Unexpected cell type-dependent effects of autophagy on polyglutamine aggregation revealed by natural genetic variation in C. elegans.

BACKGROUND: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown. RESULTS: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells. CONCLUSIONS: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal.

Neuroendocrine mechanisms underlying social inhibition of puberty are not well understood. Here, we use a model exhibiting the most profound case of pubertal suppression among mammals to explore a role for RFamide-related peptide-3 [RFRP-3; mammalian ortholog to gonadotropin-inhibitory hormone (GnIH)] in neuroendocrine control of reproductive development. Naked mole rats (NMRs) live in sizable colonies where breeding is monopolized by two to four dominant animals, and no other members exhibit signs of puberty throughout their lives unless they are removed from the colony. Because of its inhibitory action on the reproductive axis in other vertebrates, we investigated the role of RFRP-3 in social reproductive suppression in NMRs. We report that RFRP-3 immunofluorescence expression patterns and RFRP-3/GnRH cross-talk are largely conserved in the NMR brain, with the exception of the unique presence of RFRP-3 cell bodies in the arcuate nucleus (Arc). Immunofluorescence comparisons revealed that central expression of RFRP-3 is altered by reproductive status, with RFRP-3 immunoreactivity enhanced in the paraventricular nucleus, dorsomedial nucleus, and Arc of reproductively quiescent NMRs. We further observed that exogenous RFRP-3 suppresses gonadal steroidogenesis and mating behavior in NMRs given the opportunity to undergo puberty. Together, our findings establish a role for RFRP-3 in preserving reproductive immaturity, and challenge the view that stimulatory peptides are the ultimate gatekeepers of puberty.

Persistent Mild Anemia and Hypercalcemia were Ignored as Normal Reaction Secondary to Oral Calcium Supplementation in a Steroid-Dependent Asthma Patient Ultimately Diagnosed as Multiple Myeloma: a Case Report and Literature Review.

BACKGROUND: Anemia can be secondary to many diseases and hypercalcemia can be secondary to oral calcium supplementation. For non-hematologists, anemia and hypercalcemia are usually ignored. Here we report a case of persistent mild anemia and hypercalcemia which were ignored as a normal reaction secondary to oral calcium supplementation in a steroid-dependent asthma patient; it was ultimately diagnosed as multiple myeloma. METHODS: Bone marrow puncture, combined serum, and urine laboratory indexes were performed for diagnosis. RESULTS: A bone marrow puncture specimen comprised 31.5% plasma cells. The serum and urine immunoelectrophoresis showed monoclonal kappa light chains. CONCLUSIONS: When anemia and hypercalcemia occur in an elderly patient, physicians should pay attention to multiple myeloma, especially when accompanied with vertebral and flat bone fractures.

Normal Tumor Markers and Increased Adenosine Deaminase in Pericardial Effusion Misdiagnosed as Tuberculous Pericarditis Ultimately Proven as Lung Adenocarcinoma with Pericardial Metastasis: a Case Report and Literature Review.

BACKGROUND: Elevated adenosine deaminase (ADA) and normal tumor markers in pericardial or pleural effusion are usually considered to be a specific manifestation of benign pericardial or pleural effusion. Here we report a case of lung adenocarcinoma with pericardial metastasis with elevated ADA and normal tumor markers in pericardial effusion. METHODS: Pericardiocentesis and lung puncture combined laboratory indexes and pathology were performed for diagnosis. RESULTS: Analysis of pericardial fluid revealed a white blood cell (WBC) count of 2,000 x 106/L (70% for lymphocytes) with an ADA level of 72.8 U/mL. Pathology of pericardial effusion found no malignant cells. Histopathology of percutaneous lung puncture showed adenocarcinoma. CONCLUSIONS: ADA and tumor markers were not a specific index in differential diagnosis between tuberculosis and metastasis in pericardial effusion.

[Relationship between c-kit mRNA expression and prognosis in patients with rectal carcinoma].

Objective: To investigate the correlation between c-kit mRNA expression and prognosis in patients with rectal carcinoma. Methods: The expression of c-kit mRNA in rectal carcinoma tissues(n=66) was detected by multiplex branched-DNA liquid chip method. According to the expression level, the patients were classified into the c-kit mRNA high expression group and the low group. We analyzed the relationship between the c-kit mRNA expression and the clinicopathological characteristics of patients, as well as the factors affecting patients'prognosis. Results: Of the 66 rectal carcinoma patients, 18(27.3%)cases were c-kit mRNA high expression. No significant correlation was found between the c-kit mRNA expression and gender, age, preoperative carcinoembryonic antigen, preoperative hemoglobin, distance to verge, lymph node metastasis, tumor thrombus, T stage, TNM stage and tumor differentiation (P>0.05). In follow-up, 34 patients died, 32 patients and 36 patients were recurrence or metastasis. The 1-, 3-, 5-year overall survival(OS) of c-kit mRNA high expression group were 100.0%, 77.8%, 77.8%, respectively, while those of the low one were 93.8%, 56.3%, 45.8%, respectively. The difference was statistically significant(P=0.025). Lymph node metastasis, T stage and TNM stage were also significant associated with OS(P<0.05). The 1-, 3-, 5-year disease free rate (DFS)of the c-kit mRNA high expression group were 100.0%, 77.8% and 77.8%, respectively, while those of the low one were 77.1%、43.8% and 41.7%, respectively, and the difference between the two groups was significant (P=0.044). As a reslut, c-kit mRNA expression (P=0.038) and TNM stage (P=0.039) were the independent prognostic factors affecting the OS in rectal cancer patients. Conclusions: Low expression of c-kit was associated with poor prognosis of rectal carcinoma. And the mechanism underlying this phenomenon deserves further exploration.

The circadian molecular clock creates epidermal stem cell heterogeneity.

