All-aqueous droplets-templated tailorable core-shell alginate microspheres for constructing vascularized intestinal mucosain vitromodels.

Recently,in vitromodels of intestinal mucosa have become important tools for drug screening and studying the physiology and pathology of the intestine. These models enable the examination of cellular behavior in diseased states or in reaction to alterations in the microenvironment, potentially serving as alternatives to animal models. One of the major challenges in constructing physiologically relevantin vitromodels of intestinal mucosa is the creation of three-dimensional microstructures that accurately mimic the integration of intestinal epithelium and vascularized stroma. Here, core-shell alginate (Alg) microspheres were generated to create the compartmentalized extracellular matrix microenvironment needed to simulate the epithelial and vascularized stromal compartments of the intestinal mucosa. We demonstrated that NIH-3T3 and human umbilical vein endothelial cells embedded in the core of the microspheres can proliferate and develop a vascular network, while human colorectal adenocarcinoma cells (Caco-2) can form an epithelial monolayer in the shell. Compared to Caco-2 monolayer encapsulated within the shell, the presence of the vascularized stroma enhances their proliferation and functionality. As such, our core-shell Alg microspheres provide a valuable method for generatingin vitromodels of vascularized intestinal mucosa with epithelial and vascularized stroma arranged in a spatially relevant manner and demonstrating near-physiological functionality.

MRE11A: a novel negative regulator of human DNA mismatch repair.

BACKGROUND: DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3'-5' exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear. METHODS: Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used. RESULTS: We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity. CONCLUSIONS: Our findings reveal that MRE11A is a negative regulator of human MMR.

Elucidating the Mass Transportation Behavior of Gas Diffusion Layers via a H2 Limiting Current Test.

The gas diffusion layer (GDL), as a key component of proton exchange membrane fuel cells (PEMFCs), plays a crucial role in PEMFC's polarization performance, particularly in mass transport properties at high current densities. To elucidate the correlation between GDLs' structure and their mass transport properties, a limiting current test with the H2 molecular probe was established and employed to investigate three representative GDLs with and without the microporous layer (MPL). By varying humidity and back pressure, the mass transport resistance of three GDLs was measured in an operating fuel cell, and an elaborate analysis of H2 transport was conducted. The results showed that the transport resistance (RDM) of GDLs was affected by the thickness and pore size distribution of the macroporous substrate (MPS) and the MPL. In the process of gas transport, the smaller pore size and thicker MPL increase the force of gas on the pore wall, resulting in an increase in transmission resistance. Through further calculation and analysis, the total transport resistance can be divided into pressure-related resistance (RP) and pressure-independent resistance (RNP). RP mainly originates from the transport resistance in both MPLs and the substrate layers of GDLs, exhibiting a linear relationship to the pressure; RNP mainly originates from the transport resistance in the MPLs. 29BC with thick MPL shows the largest RNP, and T060 without MPL shows the RNP = 0. This methodology enables in situ measurements of mass transport resistances for gas diffusion media, which can be easily applied for developing and deploying PEMFCs.

[Genetic analysis of a patient with familial hypercholesterolemia due to variant of LDLR gene].

OBJECTIVE: To analyze variant of LDLR gene in a patient with familial hypercholesterolemia (FH) in order to provide a basis for the clinical diagnosis and genetic counseling. METHODS: A patient who had visited the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University in June 2020 was selected as the study subject. Clinical data of the patient was collected. Whole exome sequencing (WES) was applied to the patient. Candidate variant was verified by Sanger sequencing. Conservation of the variant site was analyzed by searching the UCSC database. RESULTS: The total cholesterol level of the patient was increased, especially low density lipoprotein cholesterol. A heterozygous c.2344A>T (p.Lys782*) variant was detected in the LDLR gene. Sanger sequencing confirmed that the variant was inherited from the father. CONCLUSION: The heterozygous c.2344A>T (p.Lys782*) variant of the LDLR gene probably underlay the FH in this patient. Above finding has provided a basis for genetic counseling and prenatal diagnosis for this family.

