organoids, and LSCC patient-derived xenograft models. RESULTS: ERO1α is a functional downstream target of mTORC1. Elevated ERO1α induced ferroptosis resistance and exerted pro-oncogenic roles in mTORC1-activated cells via upregulation of SLC7A11. Mechanically, ERO1α stimulated the transcription of SLC7A11 by activating the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway. Moreover, ERO1α inhibition combined with treatment using the ferroptosis inducer IKE exhibited synergistic antitumor effects on mTORC1-activated tumors. CONCLUSIONS: The ERO1α/IL-6/STAT3/SLC7A11 pathway is crucial for mTORC1-mediated ferroptosis resistance and tumor growth, and combining ERO1α inhibition with ferroptosis inducers is a novel and effective treatment for mTORC1-related tumors.

Pan-cancer analysis reveals the associations between MMP13 high expression and carcinogenesis and its value as a serum diagnostic marker.

BACKGROUND: Matrix metalloproteinase-13 (MMP13) is a member of the endopeptidase matrix metalloproteinase family, which is involved in many normal physiological processes and even tumorigenesis. However, its co-carcinogenic signature in different cancers is not fully understood. METHODS: In this study, we first analyzed the expression of MMP13 in pan-cancer and its association with prognosis, immune infiltration, and cancer-related signaling pathways through integrated bioinformatics. Furthermore, western blotting (WB) was used to verify the expression of MMP13 and epithelial-mesenchymal transition (EMT) factors in cancer tissues. Finally, the value of MMP13 as a serum diagnostic marker was analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: MMP13 expression is frequently upregulated in multiple cancers that always indicate an adverse prognosis. MMP13 undergoes comprehensive genetic alterations and promoter methylation reduction in various tumors. Additionally, immune correlation analysis showed that MMP13 expression was significantly associated with TMB, MSI, and tumor immune infiltration. Pathway enrichment analysis showed that MMP13 upregulation was correlated with activation of the EMT signaling pathway, which was verified by WB in lung adenocarcinoma tissues. Most importantly, ELISA results showed that serum MMP13 levels could be used for the diagnosis of multiple tumors, including BRCA, HNSC, LUAD, and LUSC, with the area under the curve (AUC) values of 0.8494, 0.9259, 0.7144, and 0.8575, respectively. CONCLUSIONS: MMP13 is often overexpressed across cancers and predicts poor prognosis, with the potential as a therapeutic target. Furthermore, the up-regulation of its expression can be effectively reflected in the serum protein level, thus serving as a valuable diagnostic marker.

Effects of obstetric critical care simulation training on core competency and learning experience of midwives: A pilot quasi-experimental study.

AIM: This study aimed to investigate the effects of maternal critical care simulation training on the core competency and satisfaction of midwives in China. BACKGROUND: Midwives play an important role during the peripartum period. Simulation-based training could be an effective tool in improving the core competency of midwives when managing critical obstetric illnesses. DESIGN: A pilot pre- and post-course, quasi-experimental study in China. METHOD: In July 2022, 82 midwives completed a 2-day obstetric critical care simulation training and survey. Core competency was evaluated by a comprehensive score system, including response ability, communication ability, site control ability, critical thinking ability, team cooperation ability, forward-thinking ability, midwifery specialty ability, and error correction ability. We used the Simulation Effectiveness Tool-Modified (SET-M) to evaluate the learning experience and satisfaction. Descriptive analysis, McNemar χ2 test, and subject content analysis were used for data analysis. RESULTS: After the training, the core competency scores showed significant improvements in the case scenarios simulating shoulder dystocia, amniotic fluid embolism, and eclampsia (P < 0.05) but not postpartum hemorrhage (P > 0.05). The scores evaluated by the SET-M were all above 2.5 points. Some midwives preferred extended course duration, expanded course materials, and more active involvement in the simulation exercises. The midwives were generally highly satisfied with the training, but some expressed certain negative emotions, such as anxiety and nervousness. CONCLUSION: The high quality of scientifically constructed and implemented obstetric critical care simulation training courses could improve the core competency and satisfaction of midwives. Appropriate preparation and professional simulation teachers are required to reduce negative emotions and improve learning outcomes and experience.

Effect of Y2O3 Addition on Microstructure and Properties of Laser Cladded Al-Si Coatings on AZ91D Magnesium Alloy.

The effect of Y2O3 addition on the microstructure and properties of the laser cladded Al-Si alloy coating on the surface of AZ91D magnesium alloy was investigated in this study. The experimental results showed that the Al-Si + Y2O3 cladding layers contained α-Mg, Mg2Si, Al4MgY and a small amount of Al12Mg17 phases. The coarse dendrites, reticulated eutectic structures and massive phases in the coatings tended to be refined and gradually uniformly distributed with the increased amount of Y2O3. The introduction of Y2O3 into the cladding layer favored the improvement of microhardness and wear resistance due to the grain refinement strengthening and dispersion strengthening. The addition of Y2O3 also promoted the reduction of localized corrosion sites and made the corrosion surface smoother, implying that the corrosion resistance of the Y2O3-modified coatings was better than that of the unmodified cladding layer.

