Construction and analysis of a joint diagnostic model of machine learning for cryptorchidism based on single-cell sequencing.

BACKGROUND: Cryptorchidism is a condition in which one or both of a baby's testicles do not fully descend into the bottom of the scrotum. Newborns with cryptorchidism are at increased risk of developing infertility later in life. The aim of this study was to develop a novel diagnostic model for cryptorchidism and to identify new biomarkers associated with cryptorchidism. METHODS: The study data were obtained from RNA sequencing data of cryptorchid patients from Nantong University Hospital and the Gene Expression Omnibus (GEO) database. Differential expression analysis was used to obtain differentially expressed genes (DEGs) between the control and cryptorchid groups. These DEGs were analyzed for their functions by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using GSEA software. Random Forest algorithm was used to screen central genes based on these DEGs. Neuralnet software package was used to develop artificial neural network models. Based on clinical data, receiver operating characteristic (ROC) was used to validate the models. Single-cell sequencing analysis was used for the pathogenesis of cryptorchidism. RESULTS: We obtained a total of 525 important DEGs related to cryptorchidism, which are mainly associated with biological functions such as supramolecular complexes and microtubule cytoskeleton. Random forest approach screening obtained eight hub genes. A neural network based on the hub genes showed a 100% success rate of the model. Finally, single-cell sequencing analysis validated the hub genes. CONCLUSION: We developed a novel diagnostic model for cryptorchidism using artificial neural networks and validated its utility as an effective diagnostic tool.

Analysis of The Curative Effect of Tubularized Incised Plate Urethroplasty for Distal Hypospadias With The Dysplastic Corpus Spongiosum Covering Technique.

PURPOSE: To investigate the use of tubularized incised plate (TIP) urethroplasty for distal second- and third-degree hypospadias to free the dysplastic forked corpus spongiosum and Buck's fascia, which are used as a covering material for the new urethra, thereby reducing the incidence of urinary fistula and other complications in the coronal sulcus. MATERIALS AND METHODS: Clinical data of 113 patients with distal hypospadias treated with TIP urethroplasty from January 2017 to December 2020 were retrospectively analyzed. The study group comprised 58 patients (use of dysplastic corpus spongiosum and Buck's fascia to cover the new urethra), and the control group comprised 55 patients (use of dorsal Dartos fascia to cover the new urethra). RESULTS: All children were followed up for more than 12 months. In the study group, 4 patients developed urinary fistulas, 4 developed a urethral stricture, and no case developed glans fissure. In the control group, 11 patients developed urinary fistulas, 2 developed a urethral stricture, 3 developed a glans cracking. CONCLUSION: Using the dysplastic corpus spongiosum to cover the new urethra increases the amount of tissue in the coronal sulcus and reduces the incidence of urethral fistula, but it may increase the incidence of urethral stricture.

Fluorescence-based immunochromatographic test strip for the detection of hyoscyamine.

Hyoscyamine (HSM), which acts as an antagonist of the acetylcholine muscarinic receptor and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning), is hazardous to human health. Therefore, it is urgent to develop a rapid, sensitive, and cost-effective method to determine HSM. A fluorescent microsphere based immunochromatographic assay was developed for this analyte and gold nanoparticles (AuNPs) were used as a comparison. A monoclonal antibody against HSM was prepared with a 50% inhibition concentration (IC50) of 1.17 ng mL-1, with no cross-reactivity with five drugs. Under optimized conditions, the cut off limits using the fluorescence-labeled monoclonal antibody strips were 10 ng mL-1 in 0.01 M PBS and 20 ng mL-1 in pork, pig urine, and honey samples, and the assay could be completed within 10 min. In comparison with a AuNP immunochromatographic assay, the developed method offered a higher coupling rate and lower amounts of antibodies. This approach could be used for simple, sensitive and rapid screening, and is suitable for on-site screening applications.

A paper-based sensor for rapid and ultrasensitive detection of ibuprofen in water and herbal tea.

As the use of non-steroidal anti-inflammatory drugs (NSAIDS) increases, their side effects have also attracted attention. Ibuprofen is one of the most widely-used NSAIDs. In this study, we screened the highly-sensitive and specific antibody 6E10, with an IC50 of 1.92 ng mL-1, and a linear range of 0.53-6.97 ng mL-1. In this study, we developed a rapid lateral flow immunochromatographic assay (ICA) strip method to detect ibuprofen in water or herbal tea. The cut-off limit of the strip is 10 ng mL-1 in water, and concentrations as low as 1 ng mL-1 can be detected in herbal tea samples, with the results obtained by the naked eye within 6 min. All the data were confirmed by high performance liquid chromatography-quadrupole time of flight-mass spectrometry (HPLC-QTOF-MS). This lateral-flow ICA strip is thus a rapid tool for on-site detection and screening of ibuprofen in water and herbal tea.

