RNF216 regulates meiosis and PKA stability in the testes.

Spermatogenesis is a highly sophisticated process that comprises of mitosis, meiosis, and spermiogenesis. RNF216 (ring finger protein 216), an E3 ubiquitin ligase, has been reported to be essential for spermatogenesis and male fertility in mice. However, the stages affected by Rnf216 deficiency and its underlying molecular pathological mechanisms are still unknown. In this study, we generated Rnf216-deficient mice (Rnf216-/- ) using CRISPR-Cas9 technology. Knockout of Rnf216 led to infertility in male but not female mice. Rnf216 knockout affected the prophase of meiosis I, as no genotypic difference was observed until 12 dpp (days postpartum). Rnf216-/- spermatocytes were incompletely arrested at the zygotene stage and underwent apoptosis at approximately the pachytene stage. The proportion of zygotene spermatocytes was significantly increased, whereas the proportion of pachytene spermatocytes was significantly decreased in Rnf216-/- testes. Nevertheless, there was no significantly genotypic difference in the number of diplotene spermatocytes. We further revealed that the PKA catalytic subunit ß (PRKACB) was significantly increased, which subsequently resulted in elevated PKA activity in testes from adult as well as 9 dpp Rnf216-/- mice. RNF216 interacts with PRKACB and promotes its degradation through the ubiquitin-lysosome pathway. Collectively, our results revealed an important role for RNF216 in regulation of meiosis and PKA stability in the testes.

Functional analysis of SEMA3A variants identified in Chinese patients with isolated hypogonadotropic hypogonadism.

Isolated hypogonadotropic hypogonadism (IHH) is a rare disorder characterized by impaired sexual development and infertility, caused by the deficiency of hypothalamic gonadotropin-releasing hormone neurons. IHH is named Kallmann's syndrome (KS) or normosmic IHH (nIHH) when associated with a defective or normal sense of smell. Variants in SEMA3A have been recently identified in patients with KS. In this study, we screened SEMA3A variants in a cohort of Chinese patients with IHH by whole exome sequencing. Three novel heterozygous SEMA3A variants (R197Q, R617Q and V458I) were identified in two nIHH and one KS patients, respectively. Functional studies indicated that R197Q and R617Q variants were ineffective in activating the phosphorylation of FAK (focal adhesion kinase) in GN11 cells, despite normal production and secretion in HEK293T cells. The V458I SEMA3A had defect in secretion as it was not detected in the conditioned medium from HEK293T cells. Compared with wild type SEMA3A protein, all three SEMA3A mutant proteins were ineffective in inducing the migration of GN11 cells. Our study further showed the contribution of SEMA3A loss-of-function variants to the pathogenesis of IHH.

PROKR2 mutations in idiopathic hypogonadotropic hypogonadism: selective disruption of the binding to a Gα-protein leads to biased signaling.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare disorder caused by the deficient production, secretion, or action of gonadotropin-releasing hormone. Prokineticin (PROK) receptor 2 ( PROKR2), a causative gene for IHH, encodes a GPCR PROKR2. When PROKR2 binds to its ligands PROKs, it may activate several signaling pathways, including IP3/Ca2+, MAPK, and cAMP pathways. However, the mutational spectrum of PROKR2 in Chinese patients with IHH has not been established. In the present study, we found that up to 13.3% (18/135) of patients with IHH in China carried mutations in PROKR2. Most of the variants in this study were private; however, a PROKR2 (c.533G > C; p.W178S) mutation was identified in 10 independent patients, implying a possible founder mutation. Functional studies indicated that 6 novel PROKR2 mutations led to decreased signaling to various extents. Two IHH-associated mutations (L218P and R270H) disrupted Gαq-dependent signaling but maintained normal Gαs and ERK1/2 signaling. A glutathione S-transferase pull-down experiment demonstrated that R270H mutation disrupted the interaction of intracellular loop 3 of PROKR2 to Gαq protein but not Gαs protein. Our results indicated that selective disruption of the interaction with a specific Gα-protein might underlie the biased signaling for certain IHH-associated PROKR2 mutations.-Zhao, Y., Wu, J., Jia, H., Wang, X., Zheng, R., Jiang, F., Chen, D.-N., Chen, Z., Li, J.-D. PROKR2 mutations in idiopathic hypogonadotropic hypogonadism: selective disruption of the binding to a Gα-protein leads to biased signaling.