Nanopore-based metagenomic sequencing for the rapid and precise detection of pathogens among immunocompromised cancer patients with suspected infections.

Cancer patients are at high risk of infections and infection-related mortality; thereby, prompt diagnosis and precise anti-infectives treatment are critical. This study aimed to evaluate the performance of nanopore amplicon sequencing in identifying microbial agents among immunocompromised cancer patients with suspected infections. This prospective study enlisted 56 immunocompromised cancer patients with suspected infections. Their body fluid samples such as sputum and blood were collected, and potential microbial agents were detected in parallel by nanopore amplicon sequencing and the conventional culture method. Among the 56 body fluid samples, 47 (83.9%) samples were identified to have at least one pathogen by nanopore amplicon sequencing, but only 25 (44.6%) samples exhibited a positive finding by culture. Among 31 culture-negative samples, nanopore amplicon sequencing successfully detected pathogens in 22 samples (71.0%). Nanopore amplicon sequencing showed a higher sensitivity in pathogen detection than that of the conventional culture method (83.9% vs. 44.6%, P<0.001), and this advantage both existed in blood samples (38.5% vs. 0%, P=0.039) and non-blood samples (97.7% vs. 58.1%, P<0.001). Compared with the culture method, nanopore amplicon sequencing illustrated more samples with bacterial infections (P<0.001), infections from fastidious pathogens (P=0.006), and co-infections (P<0.001). The mean turnaround time for nanopore amplicon sequencing was about 17.5 hours, which was shorter than that of the conventional culture assay. This study suggested nanopore amplicon sequencing as a rapid and precise method for detecting pathogens among immunocompromised cancer patients with suspected infections. The novel and high-sensitive method will improve the outcomes of immunocompromised cancer patients by facilitating the prompt diagnosis of infections and precise anti-infectives treatment.

EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis.

AIMS: Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis. MAIN METHODS: Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology. KEY FINDINGS: EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1f/f mice. SIGNIFICANCE: This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health.

[Cathepsin-B involved in effect of electroacupuncture by inhibiting the activation of NLRP3 inflammasome in rats with acute gouty arthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on Cathepsin-B in the synovium of the knee joint of acute gouty arthritis(AGA) rats, so as to explore the mechanism of EA in the treatment of AGA. METHODS: A total of 60 male Wistar rats were randomly divided into normal control,model, medication and EA groups, with 15 rats in each group. Rat model of AGA was established by injection of 0.2 mL sodium urate crystal suspension into the left knee joint cavity. The rats in the medication group were treated with colchicine by gavage(0.3 mg·kg-1·d-1), and the rats in the EA group were treated with EA at the left "Sanyinjiao" (SP6) and "Zusanli"(ST36) for 10 min each time, once a day for a week. The Coderre gait grading standard was used to score the gait of rats. The pathological morphology of synovial tissue of the left knee joint was observed by H.E. staining. The expression levels of Cathepsin-B protein and Nod-like receptor pyrin domain 3(NLRP3), apoptosis-associated speck-like protein containing CARD (ASC),Caspase-1, interleukin-1ß(IL-1ß) and IL-18 mRNAs were detected by Western blot and real-time fluorescence quantitative PCR, respectively. RESULTS: Compared with the normal control group, the degree of synovitis infiltration in the model group was more serious. And the gait score，the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3,ASC,Caspase-1, IL-1ß,IL-18 were significantly increased (P<0.01).After the interventions, the degree of inflammatory infiltration was mild, The gait score, the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3 and ASC,Caspase-1，IL-1ß,IL-18 were significantly decreased in both medication and EA groups in contrast to the model group (P<0.01, P<0.05). Compared with medication group, the mRNA expression levels of Caspase-1 and IL-18 in the EA group were increased (P<0.05). CONCLUSION: EA may inhibit the activation of NLRP3 inflammasome by reducing the activity of Cathepsin-B in the synovium of the knee joint, so as to treat AGA.

RUNX1/EGFR pathway contributes to STAT3 activation and tumor growth caused by hyperactivated mTORC1.

Loss of function of tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) leads to the activation of mammalian target of rapamycin complex 1 (mTORC1). Hyperactivated mTORC1 plays a critical role in tumor growth, but the underlying mechanism is still not completely elucidated. Here, by analyzing Tsc1- or Tsc2-null mouse embryonic fibroblasts, rat Tsc2-null ELT3 cells, and human cancer cells, we present evidence for the involvement of epidermal growth factor receptor (EGFR) as a downstream target of mTORC1 in tumor growth. We show that mTORC1 leads to increased EGFR expression through upregulation of runt-related transcriptional factor 1 (RUNX1). Knockdown of EGFR impairs proliferation and tumoral growth of Tsc-deficient cells, while overexpression of EGFR promotes the proliferation of the control cells. Moreover, the mTOR signaling pathway has been shown to be positively correlated with EGFR in human cancers. In addition, we demonstrated that EGFR enhances cell growth through activation of signal transducer and activator of transcription 3 (STAT3). We conclude that activation of the RUNX1/EGFR/STAT3 signaling pathway contributes to tumorigenesis caused by hyperactivated mTORC1 and should be targeted for the treatment of mTORC1-related tumors, particularly TSC.