[Clinical features and Y chromosome abnormalities in children with 45, X/46, XY mosaicism].

Objective: To investigate the clinical and genetic characteristics of children with 45, X/46, XY mosaicism. Methods: The retrospective study included 20 children diagnosed with 45, X/46, XY and 45, X/46, X,+mar mosaicism in the First Affiliated Hospital of Zhengzhou University from 2018 to 2022. The clinical features, gonadal pathology, treatment and follow-up were summarized. Genetic tests were performed by SRY gene test, azoospermia factor region (AZF) deletion test, copy number variation-sequencing (CNV-seq). Age at first diagnosis was compared between boys and girls using independent sample t-test. Results: The 20 patients included 3 boys and 17 girls, and the age at first diagnosis were (7.6±5.5) years, it is (2.1±1.9) years in boys, (8.7±5.4) years in girls, significantly younger for boys (t=-3.86, P=0.004). The chief complaint was external genitalia malformation for boys, and short stature (13 cases) and dysplastic external genital for girls (4 cases). Five girls presented with features of Turner syndrome. The gonadal phenotypes included mixed gonadal dysplasia (MGD, 6 cases), complete gonadal dysplasia (CGD, 10 cases), unilateral ovotestis (2 cases), possible ovaries (1 case) and undetermined gonad (1 case). One female with dysplastic genital was reassigned to male, and the gender of the remaining cases remained unchanged. Seven females were treated with recombinant human growth hormone. The height increased by (17±7) cm during the (2.9±1.2) years follow-up. No gonadal malignancy was observed. The karyotype was 45, X/46, XY in 16 cases, and 45, X/46, X,+mar in 4 cases. All of the 4 marker chromosomes were derived from Y chromosome confirmed by CNV-seq. SRY gene was detected in all 20 patients genome, and AZF deletion was found in 7 girls. Conclusions: 45, X/46, XY mosaicism presented with dysplastic external genital or female with remarkable short stature. Gonadal phenotypes included MGD, CGD and ovotestis. AZF microdeletions were found in the majority of female cases.

[miR-148b inhibits M2 polarization of LPS-stimulated macrophages by targeting DcR3].

Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.

[Clinical and genetic characteristic in patients with disorders of sex development caused by Y chromosome copy number variant].

Objective: To investigate the clinical phenotype and genetic characteristics of disorders of sex development (DSD) caused by Y chromosome copy number variant (CNV). Methods: A retrospective analysis was performed on 3 patients diagnosed with DSD caused by Y chromosome CNV admitted to the First Affiliated Hospital of Zhengzhou University from January, 2018 to September, 2022. Clinical data were collected. Clinical study and genetic test were performed by karyotyping, whole exome sequencing (WES), low coverage whole genome copy number variant sequencing (CNV-seq), fluorescence in situ hybridization (FISH) and gonadal biopsy. Results: The 3 children, aged 12, 9, 9 years, the social gender were all female, presented with short stature, gonadal dysplasia and normal female external genital. No other phenotypic abnormality was found except for case 1 with scoliosis. The karyotype of all cases were identified as 46, XY. No pathogenic vraiants were found by WES. CNV-seq determined that case 1 was 47, XYY,+Y(2.12) and case 2 was 46, XY,+Y(1.6). FISH concluded that the long arm of Y chromosome was broken and recombined near Yq11.2, and then produced a pseudodicentric chromosome idic(Y). The karyotype was reinterpreted as mos 47, X, idic(Y)(q11.23)×2(10)/46, X, idic(Y)(q11.23)(50) in case 1. The karyotype was redefined as 45, XO(6)/46, X, idic(Y)(q11.22)(23)/46, X, del(Y)(q11.22)(1) in case 2. 46, XY, -Y(mos) was found by CNV-seq in case 3, and the karyotype of 45, XO/46, XY was speculated. Conclusions: The clinical manifestations of children with DSD caused by Y chromosome CNV are short stature and gonadal dysgenesis. If there is an increase of Y chromosome CNV detected by CNV-seq, FISH is recommended to classify the structural variation of Y chromosome.

Technical note: Isolation and characterization of porcine mammary epithelial cells.

Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), ß-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 µg/mL prolactin to culture medium for 3 d induced the production of ß-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

Effect of low dosage of chito-oligosaccharide supplementation on intestinal morphology, immune response, antioxidant capacity, and barrier function in weaned piglets.

This study was conducted to determine the effect of dietary supplementation of a low dose of chito-oligosaccharide (COS) on intestinal morphology, immune response, antioxidant capacity, and barrier function in weaned piglets. A total of 120 weaned pigs (21 d of age; 7.86 ± 0.22 kg average BW) were randomly assigned (6 pens/diet; 10 pigs/pen) to 2 dietary treatments consisting of a basal diet (negative control) or the basal diet supplemented with COS (30 mg/kg) for a 14-d period. Six randomly selected piglets from each treatment were killed for blood and tissue sampling. No significant differences were observed in ADG, ADFI, and G:F between treatment and the control group. Piglets fed the COS-supplemented diet had greater ( < 0.05) stomach pH than those fed the control diet on d 14 postweaning. Dietary supplementation with COS reduced villus height ( < 0.05) and villus height:crypt depth ( < 0.05) in the ileum. Dietary COS supplementation tended to reduce villus height in the duodenum ( = 0.065) and jejunum ( = 0.058). There was no effect on crypt depth in the intestinal segments of treatment group. Piglets fed the COS-supplemented diet increased ( < 0.05) the number of intraepithelial lymphocytes in duodenum or jejunum and goblet cells of ileum. However, COS decreased ( < 0.05) the number of intraepithelial lymphocytes in ileum of weaned piglets. The concentrations of IL-10 (duodenum, jejunum, and ileum) and secretory immunoglobulin (SIgA; duodenum and ileum) were higher in piglets fed the COS-supplemented diet compared with control ( < 0.05). Dietary COS supplementation reduced ( < 0.05) the concentration of total antioxidant capacity and superoxide dismutase of the jejunum or ileum. The mRNA expression of occludin in the ileum and ZO-1 in jejunum and ileum had a significant change in piglets fed the COS-supplemented diet compared with the control group ( < 0.05). In conclusion, these results indicated that dietary COS supplementation at 30 mg/kg had no effects on promoting growth performance and tended to reduce villus height in the duodenum or jejunum of weaned piglets. The results further showed that supplemental COS at this level may cause an immune and oxidative stress response in small intestine and have compromised the intestinal barrier integrity in weaned piglets. The research will provide guidance on the low dosage of COS supplementation on weaning pigs.