Two new stilbenoids from aerial parts of Flickingeria fimbriata.

Two new stilbenoids, named 2,3 -dimethoxyl-7-hydroxyl-1,4-phenanthrenedione (1) and 2-methoxyl-3-methyl-7-hydroxyl-9,10-dihydro-1,4-phenanthrenedione (2), together with two known stilbenoids including densiflorol B (3) and ephemeranthoquinone (4), were isolated from aerial parts of Flickingeria fimbriata (Bl.) Hawkes. The structures of two new compounds were elucidated by extensive spectroscopic analysis, including HRESIMS, 1H and 13C NMR, DEPT, HMBC, COSY, HMQC, NOESY. All the compounds were obtained from this genus for the first time. In addition, they all exhibited moderate cytotoxic activities against HepG2 cell lines.

[A study on identification of edible bird's nests by DNA barcodes].

To provide theoretical basis for the traceability and quality evaluation of edible bird's nests (EBNs), the Cytb sequence was applied to identify the origin of EBNs. A total of 39 experiment samples were collected from Malaysia, Indonesia, Vietnam and Thailand. Genomic DNA was extracted for the PCR reaction. The amplified products were sequenced. 36 sequences were downloaded from Gen Bank including edible nest swiftlet, black nest swiftlet, mascarene swiftlet, pacific swiftlet and germain's swiftlet. MEGA 7.0 was used to analyze the distinction of sequences by the method of calculating the distances in intraspecific and interspecific divergences and constructing NJ and UPMGA phylogenetic tree based on Kimera-2-parameter model. The results showed that 39 samples were from three kinds of EBNs. Interspecific divergences were significantly greater than the intraspecific one. Samples could be successfully distinguished by NJ and UPMGA phylogenetic tree. In conclusion, Cytb sequence could be used to distinguish the origin of EBNs and it is efficient for tracing the origin species of EBNs.

Functional rescue of Kallmann syndrome-associated prokineticin receptor 2 (PKR2) mutants deficient in trafficking.

Mutations in the G protein-coupled prokineticin receptor 2 (PKR2) are known to cause Kallmann syndrome and idiopathic hypogonadotropic hypogonadism manifesting with delayed puberty and infertility. Some of the mutant receptors are not routed to the cell surface; instead, they are trapped in the cellular secretory pathway. The cell-permeant agonists/antagonists have been used to rescue some membrane receptors that are not targeted onto the cell membrane. Here, we chose three disease-associated mutations (W178S, G234D, and P290S), which all resulted in retention of PKR2 intracellularly. We show that a small molecule PKR2 antagonist (A457) dramatically increased cell surface expression and rescued the function of P290S PKR2, but had no effect on W178S and G234D PKR2. Furthermore, we also tested chemical chaperone glycerol on the cell surface expression and function of PKR2 mutants. Treatment with 10% glycerol significantly increased the cell surface expression and signaling of P290S and W178S PKR2. These data demonstrate that some Kallmann syndrome-associated, intracellularly retained mutant PKR2 receptors can be functionally rescued, suggesting a potential treatment strategy for patients bearing such mutations.

Development of a capillary zone electrophoresis method for rapid determination of human globin chains in α and ß-thalassemia subjects.

