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The article discusses how President of the People's Republic of China, Xi Jinping has consolidated power within China since assuming leadership in 2012. It examines his crackdown on dissenting voices and ideologies, his anti-corruption campaign, and military reforms as a means to solidify his position within the Communist Party. It also explores the risks of miscalculation in China's foreign policies without dissenting voices to offer alternative perspectives.

GNAQ and GNA11 are heterotrimeric G protein alpha subunits, which are mutated in a mutually exclusive pattern in most cases of uveal melanoma, one of the most aggressive cancers. Here we introduce the first transgenic mouse model of uveal melanoma, which develops cancers induced by expression of oncogenic GNAQQ209L under control of the Rosa26 promoter. Disease penetrance is 100% by 3 months of age, with 94% of mice also developing lung tumors. In this model, the Yap protein of the Hippo pathway is activated in the eyes, and blood vessels near the lesions in the head and lungs exhibit melanocytic invasion. While full transcription levels are not necessary for GNAQQ209L to transform mouse melanocytes, we obtained suggestive evidence of a selective advantage for increased GNAQQ209L expression in human tumors. Intriguingly, enforced expression of GNAQQ209L progressively eliminated melanocytes from the interfollicular epidermis in adults, possibly explaining the near absence of GNAQQ209 mutations in human epithelial melanomas. The mouse model also exhibited dermal nevi and melanocytic neoplasms of the central nervous system, accompanied by impaired hearing and balance, identifying a novel role for GNAQ inmelanocyte-like cells of the inner ear. Overall, this model offers a new tool to dissect signaling by oncogenic GNAQ and to test potential therapeutics in an in vivo setting where GNAQQ209L mutations contribute to both the initiation and metastatic progression of uveal melanoma. [ABSTRACT FROM AUTHOR]

When mutations in two different genes produce the same mutant phenotype, it suggests that the encoded proteins either interact with each other, or act in parallel to fulfill a similar purpose. Haploinsufficiency of Neurofibromin and over-expression of Endothelin 3 both cause increased numbers of melanocytes to populate the dermis during mouse development, and thus we are interested in how these two signaling pathways might intersect. Neurofibromin is mutated in the human genetic disease, neurofibromatosis type 1, which is characterized by the development of Schwann cell based tumors and skin hyper-pigmentation. Neurofibromin is a GTPase activating protein, while the Endothelin 3 ligand activates Endothelin receptor B, a G protein coupled receptor. In order to study the genetic interactions between endothelin and neurofibromin, we defined the deletion breakpoints of the classical Ednrb piebald lethal allele (Ednrbs-l) and crossed these mice to mice with a loss-of-function mutation in neurofibromin, Dark skin 9 (Dsk9). We found that Neurofibromin haploinsufficiency requires Endothelin receptor B to darken the tail dermis. In contrast, Neurofibromin haploinsufficiency increases the area of the coat that is pigmented in Endothelin receptor B null mice. We also found an oncogenic mutation in the G protein alpha subunit, GNAQ, which couples to Endothelin receptor B, in a uveal melanoma from a patient with neurofibromatosis type 1. Thus, this data suggests that there is a complex relationship between Neurofibromin and Endothelin receptor B. [ABSTRACT FROM AUTHOR]

Abstract: The intercalated disc (ICD) is a unique structure to the heart and plays vital roles in communication and signaling among cardiomyocytes. ICDs are formed and matured during postnatal development through a profound redistribution of the intercellular junctions, as well as recruitment and assembly of more than 200 proteins at the termini of cardiomyocytes. The molecular mechanism underlying this process is not completely understood. The mouse orthologs (mXinα and mXinβ) of human cardiomyopathy-associated (CMYA)/Xin actin-binding repeat-containing protein (XIRP) genes (CMYA1/XIRP1 and CMYA3/XIRP2, respectively) encode proteins localized to ICDs. Ablation of mXinα results in adult late-onset cardiomyopathy with conduction defects and up-regulation of mXinβ. ICD structural defects are found in adult but not juvenile mXinα-null hearts. On the other hand, loss of mXinβ leads to ICD defects at postnatal day 16.5, a developmental stage when the heart is forming ICDs, suggesting mXinβ is required for ICD formation. Using quantitative Western blot, we showed in this study that mXinβ but not mXinα was uniquely up-regulated during the redistribution of intercellular junction from the lateral membrane of cardiomyocytes to their termini. In the absence of mXinβ, the intercellular junctions failed to be restricted to the termini of the cells, and the onset of such defect correlated with the peak expression of mXinβ. Immunofluorescence staining and subcellular fractionation showed that mXinβ preferentially associated with the forming ICDs, further suggesting that mXinβ functioned locally to promote ICD maturation. In contrast, the spatiotemporal expression profile of mXinα and the lack of more severe ICD defects in mXinα−/−;mXinβ−/− double knockout hearts than in mXinβ−/− hearts suggested that mXinα was not essential for the postnatal formation of ICDs. A two-step model for the development of ICD is proposed where mXinβ is essential for the redistribution of intercellular junction components from the lateral puncta to the cell termini. [Copyright &y& Elsevier]