Exploration of molecular features of PCOS with different androgen levels and immune-related prognostic biomarkers associated with implantation failure.

Background: Polycystic ovary syndrome (PCOS), the most common heterogeneous reproductive disease afflicting women of childbearing age, has been recognized as a chronic inflammatory disease recently. Most PCOS patients have hyperandrogenism, indicating a poor prognosis and poor pregnancy outcomes. The molecular mechanism underlying PCOS development is still unknown. In the present study, we investigated the gene expression profiling characteristics of PCOS with hyperandrogenism (HA) or without hyperandrogenism (NHA) and identified immune-related factors that correlated with embryo implantation failure. Methods: PCOS and recurrent implantation failure (RIF) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. ClueGO software was used to perform enrichment analysis of differentially expressed genes (DEGs) in PCOS with varying androgen levels. The Weighted Co-Expression Network Analysis (WGCNA) was used to identify co-expressed modules and shared gene signatures between HA PCOS and RIF. Moreover, the upregulated DEGs of HA PCOS and RIF were intersected with shared gene signatures screening by WGCNA to excavate further key prognostic biomarkers related to implantation failure of HA PCOS. The selected biomarker was verified by qRT-PCR. Results: A total of 271 DEGs were found in HA PCOS granulosa cell samples, and 720 DEGs were found in NHA PCOS. According to CuleGO enrichment analysis, DEGs in HA PCOS are enriched in immune activation and inflammatory response. In contrast, DEGs in NHA PCOS are enriched in mesenchymal cell development and extracellular space. Using WGCNA analysis, we discovered 26 shared gene signatures between HA PCOS and RIF, which were involved in corticosteroid metabolism, bone maturation and immune regulation. DAPK2 was furtherly screened out and verified to be closely related with the development of HA PCOS, acting as an independent predictor biomarker of the embryo implantation failure. DAPK2 expression was negatively correlated to the embryo implantation rate (r=-0.474, P=0.003). The immune infiltration results suggested that upregulated DAPK2 expression was closely related with NK cell infiltration and macrophage M2, playing an essential role in the pathogenesis of implantation failure in HA PCOS. Conclusion: Our research revealed the expression profiling of PCOS with different androgen levels and identified DAPK2 as a critical prognostic biomarker for implantation failure in PCOS.

Biallelic Variants in CCDC39 Gene Lead to Primary Ciliary Dyskinesia and Kartagener Syndrome.

Background: Primary ciliary dyskinesia (PCD) is a clinical syndrome characterized by cilia with an abnormal structure or function. Its main clinical manifestations comprise chronic bronchitis, cough, recurrent respiratory infections, situs inversus, and male infertility. Single-gene variants are widely assumed to be the main cause of this rare disease, and more than 40 genes have been described to be associated with its onset. CCDC39 is essential for assembling the inner dynein arms and dynein regulatory complex and is important in cilia motility. CCDC39 variants were reported as a monogenic etiology of PCD. Methods: This study investigated two unrelated Chinese patients diagnosed as PCD. The chest computed tomography scan was performed to identify PCD phenotypes of the two probands. Considering the effect of PCD on male fertility, routine semen analysis, sperm morphology examination, and scanning electron microscopy were performed to assess the semen characteristics of male proband in family 2 (F2 II-1), who had a history of infertility. Subsequently, the peripheral blood samples of probands were collected to perform whole-exome sequencing (WES) to explore the possible genetic causes of this disease. Results: Whole-exome sequencing revealed a homozygous CCDC39 variant in the female proband of family 1 (F1 II-1: c.286C>T:p.Arg96Ter) and two compound heterozygous CCDC39 variants in the male proband of family 2 (F2 II-1: c.732_733del: p.Ala245PhefsTer18; c.2800_2802dup:p.Val934dup). The two probands showed the typical PCD phenotypes, including chronic bronchitis, recurrent respiratory infections, and situs inversus. The male proband also showed oligoasthenoteratospermia with multiple morphological abnormalities of the sperm flagella. Additionally, CCDC39 protein level was significantly lower in the sperm of male proband than in the sperm from normal controls. Conclusion: We identified a homozygous variant reported previously and two compound heterozygous variants of CCDC39 possibly responsible for PCD pathogenesis, expanding the variant spectrum of Chinese PCD, Kartagener syndrome, and morphological abnormalities of the sperm flagella involving CCDC39.