Thalassemia is an inherited autosomal recessive blood disorder characterized by the underproduction of globin chains as a consequence of globin gene defects, resulting in malfunctioning red blood cells and oxygen transport. Analysis of globin chains is an important aspect of thalassemia research. In this study we developed a capillary zone electrophoresis (CZE) method for human globin determination in the diagnosis of thalassemia and hemoglobin variants. To demonstrate the utility of this approach, α/ß area ratios were determined for samples from 310 thalassemia patients and healthy controls. The separation was performed on uncoated capillary with simple preparation. Distinct globin peaks were resolved in 17 min, and coefficients of variation (CV) for migration time and areas ranged from 0.37%-1.69% and 0.46%-6.71%, respectively. Receiver operating characteristic (ROC) curve analysis of the α/ß area ratios gave 100% sensitivity and specificity for indicating ß-TI/TM, and 100% sensitivity and 97.4% specificity for Hb H disease. Hemoglobin G-Honolulu (Hb G-Honolulu) and Hb Westmead (Hb WS) were successfully detected using this CZE method. This automated methodology is simple, rapid and cost-effective for the fast determination of human globin chains, which could be an important diagnostic tool in the field of hemoglobinopathies.

Photocatalytic Unsymmetrical Diamination of Styrenes, Indoles, and Benzofurans Facilitated by Benzotriazolyl and Iminyl Radicals.

Utilizing energy transfer catalysis, this research employed the bifunctional reagents benzotriazole carboxylic acid oxime esters to simultaneously generate benzotriazole and imine radicals. The synthesis of two distinct C-N bonds in a single conversion is showcased through radical addition and radical-radical cross-coupling processes between benzotriazole carboxylic acid oxime ester and olefins. This process facilitates the intermolecular two-component unsymmetrical diamination reaction of olefins. Using this approach, more than 40 benzotriazole-containing molecules were successfully synthesized using styrene, indole, and benzofuran as acceptors, with yields ranging from moderate to excellent.

Functional analysis of the distal region of the third intracellular loop of PROKR2.

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270-272) resulted in undetectable cell surface expression, whereas RKR (264-266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.

Photoinduced Difunctionalization of Diazenes Enabled by N-N Radical Coupling.

In this study, a metal-free difunctionalization strategy for diazenes was developed using a range of bifunctionalization reagents. This strategy involves a unique N(sp3)-N(sp2) radical coupling between the hydrazine radical and the imine radical. More than 30 triazane core motifs were constructed by installing imines and various functional groups, including alkyl, phenyl, cyanoalkyl, and sulfonyl groups, on both ends of the nitrogen-nitrogen bond of diazenes in an efficient manner.

The Merger of Halogen Atom Transfer (XAT) and Energy Transfer Catalysis (EnT) for the Modular 1,2-Iminylalkylation of Diazenes.

The 1,2-iminylalkylation of diazenes using alkyl iodides in combination with an O-benzoyl oxime is reported. In this transformation, O-benzoyl oxime acted as a radical precursor and XAT mediator. In addition to common alkyl iodides, other alkyl iodides such as iodomethane, iodomethane-d3, trifluoroiodomethane, ethyl difluoroiodoacetate, and iodoalkanes containing unprotected hydroxyl and amide groups can also serve as C-radical precursors in the 1,2-iminylalkylation with electrophilic diazenes as radical acceptors.

SLIT2 Rare Sequencing Variants Identified in Idiopathic Hypogonadotropic Hypogonadism.

INTRODUCTION: Idiopathic hypogonadotropic hypogonadism (IHH) is a rare reproductive disorder resulting from gonadotropin-releasing hormone (GnRH) deficiency. However, in only approximately half of patients with IHH is it possible to identify a likely molecular diagnosis. Mice lacking Slit2 have a reduced number or altered patterning of GnRH neurons in the brain. In order to assess the contribution of SLIT2 to IHH, we carried out a candidate gene burden test analysis. METHODS: A total of 196 IHH probands and 2,362 ethic-matched controls were recruited for this study. The IHH probands and controls were subjected to whole-exome sequencing. In the IHH patients with SLIT2 variants and their available family members, detailed phenotyping and segregation analysis were performed. RESULTS: Nine heterozygous SLIT2 rare sequencing variants (RSVs) were identified in 13 probands, with a prevalence of 6.6%. Furthermore, we identified an increased mutational burden for SLIT2 in this cohort (odds ratio = 2.2, p = 0.021). The segregation analysis of available IHH families revealed that the majority of SLIT2 RSVs were inherited from unaffected or partially affected parents. CONCLUSION: Our study suggests SLIT2 as a new IHH-associated gene and expands the clinical and genetic spectrum of IHH. Furthermore, SLIT2 alone does not appear to be sufficient to cause the disorder, and it may interact with other IHH-associated genes to induce a clinical phenotype.