Mutations in neurofibromin (NF1) cause the dominant genetic disorder neurofibromatosis type 1. Neurofibromatosis is characterized by Schwann cell-based tumors and skin hyperpigmentation, resulting from both haploinsufficiency and loss of heterozygosity. The fact that some pigment cells (melanocytes) arise from Schwann cell precursors suggests that neurofibromin could be required during the common precursor stage. In this study, we found a missense mutation in neurofibromin in Dark skin 9 (Dsk9) mutant mice, revealing that Nf1 mutations cause skin hyperpigmentation in mice, as they do in humans. Using tissue-specific knockouts, we found that haploinsufficiency of neurofibromin in melanocytes via Mitf-cre is insufficient to cause darker skin, whereas haploinsufficiency in bipotential Schwann cell-melanoblast precursors via Plp1-creER is sufficient. These findings suggest that there is a narrow developmental window during which Nf1 haploinsufficiency acts on pigment cells. Using fate mapping, we discovered differences in the colonization of the dermis and epidermis by melanocytes that arise from Schwann cell precursors, an unexpected complexity of melanocyte development. As homozygous knockout of Nf1 via Mitf-cre is sufficient to cause darker skin, we conclude that reduced gene dosage can act by a mechanism different from complete gene loss, even when the end result of both is very similar. [ABSTRACT FROM AUTHOR]

SATB2-associated syndrome (SAS) is a multisystemic disorder characterized by developmental delay often with concurrent autistic tendencies. This study aimed to characterize cellular metabolic pathways and energy metabolism from cells derived from individuals with SAS. The cellular production of NADH (nicotinamide adenine dinucleotide, reduced form) as determined by the Phenotype Mammalian MicroArrays was measured in lymphoblastoid cell lines derived from 11 subjects with a molecularly confirmed diagnosis of SAS and compared to a control population of 50 age-matched typically developing individuals. All patients were evaluated clinically by a multidisciplinary team. Eleven individuals (five in a screening cohort and six in the validation cohort, mean age 6.1 years) were recruited to the study. All individuals had developmental delay and the diagnosis of autism was previously established in five of them. Key metabolic findings included reduced NADH production in the presence of phosphorylated carbohydrates (with corresponding increased production in the presence of alternative carbon-based energy sources), increased response to certain hormones (β-estradiol in particular), and significantly reduced levels of NADH in wells containing tryptophan. The individual analysis revealed no particular differences among the SAS subjects based on molecular findings or phenotypic features. In conclusion, individuals with SAS have a common and recognizable metabolic profile. A lower capacity to utilize glucose as an energy substrate could be contributing to the neurodevelopment phenotype of SAS. The identified abnormalities offer previously unexplored insight into the potential pathophysiology of common SAS phenotypic features. [ABSTRACT FROM AUTHOR]

Rationale: Nav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage-gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods: Unique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Results: Nav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/µg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/µg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/µg membrane proteins. Conclusions: Multiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain. [ABSTRACT FROM AUTHOR]

Background: Seizures are an under-reported feature of the SATB2-associated syndrome phenotype. We describe the electroencephalographic findings and seizure semiology and treatment in a population of individuals with SATB2-associated syndrome.Methods: We performed a retrospective review of 101 individuals with SATB2-associated syndrome who were reported to have had a previous electroencephalographic study to identify those who had at least one reported abnormal result. For completeness, a supplemental survey was distributed to the caregivers and input from the treating neurologist was obtained whenever possible.Results: Forty-one subjects were identified as having at least one prior abnormal electroencephalography. Thirty-eight individuals (93%) had epileptiform discharges, 28 (74%) with central localization. Sleep stages were included as part of the electroencephalographies performed in 31 individuals (76%), and epileptiform activity was recorded during sleep in all instances (100%). Definite clinical seizures were diagnosed in 17 individuals (42%) with a mean age of onset of 3.2 years (four months to six years), and focal seizures were the most common type of seizure observed (42%). Six subjects with definite clinical seizures needed polytherapy (35%). Delayed myelination and/or abnormal white matter hyperintensities were seen on neuroimaging in 19 individuals (61%).Conclusions: Epileptiform abnormalities are commonly seen in individuals with SATB2-associated syndrome. A baseline electroencephalography that preferably includes sleep stages is recommended during the initial evaluation of all individuals with SATB2-associated syndrome, regardless of clinical suspicion of epilepsy. [ABSTRACT FROM AUTHOR]