Identification of deleterious variants in patients with male infertility due to idiopathic non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe type of male infertility, affecting 1% of men worldwide. Most of its etiologies remain idiopathic. Although genetic studies have identified dozens of NOA genes, monogenic mutations can also account for a small proportion of idiopathic NOA cases. Hence, this genetic study was conducted to explore the causes of monogenic variants of NOA in a cohort of Chinese patients. METHODS: Following the screening using chromosomal karyotyping, Y chromosome microdeletion analyses, and sex hormone assessments, subsequent whole-exome sequencing analysis was performed in 55 unrelated idiopathic NOA patients with male infertility to explore potential deleterious variants associated with spermatogenesis. We also performed Sanger sequencing to demonstrate the variants. Testicular biopsy or microsurgical testicular sperm extraction was also performed to confirm the diagnosis of NOA and identify spermatozoa. Hematoxylin and eosin staining was performed to assess the histopathology of spermatogenesis. RESULTS: Abnormal testicular pathological phenotypes included Sertoli cell-only syndrome, maturation arrest, and hypospermatogenesis. Using bioinformatics analysis, we detected novel variants in two recessive genes, FANCA (NM_000135, c.3263C > T, c.1729C > G) and SYCE1 (NM_001143763, c.689_690del); one X-linked gene, TEX11 (NM_031276, c.466A > G, c.559_560del); and two dominant genes, DMRT1 (NM_021951, c.425C > T, c.340G > A) and PLK4 (NM_001190799, c.2785A > G), in eight patients, which corresponded to 14.55% (8/55) of the patients. CONCLUSION: This study presented some novel variants of known pathogenic genes for NOA. Further, it expanded the variant spectrum of NOA patients, which might advance clinical genetic counseling in the future.

Homozygous SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF.

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a subtype of severe asthenoteratozoospermia with poorly understood genetic etiology. SPAG6 is a core axonemal component that plays a critical role in the formation of cilia and sperm flagella. Previous studies have reported that mutations in SPAG6 cause primary ciliary dyskinesia (PCD), but the association between SPAG6 gene variants and the MMAF phenotype has not yet been described. METHODS: We performed whole-exome sequencing (WES) in two unrelated Han Chinese men with MMAF. Sanger sequencing was used to validate the candidate variants. Routine semen analysis was carried out according to the WHO guidelines (5th Edition). Sperm morphology was assessed using modified Papanicolaou staining. Scanning and transmission electron microscopy (S/TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to examine the expression of SPAG6 protein. Assisted fertilization with intracytoplasmic sperm injection (ICSI) was applied. RESULTS: Two homozygous SPAG6 variants were identified by WES and Sanger validation in two patients with MMAF phenotype (F1 II-1: c.308C > A, p. A103D; F2 II-1: c. 585delA, p. K196Sfs*6). Semen analysis showed progressive rates of less than 1%, and most of the spermatozoa presented MMAF by Papanicolaou staining. TEM revealed that the overall axonemal ultrastructure was disrupted and primarily presented an abnormal "9 + 0" configuration. No other PCD-related symptoms were found on physical examination and medical consultations, as well as lung CT screening. The level of SPAG6 protein was significantly decreased in the spermatozoa, and IF analysis revealed that SPAG6 staining was extremely weak and discontinuous in the sperm flagella of the two patients. Notably, F1 II-1 and his wife conceived successfully after undergoing ICSI. CONCLUSIONS: Our research provides new evidence for a potential correlation between SPAG6 variants and the MMAF phenotype.