Analysis of PLXNA1, NRP1, and NRP2 variants in a cohort of patients with isolated hypogonadotropic hypogonadism.

BACKGROUND: Isolated hypogonadotropic hypogonadism (IHH) is a clinical syndrome described by failure of gonadal function secondary to defects on the synthesis, secretion, or action of the gonadotropin-releasing hormone (GnRH). The secreted glycoprotein SEMA3A binds its receptors NRP1 or NRP2 and PLXNA to participate in axonal projection, dendritic branching, synaptic formation, and neuronal migration. Deficiency in SEMA3A, NRP1, NRP2, and PLXNA1 have been related to abnormal GnRH neuron development in mice and IHH in humans. METHODS: The aim of this study was to examine the genotypic and phenotypic spectra of the NRP1, NRP2, and PLXNA1 genes in a large cohort of IHH probands from China. We screened NRP1, NRP2, and PLXNA1 variants in Chinese IHH patients by whole exome sequencing and pedigree analysis. RESULTS: We identified 10 heterozygous missense variants in PLXNA1, five heterozygous missense variants in NRP1, and two heterozygous missense variants in NRP2. NRP1 variants were found only in IHH patients with defective olfaction (i.e., Kallmann syndrome, KS). In addition, 85% (17/20) of patients harbored variants in other IHH-associated genes. CONCLUSION: Our study greatly enriched the genotypic and phenotypic spectra of PLXNA1, NRP1, and NRP2 in IHH. It may be conducive to the genetic counseling, diagnosis, and treatment of IHH with mutations in the PLXNA1, NRP1, and NRP2 genes. Furthermore, our results indicated that NRP1 were strongly linked to hearing loss.

Genotypic and phenotypic spectrum of CCDC141 variants in a Chinese cohort with congenital hypogonadotropic hypogonadism.

OBJECTIVE: To identify CCDC141 variants in a large Chinese cohort with congenital hypogonadotropic hypogonadism (CHH) and to assess the contribution of CCDC141 to CHH. DESIGN: Detailed phenotyping was conducted in CHH patients with CCDC141 variants and co-segregation analysis was performed, when possible. METHODS: Whole-exome sequencing was performed in 177 CHH patients and 450 unrelated, ethnically matched controls from China. RESULTS: Seven novel CCDC141 rare sequencing variants (RSVs) were identified in 12 CHH pedigrees. Four of the variants were private mutations; however, p.Q409X, p.Q871X and p.G1488S were identified in more than one patient. Up to 75% (9/12) of patients had mutations in other CHH-associated genes, which is significantly higher than CHH patients without CCDC141 RSVs. The co-segregation analysis for eight CHH families showed that 75% (6/8) CCDC141 RSVs were inherited from their fertile parents. Over half (58.3%, 8/18) of the patients exhibited other clinical deformities in addition to hypogonadism. One patient harbouring a CCDC141 RSV showed a reversal of CHH after sex-steroid replacement. CONCLUSIONS: Our results broaden the genotypic spectrum of CCDC141 in CHH, as CCDC141 RSVs alone do not appear sufficient to cause CHH. The phenotypic spectrum in patients with CCDC141 RSVs is much wider than originally believed.

Stilbenoids from aerial parts of Dendrobium plicatile.