The G‐protein‐coupled receptor, endothelin receptor B (EDNRB), is an important regulator of melanocyte survival and proliferation. It acts by stimulating downstream heterotrimeric G proteins, such as Gαq and Gα1. Constitutively active, oncogenic versions of Gαq and Gα11 drive melanomagenesis, but the role of Ednrb in the context of these mutant G proteins has not been previously examined. In this paper, we used a knock‐in mouse allele at the Rosa26 locus to force oncogenic GNAQQ209L expression in melanocytes in combination with Ednrb gene knockout. The resulting pathological analysis revealed that every aspect of melanomagenesis driven by GNAQQ209L was inhibited. We conclude that even in the presence of oncogenic Gαq, the Ednrb receptor activates normal Gαq and Gα11 proteins. This likely promotes tumorigenesis by activating phospholipase C‐beta, the immediate effector of Gαq/11. These findings suggest that it might be possible to target upstream receptors to offset the effects of hyperactive G proteins, recognized as the cause of a growing number of human disorders. [ABSTRACT FROM AUTHOR]

Test coverage is sometimes used to measure how thoroughly software is tested and developers and vendors sometimes use it to indicate their confidence in the readiness of their software. This survey studies and compares 17 coverage-based testing tools primarily focusing on, but not restricted to, coverage measurement. We also survey features such as program prioritization for testing, assistance in debugging, automatic generation of test cases and customization of test reports. Such features make tools more useful and practical, especially for large-scale, commercial software applications. Our initial motivations were both to understand the available test coverage tools and to compare them to a tool that we have developed, called eXVantage (a tool suite that includes code coverage testing, debugging, performance profiling and reporting). Our study shows that each tool has some unique features tailored to its application domains. The readers may use this study to help pick the right coverage testing tools for their needs and environment. This paper is also valuable to those who are new to the practice and the art of software coverage testing, as well as those who want to understand the gap between industry and academia. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae. NiV is deadly to humans, infecting host cells by direct fusion of the viral and host cell plasma membranes. This membrane fusion process is coordinated by the receptorbinding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scanning mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion pore expansion during syncytium formation, also uncovering a link between F-G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F-triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections. [ABSTRACT FROM AUTHOR]

Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members. [ABSTRACT FROM AUTHOR]

Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are BSL-4 paramyxoviruses in the growing genus, Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, pathognomonic of henipaviral infections. Here we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (HA tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F/G interactions, supporting the F/G dissociation model of henipaviral-induced membrane fusion, even in the context of heterologous glycoprotein pairs. [ABSTRACT FROM AUTHOR]

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell--specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell--specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies. [ABSTRACT FROM AUTHOR]

Background: Many sexually transmitted diseases are asymptomatic in the lower genital tract and can cause upper tract complications if left untreated. Self-collected vaginal (SCV) swabs enable the accurate detection of many sexually transmitted infections and give women the option of collecting their own samples while providing them with privacy and convenience.Methods: We compared SCV samples collected and transported dry using the HerSwab device to physician-collected vaginal (PCV) Aptima swabs for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), and measured patients' ease and comfort with self-collection. A total of 189 women aged 16 to 41 years were consented into the study and answered a standardized anonymized questionnaire regarding self-collection with the HerSwab device.Results: Women reported self-collection with HerSwab to be easy (97.1%) and comfortable (88.3%). They preferred self-collection over physician collection (80.9%) and would consider using HerSwab for self-collection at home (79.7%). Samples of SCV and PCV showed an overall agreement of 94.7% (κ = 0.64) for CT and of 98.4% (κ = 0.56) for NG, and HerSwab collection detected 7 more positive patients than PCV collection. The overall prevalence of infection was 10.6% for CT and 2.6% for NG.Conclusion: HerSwab SCV samples are suitable for the diagnosis of CT and NG. [ABSTRACT FROM AUTHOR]

Abstract: The Xin repeat-containing proteins, Xinα (Xirp1) and Xinβ (Xirp2), localize to the intercalated discs (ICDs) of mammalian hearts. Mouse Xinα (mXinα) directly interacts with β-catenin and actin filaments, potentially coupling the N-cadherin/β-catenin complexes to the underlying actin cytoskeleton and modulating ICD integrity and function. Supporting this possibility, mXinα-null hearts develop ICD structural defects and cardiomyopathy with conduction defects. However, the underlying mechanisms leading to these defects remain unclear. Here, we showed that mXinα also interacted with p120-catenin and cortactin. Different from the β-catenin binding domain, there existed multiple p120-catenin binding sites on mXinα, while only the extreme N-terminus of mXinα containing a SH3-binding motif could interact with cortactin. In mouse heart, a significant fraction of cortactin was co-localized with N-cadherin to ICDs, whereas in mXinα-null heart, this fraction of cortactin was drastically reduced. Therefore, mXinα may modulate ICD integrity and function through its interactions with catenins and cortactin. Analyses of the in vivo consequence of p120-catenin and mXinα interaction revealed that force-expressed mXinα or its fragments significantly suppressed the p120-catenin-induced branching phenotypes. It is known that p120-catenin directly regulates Rho GTPases, leading to the branching phenotype. Thus, mXinα may sequester the p120-catenin from inhibiting RhoA activity and/or from activating Rac1 activity. [Copyright &y& Elsevier]