Global 3'UTR shortening and down-regulation of repeated element related piRNA play crucial roles in boys with cryptorchidism.

BACKGROUND: Cryptorchidism is the most common congenital defect in children's genitourinary system. Decades of research have identified both environmental and genetic factors contribute to the etiology. METHODS: Small-RNA/mRNA-seq were performed on testicular tissues from cryptorchidism patients. Downstream analysis included mRNA expression, piRNA expression and miRNA expression. RESULTS: We find a global downregulation of repeated element related piRNA expression as well as a global 3'UTR shortening of mRNAs in patients with cryptorchidism. We also find that genes with shortened 3'UTR which are highly enriched in vascular endothelial growth and protein ubiquitination, tend to be up-regulated in cryptorchidism. These results indicate that boys with cryptorchidism may not have normal piRNA functions to protect developmental tissues from transposon invasion. Dysregulated shortened 3'UTR genes may affect normal testicular tissue development. CONCLUSION: In summary, our findings also provided the first landscape of gene regulation in cryptorchidism, especially in terms of post-transcriptional regulations.

Dysfunction of Tregs contributes to FGR pathogenesis via regulating Smads signalling pathway.

Fetal growth restriction (FGR) is ranked number two of most common complication of abnormal pregnancy worldwide. The pathogenesis of FGR is complicated due to multiple aetiologies and the exact mechanism for FGR development is currently unknown. T regulatory cells (Tregs) are proven to play central roles in the maintenance of normal pregnancy. Peripheral blood samples of 102 pregnant human were collected analysed using flow cytometry to identify Tregs. We found that reduced Tregs and down-regulation of Foxp3 were observed in peripheral blood of FGR patients. In FGR mouse model, we have found that Tregs were not only reduced in spleen but also in placenta. In vitro, Foxp3 and its transcription regulatory signalling molecules, including P-Smad2, P-Smad3 and Smad4, were diminished as well. Inhibition on Foxp3 expression was partially reversed by overexpression of Smad2 and Smad4. In FGR patients, Western blot results revealed that Foxp3, P-Smad2, P-Smad3 and Smad4 expression was inhibited in placenta. Our preliminary result suggests that maternal-foetal immune tolerance mediated by Tregs plays an essential role in the development of FGR. The inhibited expression of Foxp3 and down-regulated Smad2/Smad3/Smad4 signalling pathway were involved in the FGR pathogenesis. Targeting maternal-foetal immune tolerance through Tregs might represent a novel therapeutic option for FGR.

Reduced GRAMD1C expression correlates to poor prognosis and immune infiltrates in kidney renal clear cell carcinoma.

There has been an increase in the mortality rate and morbidity of kidney cancer (KC) with kidney renal clear cell carcinoma (KIRC) being the most common subtype of KC. GRAMD1C (GRAM Domain Containing 1C) has not been reported to relate to prognosis and immunotherapy in any cancers. Using bioinformatics methods, we judged the prognostic value of GRAMD1C expression in KIRC and investigated the underlying mechanisms of GRAMD1C affecting the overall survival of KIRC based on data downloaded from The Cancer Genome Atlas (TCGA). The outcome revealed that reduced GRAMD1C expression could be a promising predicting factor of poor prognosis in kidney renal clear cell carcinoma. Meanwhile, GRAMDIC expression was significantly correlated to several tumor-infiltrating immune cells (TIICs), particularly the regulatory T cells (Tregs). Furthermore, GRAMD1C was most significantly associated with the mTOR signaling pathway, RNA degradation, WNT signaling pathway, toll pathway and AKT pathway in KIRC. Thus, GRAMD1C has the potential to become a novel predictor to evaluate prognosis and immune infiltration for KIRC patients.

PKM2 Involved in Neuronal Apoptosis on Hypoxic-ischemic Encephalopathy in Neonatal Rats.