Chemical investigation of Dendrobium plicatile Lindl resulted in the isolation and identification of one new bibenzyl, 2-chloro-3, 4'-dihydroxy-3',5-dimethoxybibenzyl (1), as well as 15 known stilbenoids. The structures of this new compound was elucidated by extensive spectroscopic analysis, including HRESIMS, 1H and 13C NMR, DEPT, HMBC, COSY, HMQC, NOESY. Compounds 2, 3 and 5 were obtained from this genus for the first time, compounds 8, 10, 13 and 14 were obtained from this plant for the first time. In addition, the new compound exhibited potent cytotoxic activities against the human breast cancer (MDA-MB231) cell line, the hepatocellular carcinoma (HepG2) cell line and the human lung carcinoma (A549) cell line, with IC50 3.41, 3.02, 2.80 µM, respectively.

Phenotypic Spectrum of Idiopathic Hypogonadotropic Hypogonadism Patients With CHD7 Variants From a Large Chinese Cohort.

PURPOSE: Idiopathic hypogonadotropic hypogonadism (IHH) and CHARGE (C, coloboma; H, heart abnormalities; A, choanal atresia, R, retardation of growth and/or development; G, gonadal defects; E, ear deformities and deafness) syndrome are 2 distinct developmental disorders sharing features of hypogonadism and/or impaired olfaction. CHD7 variants contribute to >60% CHARGE syndrome and ~10% IHH patients. A variety of extended CHARGE-like features are frequently reported in CHARGE patients harboring CHD7 variants. In this study, we aimed to systematically analyze the diagnostic CHARGE features and the extended CHARGE-like features in patients with IHH with CHD7 variants. METHODS: Rare sequencing variants (RSVs) in CHD7 were identified through exome sequencing in 177 IHH probands. Detailed phenotyping was performed in the IHH patients harboring CHD7 variants and their available family members. RESULTS: CHD7 RSVs were identified in 10.2% (18/177) of the IHH probands. Two diagnostic CHARGE features, hearing loss and ear deformities, were significantly enriched in patients with CHD7 variants. Furthermore, CHD7 variants were significantly associated with a panel of extended CHARGE-like phenotypes, including mild ocular defects, dyspepsia/gastroesophageal reflux disease and skeletal defects. We also developed a predictive model for prioritizing CHD7 genetic testing in IHH patients. CONCLUSION: CHD7 variants rarely cause isolated IHH. Surveillance of symptoms in CHARGE syndrome-affected organs will facilitate the proper treatment for these patients. Certain clinical features can be useful for prioritizing CHD7 genetic screening.

[Analysis of Human Herpes Viruses-Activated Infection Spectra in Patients with Various Immunodeficiencies].

OBJECTIVE: To study the epidemiologic characteristics of human herpes virus (HHV) activated infection in the diseases of blood system and patients received allo-HSCT by statistically analyzing the screening results of 8 human herpes viruses (HHVs) of 4164 patients in Hebei Yanda LU Dao-Pei Hospital from 2012 to 2017. METHODS: PCR was used to screen 8 HHVs. RESULTS: Two thousand and fifty-two patients (49.28%) were HHV-positive among 4164 patients screened. Among these patients screened, the infection spectra of 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation of totally 2994 patients were summarized as follows: the positive rate of EBV (29.49%) was the highest, that of HCMV (23.15%), HHV-6 was 18.77% and HHV-7 was 17.64%, while the remaining 4 HHVs all≤2.1%. The rate of co-infection of various HHVs was significantly higher than that of single infection of HHV among all these disease groups except familial hemophagocytic lymphohistiocytosis, for which single EBV infection was the most common. The differences of positive rates among these 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation were statistically significant by Chi-square test of R*C tables (χ2=54.99, P＜0.05). For each HHV, the differences of positive rates among the above-mentioned disease groups were also statistically significant except HHV-8 (P＜0.05). CONCLUSION: The patients with various blood diseases have different activated infection spectra of HHVs. EBV, HCMV, HHV-6 and HHV-7 are most common in HHVs infection. Different HHVs infections correlate with different hematologion diseases.

