The epithelial Na+ channel (ENaC) in ovarian granulosa cells modulates Ca2+ mobilization and gonadotrophin signaling for estrogen homeostasis and female fertility.

Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na+ channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na+ imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca2+ imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca2+ oscillations, lowered the capacity of intracellular Ca2+ stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.

Biphasic regulation of CFTR expression by ENaC in epithelial cells: The involvement of Ca2+-modulated cAMP production.

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl- transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

[Phenotype and genetic analysis of three patients with PKHD1 associated autosomal recessive polycystic kidney disease at childhood, teenage and advanced age].

OBJECTIVE: The phenotype and genetics of three patients with autosomal recessive polycystic kidney disease (ARPKD) at childhood, teenage and advanced age were analyzed. METHODS: Next generation sequencing (NGS) was applied to all the probands. PCR and Sanger sequencing were used to verify the suspicious gene variants screened by NGS in the probands and their family members, and one of the family got prenatal diagnosis. RESULTS: Through NGS, PCR and Sanger sequencing, the 5-yr proband in pedigree 1 was shown to carry compound heterozygous variants of c.5935G>A(p.G1979R) and c.5428G>T(p.E1810X) of PKHD1, originated from his parents; In pedigree 2, the 17-ys proband was detected with c.5512T>C(p.Y1838H) and c.5935G>A(p.G1979R) variants of PKHD1 orginated from her parents, and her mother also got prenatal diagnosis during the second trimester; In pedigree 3, the 70-ys female proband was found with variants c.11314C>T (p.R3772X) and c.3860T>G (p.V1287G) of PKHD1. CONCLUSION: The three pedigrees were diagnosed as ARPKD caused by PKHD1 variants. Five types of variants were detected, c.5935G>A and c.11314C>T were the known pathogenic variants, while c.5512T>C, c.5428G>T and c.3860T>G were not reported previously. Considering the complexity of the genetics and phenotypes of the cystic renal diseases, genetic diagnosis is crucial to give accurate etiological diagnosis, which may benefit the clinic management.

[Genetic analysis of a family with recurrent hydrops fetalis and dilated cardiomyopathy].

OBJECTIVE: To carry out genetic testing for a family with two pregnancies affected with hydrops fetalis and dilated cardiomyopathy (DCM) of the fetus. METHODS: DNA was extracted from fetal tissue as well as peripheral blood samples from the couple. Single nucleotide polymorphism array (SNP array) and next-generation sequencing (NGS) were carried out to screen potential mutation. Suspected mutation was validated with PCR and Sanger sequencing. RESULTS: The manifestation of fetal echocardiography was consistent with DCM. No obvious abnormality was found by SNP array analysis. A hemizygous c.481G>A (p.G161R) mutation of the TAZ gene was detected in the male fetus by NGS and confirmed by Sanger sequencing. The mutation was inherited from his mother. CONCLUSION: Barth syndrome due to the c.481G>A mutation of the TAZ gene probably underlies the recurrent hydrops fetalis and fetal DCM in this family.

Interfacial modification of titanium dioxide to enhance photocatalytic efficiency towards H2 production.

Strong demand for affordable clean energy to support applications ranging from conventional energy supply to space propulsion places spotlight on advanced energy generation using photovoltaic and wind power. Yet, the intermittent nature of solar and wind sources drives the search for energy storage solutions that would permit the needed level of resilience and support further growth in the use of renewable sources of power. Hydrogen generation using sunlight is a promising pathway to decouple demand from supply. Herein, we show how exposure to reactive Ar-H2, Ar-H2-N2, and Ar-O2 plasma environments can notably enhance surface properties of photocatalytic TiO2 nanosheets used in advanced energy generation systems. Treatment using Ar-H2 plasmas produced highly hydrogenated, surface-disordered TiO2 nanosheets with oxygen vacancies, whereas exposure to Ar-H2-N2 plasmas resulted in N doping. Surprisingly, Ar-O2 plasma treatment did not change surface properties of TiO2. Optical emission spectroscopy was used to monitor transient species to further understand surface modification in plasma. Direct measurements demonstrated that among thus-produced samples, hydrogenated TiO2 nanosheets exhibit the highest photocatalytic H2-generation activity under visible-light irradiation, which is also greater than the activity of pure, untreated nanosheets. The mechanism of enhancing the visible-light photocatalytic H2-generation activity on hydrogenated TiO2 nanosheets is also proposed. The level of surface disorder and oxygen vacancies plays an important role in enhancing visible-light absorption and reducing the recombination of photogenerated electrons and holes.

[Phenotype and genetic analysis of a pedigree affected with progressive familial intrahepatic cholestasis].

OBJECTIVE: To explore the genetic etiology for a pedigree affected with progressive familial intrahepatic cholestasis (PFIC). METHODS: Target sequence capture and next generation sequencing (NGS) were applied for the proband. PCR and Sanger sequencing were used to verify the suspected mutation in his sister with similar symptoms and his parents. RESULTS: The proband and his sister manifested after birth with symptoms including jaundice, pruritus and developmental retardation. NGS has identified compound heterozygous mutations of ABCB11 gene, which encodes bile salt export pump protein (BSEP), namely c.2494C>T (p.Arg832Cys) and c.3223C>T (p.Gln1075*), in the proband, which were inherited from his father and mother respectively. His sister carried the same compound mutations. CONCLUSION: Based on the phenotype and genetic testing, the patients were diagnosed as PFIC2 caused by mutation of the ABCB11 gene. The c.3223C>T is a novel nonsense mutation which may cause premature termination of translation. Above results have enriched the spectrum of ABCB11 mutations and provided new evidence for the molecular basis of PFIC, which also facilitated genetic counseling for this pedigree.

[Two cases of rare diseases with abnormalities of X chromosome].

OBJECTIVE: To explore the clinical features and genetic diagnosis of two cases with rare diseases and X chromosome abnormalities. METHODS: Multiple ligation-dependent probe amplification (MLPA) and karyotype analysis were carried out on an 8-year-old girl who was diagnosed with Duchenne muscular dystrophy. Karyotype analysis and PCR assay for SRY and AZF genes were carried out for a-2-month-old male infant with short penis. RESULTS: The girl, who featured short stature and cubitus valgus, was diagnosed as Turner syndrome with a karyotype of 46,X,i(Xq). The male infant was detected with a karyotype of 45,X, with presence of SRY gene but absence of AZF gene. CONCLUSION: Both cases may be associated with abnormalities of X chromosome. Genetic testing can facilitate early diagnosis and clinical intervention for such patients.

[Progress in genetic research on non-immune hydrops fetalis].

Non-immune factors are the most common cause of hydrops fetalis, which may manifest as abnormal accumulation of fluid in the body cavities. Hydrops fetalis is a complex condition with a high mortality rate. The prognosis is depending on the underlying disease and is particularly poor in those with chromosomal aberrations or monogenic disorders. In this paper, advance in the research on non-immune hydrops fetalis is reviewed.