Bi-allelic variants in DNAH10 cause asthenoteratozoospermia and male infertility.

PURPOSE: Multiple morphological abnormalities in the sperm flagella (MMAF) comprise a severe phenotype of asthenoteratozoospermia with reduced or absent spermatozoa motility. Whereas dozens of candidate pathogenic genes for MMAF have been identified, the genetic cause in a large proportion of patients is unknown. We attempted to identify novel genetic explanations for MMAF. METHODS: We performed whole-exome sequencing of patients with MMAF to identify pathogenic variants. The phenotypes of spermatozoa in patients carrying DNAH10 variants were investigated using haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy. The expression and location of DNAH10 and other spermatozoa structure-related proteins were analyzed using immunofluorescence assays. RESULTS: We found one homozygous frameshift DNAH10 variant (NM_207437: c.2514delG:p.L839*) and one compound heterozygous DNAH10 variant (NM_207437: c.10820 T > C:p.M3607T; c.12692C > T:p.T4231I) in two patients with MMAF. These variants were absent or rare in the general population. Haematoxylin and eosin staining and scanning electron microscopy revealed the significant disruption of sperm flagella in the patients. In addition, ultrastructural analysis by transmission electron microscopy showed significant inner dynein arm (IDA) deficiency in sperm flagella. Using immunofluorescence assays, we found a significant reduction in IDA-related proteins including DNAH10 and DNAH1. CONCLUSIONS: We identified putative novel pathogenic variants in DNAH10 for MMAF, which might advance the genetic diagnosis and clinical genetic counselling for male infertility.

GNS561, a New Autophagy Inhibitor Active against Cancer Stem Cells in Hepatocellular Carcinoma and Hepatic Metastasis from Colorectal Cancer.

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and can regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Thus, GNS561 offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population. Accordingly, a global phase 1b clinical trial in liver cancers was recently completed.

Regulation of tubular recycling endosome biogenesis by the p53-MICALL1 pathway.

p53, one of the most frequently mutated genes in colon cancer, suppresses cancer development through transactivation of its targets. Herein, we conducted a comprehensive analysis of the p53 downstream pathway in colorectal cancer by using multi-omics analysis. Mass spectrometric analysis of HCT116 p53+/+ and HCT116 p53-/- cells treated with adriamycin identified 124 proteins increased by DNA damage in a p53-dependent manner. Further screening using a cDNA microarray and the TCGA database revealed MICALL1 as a novel p53 target, and we identified functional p53 binding motifs located approximately 3000 base pairs upstream of the MICALL1 gene. MICALL1 expression was significantly decreased in colorectal cancer tissues with p53 mutation compared with those without p53 mutation. In response to DNA damage, MICALL1 co-localized with RAB8A and CD2AP at tubular recycling endosomes, whereas these proteins hardly localized at tubular recycling endosomes when p53 or MICALL1 expression was inhibited by siRNA. Our findings show that p53 regulates tubular recycling endosome biogenesis via transcriptional regulation of MICALL1, whose expression is frequently suppressed in colorectal cancer tissues.

Immunophilin FK506 loaded in chitosan guide promotes peripheral nerve regeneration.

Immunophilin ligand FK506 has been treated as adjunct therapy for nerve repair due to its potent neurotrophic and neuroprotective actions. It was hypothesized that FK506 releasing from biodegradable chitosan guide provided better nerve regenerative response than the guide with no FK506. The drug was entrapped in the semi-permeable wall of chitosan guide with the drug-loading of 647 microg/g. Rat sciatic nerve defect model treated with FK506-releasing chitosan guide showed more mature appearance of myelinated fibers 8 weeks after surgery; furthermore, the motor functional reinnervation occurred, the amplitude and velocity of compound muscle action potentials reached 60% and 73% with respect to the normal. Thus, FK506-releasing chitosan guide should be acted as a long-lasting delivery device of immunosuppressive and neuroregenerative agent for peripheral nerve repair.