Prokineticins and their G protein-coupled receptors in health and disease.

Prokineticins are two conserved small proteins (~8kDa), prokineticin 1 (PROK1; also called EG-VEGF) and prokineticin 2 (PROK2; also called Bv8), with an N-terminal AVITGA sequence and 10 cysteines forming 5 disulfide bridges. PROK1 and PROK2 bind to two highly related G protein-coupled receptors (GPCRs), prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). Prokineticins and their receptors are widely expressed. PROK1 is predominantly expressed in peripheral tissues, especially steroidogenic organs, whereas PROK2 is mainly expressed in the central nervous system and nonsteroidogenic cells of the testes. Prokineticins signaling has been implicated in several important physiological functions, including gastrointestinal smooth muscle contraction, circadian rhythm regulation, neurogenesis, angiogenesis, pain perception, mood regulation, and reproduction. Dysregulation of prokineticins signaling has been observed in a variety of diseases, such as cancer, ischemia, and neurodegeneration, in which prokineticins signaling seems to be a promising therapeutic target. Based on the phenotypes of knockout mice, PROKR2 and PROK2 have recently been identified as causative genes for idiopathic hypogonadotropic hypogonadism, a developmental disorder characterized by impaired development of gonadotropin-releasing hormone neurons and infertility. In vitro functional studies with these disease-associated PROKR2 mutations uncovered some novel features for this receptor, such as biased signaling, which may be used to understand GPCR signaling regulation in general.

Functional analysis of SOX10 mutations identified in Chinese patients with Kallmann syndrome.

Kallmann syndrome (KS) is characterized by the association of anosmia and hypogonadotropic hypogonadism. The hypogonadotropic hypogonadism is due to deficient production, secretion or action of gonadotropin-releasing hormone (GnRH). Mutations in transcription factor SOX10 have been recently identified in patients with KS and hearing loss. In this study, we identified three novel SOX10 mutations in a cohort of Chinese KS patients by using exome sequencing. Two mutations (A44G and L80V) are in heterozygous state whereas the other one (G41V) is a homozygous mutation. The patient with a homozygous G41V mutation had impaired hearing in both ears, whereas the patient with a heterozygous L80V mutation showed subtle hearing impairment in the left ear. Functional studies indicated that all three SOX10 mutations showed reduced capacity to transactivate the MITF promoter alone or in synergy with PAX3, although they showed similar subcellular localization, and DNA binding ability. Our study further highlighted the significance of SOX10 haploinsufficiency as a genetic cause of KS with hearing problem.

RNF216 Regulates the Migration of Immortalized GnRH Neurons by Suppressing Beclin1-Mediated Autophagy.

RNF216, encoding an E3 ubiquitin ligase, has been identified as a causative gene for Gordon Holmes syndrome, characterized by ataxia, dementia, and hypogonadotropic hypogonadism. However, it is still elusive how deficiency in RNF216 leads to hypogonadotropic hypogonadism. In this study, by using GN11 immature GnRH neuronal cell line, we demonstrated an important role of RNF216 in the GnRH neuron migration. RNA interference of RNF216 inhibited GN11 cell migration, but had no effect on the proliferation of GN11 cells or GnRH expression. Knockdown of RNF216 increased the protein levels of its targets, Arc and Beclin1. RNAi of Beclin1, but not Arc, normalized the suppressive effect caused by RNF216 knockdown. As Beclin1 plays a critical role in the autophagy regulation, we further demonstrated that RNAi of RNF216 led to increase in autophagy, and autophagy inhibitor CQ and 3-MA rescued the GN11 cell migration deficit caused by RNF216 knockdown. We further demonstrated that pharmacological increase autophagy by rapamycin could suppress the GN11 cell migration. We thus have identified that RNF216 regulates the migration of GnRH neuron by suppressing Beclin1 mediated autophagy, and indicated a potential contribution of autophagy to the hypogonadotropic hypogonadism.