Somatic genetics analysis of sleep in adult mice.

Classical forward and reverse mouse genetics require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct electroencephalogram/electromyogram-based mouse sleep screening owing to labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)-expression/knockout (KO) platform for somatic genetics analysis of sleep in adult male or female mice. A pilot ABC screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and/or quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy episodes, enabling one-step analysis of redundant genes in adult mice. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping mouse development and minimizing genetic crosses.SIGNIFICANCE STATEMENTThe molecular mechanisms of mammalian sleep regulation remain unclear. Classical germline mouse genetics are unwieldy to study sleep functions of essential genes or redundant pathways. The EEG/EMG-based mouse sleep screening is time-consuming owing to labor-intensive surgeries and lengthy genetic crosses. To overcome these "bottlenecks", we developed a highly accurate video-based sleep analysis system and adeno-associated virus-mediated ABC-expression/knockout platform for somatic genetics analysis of sleep in adult mice. These methodologies facilitate rapid identification of sleep regulatory genes, but also efficient mechanistic studies of the molecular pathways of sleep regulation in mice.

Constructing a general competency model for Chinese public health physicians: a qualitative and quantitative study.

BACKGROUND: The purpose of the study was to construct a general model of the competencies required by Chinese public health physicians. This study is intended to lay the foundation for promoting reform of public health education in China, and may help perfect the testing and grading system of public health courses. METHODS: The behavioral event interviews were used to collect data on knowledge, skills and performance characteristics of public health physicians for coping with public health events. A random stratified sampling survey was used to select public health professionals and workers from different public health institutions in 14 provinces (municipalities or autonomous regions) of China. We then purposively selected 150 senior public health professionals from various agencies and 85 senior public health educators at colleges and universities to take part in the checklist survey. Finally, three times expert consultations were performed to determine the dimensions and elements of the general competency model for Chinese public health physicians. RESULTS: The age range of the 3245 participants was 18-65 years old. When comparing public health professionals and public health education specialists, we found significant differences in the perceived importance of the core competencies between these groups. The model was revised several times and finalized through the expert consultations, and comprised seven competency dimensions. CONCLUSION: The final public health practitioner competency model comprised seven competency dimensions: basic knowledge of medicine, public health knowledge, health management and health promotion, specialized skills, research and development, comprehensive abilities and professionalism.

σ(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria.

Mycobacterium tuberculosis adopts various strategies to cope with oxidative stress during infection. Transcriptional regulators, including σ factors, make important contributions to this stress response, but how these proteins cooperate with each other is largely unknown. In this study, the role of RbpA and its cooperation with σ factors in response to oxidative stress are investigated. Knock down expression of rbpA in Mycobacterium smegmatis attenuated bacterial survival in the presence of H2 O2 . Additionally, transcription of the rbpA gene was induced by H2 O2 in a σ(E) -dependent manner. After induction, RbpA interacts with the principal sigma factor, σ(A) , to control the transcription of furA-katG operon, which encodes an H2 O2 scavenging enzyme. Moreover, this regulation is responsible for the role of σ(E) in oxidative response because bacterial survival was attenuated and transcription of the furA-katG operon was down-regulated with H2 O2 treatment in sigE deletion mutant (ΔsigE), and over-expression of RbpA in ΔsigE strain restored all of these phenotypes. Taken together, our study first illustrated a mechanism for σ(E) in response to oxidative stress through regulation of rbpA transcription. This study was also the first to demonstrate that RbpA is required for the full response to oxidative stress by cooperating with the principal σ(A) .

Genome-wide characterization of monomeric transcriptional regulators in Mycobacterium tuberculosis.

Gene transcription catalysed by RNA polymerase is regulated by transcriptional regulators, which play central roles in the control of gene transcription in both eukaryotes and prokaryotes. In regulating gene transcription, many regulators form dimers that bind to DNA with repeated motifs. However, some regulators function as monomers, but their mechanisms of gene expression control are largely uncharacterized. Here we systematically characterized monomeric versus dimeric regulators in the tuberculosis causative agent Mycobacterium tuberculosis. Of the >160 transcriptional regulators annotated in M. tuberculosis, 154 transcriptional regulators were tested, 22 % probably act as monomers and most are annotated as hypothetical regulators. Notably, all members of the WhiB-like protein family are classified as monomers. To further investigate mechanisms of monomeric regulators, we analysed the actions of these WhiB proteins and found that the majority interact with the principal sigma factor σA, which is also a monomeric protein within the RNA polymerase holoenzyme. Taken together, our study for the first time globally classified monomeric regulators in M. tuberculosis and suggested a mechanism for monomeric regulators in controlling gene transcription through interacting with monomeric sigma factors.

Lactate metabolism is essential in early-onset mitochondrial myopathy.

Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.

Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis.

Mitochondrial DNA (mtDNA) mutations are frequently observed in cancer, but their contribution to tumor progression is controversial. To evaluate the impact of mtDNA variants on tumor growth and metastasis, we created human melanoma cytoplasmic hybrid (cybrid) cell lines transplanted with wildtype mtDNA or pathogenic mtDNA encoding variants that partially or completely inhibit oxidative phosphorylation. Homoplasmic pathogenic mtDNA cybrids reliably established tumors despite dysfunctional oxidative phosphorylation. However, pathogenic mtDNA variants disrupted spontaneous metastasis of subcutaneous tumors and decreased the abundance of circulating melanoma cells in the blood. Pathogenic mtDNA did not induce anoikis or inhibit organ colonization of melanoma cells following intravenous injections. Instead, migration and invasion were reduced, indicating that limited circulation entry functions as a metastatic bottleneck amidst mtDNA dysfunction. Furthermore, analysis of selective pressure exerted on the mitochondrial genomes of heteroplasmic cybrid lines revealed a suppression of pathogenic mtDNA allelic frequency during melanoma growth. Collectively, these findings demonstrate that functional mtDNA is favored during melanoma growth and enables metastatic entry into the blood.