Pyruvate Kinase isozymes M2 (PKM2) is a glycolytic enzyme involved in glycolysis that decarboxylates phosphoenolpyruvate to pyruvate and generates ATP. PKM2 also plays a significant role in tumor growth, in cell division, angiogenesis, apoptosis and metastasis. In this study, we have investigated the role of PKM2 in cortical neurons which suffered hypoxic-ischemic encephalopathy (HIE) in newborn rats. Immunohistochemistry and Western blot analysis revealed the protein expression of PKM2 peaking at 24 h after HIE. Double immunofluorescence labeling showed that PKM2 was mainly located in the neurons of the ipsilateral cerebral cortex, not in astrocytes or microglia. The increased level of active caspase-3 and the decreased level of phosphorylated AKT (p-AKT) were consistent with the PKM2 expression. TUNEL staining assay showed that PKM2 may participate in neuronal apoptosis in the rat ipsilateral cerebral cortex. Silencing of PKM2 in primary cultures of cortical neurons using a specific siRNA reduced the expression of active caspase-3 and upregulated p-AKT expression. Taken together, the results indicate that PKM2 may be involved in neuronal apoptosis after HIE by a mechanism dependent on the inactivation of p-AKT.

Therapeutic effects of modified Devine surgery for concealed penis in children.

OBJECTIVE: Considerable controversy exists regarding the surgical indications for a concealed penis. We herein describe a modified technique for correction of a concealed penis. The superfluous inner plate is resected to accelerate the disappearance of the postoperative lymphedema, and the skin between the penis and scrotum is trimmed to recover the penoscrotal angle. METHODS: From January 2014 to October 2017, 79 patients with a concealed penis underwent our modified Devine penoplasty procedure. We measured the penile length preoperatively and postoperatively to confirm the improvement. A questionnaire was administered to the patients' parents to assess satisfaction regarding penile size, morphology, voiding status, and hygiene. RESULTS: The perpendicular penile length was 1.88 ± 0.76 cm preoperatively and 4.42 ± 0.48 cm postoperatively, representing a significant improvement(p < 0.05). The parents' satisfaction grades for penile size, morphology, voiding status, and hygiene were significantly improved postoperatively (p < 0.05). Almost every patient had postoperative penile lymphedema; however, this symptom had spontaneously resolved by 6 weeks. No other complications occurred, such as skin necrosis, tissue contracture, or wound infection. CONCLUSION: The herein-described modified repair technique for a concealed penis was technically feasible and safe, and excellent postoperative satisfaction was achieved. Additionally, the postoperative penis exhibited a good cosmetic appearance. Because of the successful outcomes with few complications, we believe that this surgical method for selected patients with a concealed penis is more effective than the traditional method. LEVEL OF EVIDENCE: Level IV, therapeutic study.

LAMP3 expression correlated with poor clinical outcome in human ovarian cancer.

Lysosome-associated membrane protein 3 belongs to the lysosome-associated membrane glycoprotein family, which is associated with lymph node, metastasis, poor overall survival, and resistance to chemotherapy and radiotherapy. Epithelial ovarian cancer is one of the most deadly global female gynecologic malignant tumors. Its clinical outcome is poor and most epithelial ovarian cancer patients tend to relapse because of drug resistance. However, lysosome-associated membrane protein 3 expression in epithelial ovarian cancer and its relationship between clinicopathologic factors remain poorly understood. To clarify the prognostic implications of lysosome-associated membrane protein 3 in epithelial ovarian cancer, we analyzed both messenger RNA and protein levels of lysosome-associated membrane protein 3 in ovarian carcinomas. Polymerase chain reaction results showed higher expression of lysosome-associated membrane protein 3 messenger RNA in epithelial ovarian cancer than in noncancerous tissues. Immunohistochemical results showed that high lysosome-associated membrane protein 3 cytoplasmic expression was significantly related to tumor grade ( p = 0.038), lymph node metastasis ( p = 0.049), metastasis ( p < 0.001), level of CA125 ( p = 0.030), and International Federation of Gynecology and Obstetrics (FIGO) ( p < 0.001). High lysosome-associated membrane protein 3 nuclear expression was significantly associated with tumor grade ( p = 0.046), tumor single or double (representative whether the tumor involving one or both ovaries) ( p = 0.016), metastasis ( p < 0.001), and FIGO stage ( p < 0.001). Survival analysis indicated that high lysosome-associated membrane protein 3 cytoplasmic expression (hazard ratio: 4.632, 95% confidence interval: 2.421-8.864; p < 0.001), patients' age (hazard ratio: 1.729, 95% confidence interval: 1.027-2.911; p = 0.039), and FIGO stage (hazard ratio: 2.049, 95% confidence interval: 1.113-3.774; p = 0.021) were significantly correlated with poor survival outcome of epithelial ovarian cancer patients.

Ectopic immature renal tissue in gubernaculum associated with undescended testis of a 1-year-old child: A case report.