Autosomal recessive progeroid syndrome due to homozygosity for a TOMM7 variant.

Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here, we report a 21-year-old man of Chinese ancestry who has an autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia, and partial lipodystrophy. Analyses of exome sequencing data from the entire family revealed only 1 rare homozygous missense variant (c.86C>T; p.Pro29Leu) in TOMM7 in the proband, while the parents and 2 unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex makes up the outer membrane pore, which is responsible for importing many preproteins into the mitochondria. A proteomic comparison of mitochondria from control and proband-derived cultured fibroblasts revealed an increase in abundance of several proteins involved in oxidative phosphorylation, as well as a reduction in abundance of proteins involved in phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared with control fibroblasts. We concluded that altered mitochondrial protein import due to biallelic loss-of-function TOMM7 can cause severe growth retardation and progeroid features.

Differential requirements for mitochondrial electron transport chain components in the adult murine liver.

Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue-specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.

Mitochondria are specialised structures inside cells that help to convert nutrients into energy. They take electrons from nutrients and use them to power biochemical reactions that supply chemical fuel. Previous studies of cells grown in the laboratory have found that electrons enter this process via a large assembly of proteins in mitochondria called complex I. Understanding the mechanism of energy production is important, as issues with mitochondria can lead to a variety of metabolic diseases. However, it is still unclear how complex I acts in living animals. Lesner et al. addressed this knowledge gap by genetically removing a key protein from complex I in the liver of mice. Surprisingly, the animals did not develop any detectable symptoms and maintained healthy liver function. Mice did not seem to compensate by making energy in a different way, suggesting that complex I is not normally used by the mouse liver for this process. This research suggests that biologists should reconsider the mechanism that mitochondria use to power cells in animals. While the role of Complex I in electron transfer is well established in laboratory-grown cells and some organs, like the brain, it cannot be assumed this applies to the whole body. Understanding energy production in specific organs could help researchers to develop nutrient-based therapies for metabolic diseases.

Determinate factors of mental health status in Chinese medical staff: A cross-sectional study.

Numerous previous studies have investigated the mental health status of medical staff in China and explored its associated determinate factors; however, scope and methods associated with these have introduced uncertainty regarding the results. The aim of this study was to perform a comprehensive examination of the mental health status of Chinese medical staff and its relative risk factors based on a cross-sectional survey.We conducted a broad area, cross-sectional, questionnaire-based survey of Chinese medical workers. Participants were randomly selected from 27 hospitals in the Heilongjiang province. The questionnaire that was distributed consisted of 5 parts: the demographic characteristics of the participant; questions related to the relative risk factors of psychological health; the posttraumatic stress disorder (PTSD) Checklist-Civilian Version (PCL-C); the Self-rating Depression Scale (SDS); and the Self-rating Anxiety Scale (SAS). The last 3 components were used to evaluate the mental health status of the target population. Logistic and linear regression were used to analyze the determinate factors of the mental health status of Chinese medical staff.Of the 1679 questionnaires distributed, 1557 medical workers responded (response rate: 92.73%; male: 24.1%; female 75.9%). The results of mental health status self-assessments indicated that 32.3% of participants were considered to have some degree of PTSD (based on the PCL-C). The SDS index was 0.67 and the mean score from SAS was 55.26; a result higher than found in the general population. Multivariate logistic regression analysis revealed that being female, dissatisfaction or average satisfaction with income, and good or very good self-perceived psychological endurance when faced with an emergency were associated with a reduction of PTSD symptoms. A frequency of verbal abuse incidents greater than 4 was associated with an increase in PTSD symptoms.The mental health status of Chinese medical staff is poor. While the determinate factors based on different measurement standards were not completely consistent, the overlapping major risk factors identified that influenced psychological health were the amount of education, the perceived level of respect, and psychological endurance.

Mycobacterial WhiB6 Differentially Regulates ESX-1 and the Dos Regulon to Modulate Granuloma Formation and Virulence in Zebrafish.

During the course of infection, Mycobacterium tuberculosis (Mtb) is exposed to diverse redox stresses that trigger metabolic and physiological changes. How these stressors are sensed and relayed to the Mtb transcriptional apparatus remains unclear. Here, we provide evidence that WhiB6 differentially regulates the ESX-1 and DosR regulons through its Fe-S cluster. When challenged with NO, WhiB6 continually activates expression of the DosR regulons but regulates ESX-1 expression through initial activation followed by gradual inhibition. Comparative transcriptomic analysis of the holo- and reduced apo-WhiB6 complemented strains confirms these results and also reveals that WhiB6 controls aerobic and anaerobic metabolism, cell division, and virulence. Using the Mycobacterium marinum zebrafish infection model, we find that holo- and apo-WhiB6 modulate levels of mycobacterial infection, granuloma formation, and dissemination. These findings provide fresh insight into the role of WhiB6 in mycobacterial infection, dissemination, and disease development.