RATIONALE: Ectopic immature renal tissue (EIRT) is extremely rare in congenital malformations. Moreover, the fundamental pathogenesis of EIRT is still unclear and controversial. PATIENT CONCERNS: The right scrotum of a 1-year-old man was found empty for a period of 1 month. B-ultrasonography revealed normal bilateral kidneys and a hypoechoic nodule in the right groin. DIAGNOSES: Based on B-ultrasonography, surgery and pathological examination, we concluded a case of abnormally located and EIRT in the inguinal canal. INTERVENTIONS: After pathological diagnosis, the patient was not treated with drugs. OUTCOMES: One year after the operation, the patient recovered. LESSONS: EIRT in gubernaculum is extremely rare. Because of the potential risk of malignant transformation, it is necessary to diagnose and treat it early.

Prospective identification and culture of rat enteric neural stem cells (ENSCs).

Hirschprung's disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15 % chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10 % FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.

Wogonin Induced Mitochondrial Dysfunction and Endoplasmic Reticulum Stress in Human Malignant Neuroblastoma Cells Via IRE1α-Dependent Pathway.

Wogonin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been reported to exhibit a variety of biological effects including anti-cancer effects. It has a pro-apoptotic role in many cancer types. However, the molecular mechanisms of wogonin in treating neuroblastoma remain elusive. In the present study, two malignant neuroblastoma cell lines (SK-N-BE2 and IMR-32 cells) were treated with different doses of wogonin (0-150 µM). Wogonin showed significant cytotoxic effects in SK-N-BE2 and IMR-32 cells in a dose- and time-dependent manner. Treatment of SK-N-BE2 and IMR-32 cells with 75 µΜ wogonin for 48 h significantly promoted apoptosis, the release of cytochrome c, altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), and increased the activation of caspase-3, caspase-8, caspase-9, and PARP-1, which demonstrated that the cytotoxic effect of wogonin in SK-N-BE2 and IMR-32 cells is mediated by mitochondrial dysfunction. Moreover, wogonin induced the expression of endoplasmic reticulum (ER) stress-related proteins (GRP78/Bip and GRP94/gp96) and activation of caspase-12 and caspase-4 in SK-N-BE2 and IMR-32 cells. In addition, wogonin increase the expression of IRE1α and TRAF2, and phosphorylation of ASK1 and JNK in SK-N-BE2 and IMR-32 cells. Knockdown of IRE1α by siRNA not only markedly inhibited wogonin-induced up-regulation of IRE1α and TRAF2, and phosphorylation of ASK1 and JNK but also reduced wogonin-induced cytotoxic effects and mitochondrial dysfunction in SK-N-BE2 and IMR-32 cells. These results indicated that wogonin could induce apoptosis, mitochondrial dysfunction, and ER stress in SK-N-BE2 and IMR-32 cells by modulating IRE1α-dependent pathway.

[Expression change and significance of homebox gene-A10 in rat cryptorchidism].

OBJECTIVE: To explore the effects of HOXA10 gene expression in undescended and descended testis. METHODS: Twenty-four male Sprague Dawley rats were surgically prepared for unilateral cryptorchidism model. Their undescended and descended testes were removed at days 7 (infancy period, group B7) and 60 (sexual maturity period, group B60) post-operation. And contralateral descended testis served as controls (group A7, group A60). The expression of HOXA10 with histologic changes in experimental cryptorchidism were detected with one-step real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Compared with the control group, the levels of HOXA10 gene for groups B7 and B60 decreased markedly to 0.78 ± 0.11 (P < 0.01) and 0.56 ± 0.03 (P < 0.01) respectively.However the levels of HOXA10 gene and protein in group B60 were significantly lower than those in group B7 (P < 0.01). CONCLUSIONS: There is a lower expression of HOXA10 in undescended testes than in normally developed counterparts. And the expression of HOXA10 decreases with the elapsing time of cryptorchidism.

Identification of serum microRNA-21 as a biomarker for early detection and prognosis in human epithelial ovarian cancer.

Recent investigations have confirmed up-regulation of serum miR-21 and its diagnostic and prognostic value in several human malignancies. In this study, we examined serum miR-21 levels in epithelial ovarian cancer (EOC) patients, and explored its association with clinicopathological factors and prognosis. The results showed significantly higher serum miR-21 levels in EOC patients than in healthy controls. In addition, increased serum miR-21 expression was correlated with advanced FIGO stage, high tumor grade, and shortened overall survival. These findings indicate that serum miR-21 may serve as a novel diagnostic and prognostic marker, and be used as a therapeutic target for the treatment of EOC.