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

The proteomic landscape of the suprachiasmatic nucleus clock reveals large-scale coordination of key biological processes.

The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.

Distinct Etiological Roles for Myocytes and Motor Neurons in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy.

Polyglutamine (polyQ) expansion of the androgen receptor (AR) causes Kennedy's disease/spinobulbar muscular atrophy (KD/SBMA) through poorly defined cellular mechanisms. Although KD/SBMA has been thought of as a motor neuron disease, recent evidence indicates a key role for skeletal muscle. To resolve which early aspects of the disease can be caused by neurogenic or myogenic mechanisms, we made use of the tet-On and Cre-loxP genetic systems to selectively and acutely express polyQ AR in either motor neurons (NeuroAR) or myocytes (MyoAR) of transgenic mice. After 4 weeks of transgene induction in adulthood, deficits in gross motor function were seen in NeuroAR mice, but not MyoAR mice. Conversely, reduced size of fast glycolytic fibers and alterations in expression of candidate genes were observed only in MyoAR mice. Both NeuroAR and MyoAR mice exhibited reduced oxidative capacity in skeletal muscles, as well as a shift in fast fibers from oxidative to glycolytic. Markers of oxidative stress were increased in the muscle of NeuroAR mice and were reduced in motor neurons of both NeuroAR and MyoAR mice. Despite secondary pathology in skeletal muscle and behavioral deficits, no pathological signs were observed in motor neurons of NeuroAR mice, possibly due to relatively low levels of polyQ AR expression. These results indicate that polyQ AR in motor neurons can produce secondary pathology in muscle. Results also support both neurogenic and myogenic contributions of polyQ AR to several acute aspects of pathology and provide further evidence for disordered cellular respiration in KD/SBMA skeletal muscle.

Predicting the risk of nephrotoxicity in patients receiving colistimethate sodium: a multicentre, retrospective, cohort study.

OBJECTIVES: With increasing rates of infections caused by MDR Gram-negative organisms, clinicians resort to older agents such as colistimethate sodium (CMS) despite a significant risk of nephrotoxicity. Several risk factors for CMS-associated nephrotoxicity have been reported, but they have yet to be validated. We compared the performance of published mathematical models in predicting the risk of CMS-associated nephrotoxicity. METHODS: In a multicentre, retrospective, cohort study, adult patients (≥18 years of age) were evaluated from five large academic medical centres in the USA. Patients with normal renal function (baseline serum creatinine ≤1.5 mg/dL) who received intravenous CMS for ≥72 h were followed for up to 30 days. The development of nephrotoxicity was as defined by the RIFLE criteria. Each published model was conditioned using patient-specific variables to predict the risk of nephrotoxicity. The predictive performance of the models was evaluated using the observed-to-expected (O/E) ratio. The most significant cut-off threshold for stratifying patients into high and low risk of nephrotoxicity was identified using classification and regression tree analysis. RESULTS: A total of 106 patients were examined (mean age 53.3 ±â€Š14.9 years, 66% male); the overall observed nephrotoxicity rate was 52.8%. We identified a simple model demonstrating reasonable overall nephrotoxicity risk assessment [O/E ratio of 1.07 (95% CI = 0.81-1.39)] and high sensitivity (92.9%) in predicting nephrotoxicity development in patients on CMS therapy. CONCLUSIONS: We identified a model that could be incorporated into patient management strategies to reduce the risk of nephrotoxicity in patients requiring CMS therapy.

Association between ERCC1 and ERCC2 polymorphisms and breast cancer risk in a Chinese population.

We conducted a case-control study to investigate the role of ERCC1 rs3212986 and ERCC2 rs13181 gene polymorphisms in the development of breast cancer. Between March 2012 and March 2014, a total of 242 newly diagnosed breast cancer patients with histopathologically confirmed primary breast cancer and 242 healthy controls were recruited. Genotyping of ERCC1 rs3212986 and ERCC2 rs13181 polymorphisms was carried out using polymerase chain reaction-restriction fragment length polymorphism analysis. Unconditional logistic regression analyses indicated that the TT genotype of rs3212986 was associated with a higher risk of breast cancer compared to that associated with the GG genotype (OR = 2.05, 95%CI = 1.13-3.78). In dominant and recessive models, we found that the rs3212986 polymorphism was associated with increased risk of breast cancer, and the ORs were 1.50 (95%CI = 1.03-2.18) and 1.74 (95%CI = 1.01-3.11), respectively. In summary, we found that the ERCC1 rs3212986 polymorphism was associated with the development of breast cancer.

Role of single nucleotide polymorphisms of DNA repair genes in susceptibility to pancreatic cancer in Chinese population.

We conducted a case-control study to investigate the role of ERCC1-ERCC5 gene polymorphisms in the risk of pancreatic cancer. This study included 195 patients who were newly diagnosed with histopathologically confirmed primary pancreatic cancer, and 254 controls were recruited from Sir Run Run Shaw Hospital, between January 2012 and December 2014. Genotyping of ERCC1 rs3212986 and rs11615, ERCC2 rs13181, ERCC3 rs4150441, ERCC4 rs6498486, and ERCC5 rs2094258 polymorphisms was carried out using polymerase chain reaction coupled with restriction fragment length polymorphism. Unconditional logistic regression analyses showed that the TT genotype of ERCC1 rs3212986 was associated with an increased risk of pancreatic cancer, and the OR (95%CI) was 2.26 (1.21-4.22). However, we did not find a significant association between ERCC1 rs11615, ERCC2 rs13181, ERCC3 rs4150441, ERCC4 rs6498486, and ERCC5 rs2094258 polymorphisms and risk of pancreatic cancer. In summary, we found that the presence of the ERCC1 rs3212986 polymorphism correlated with an increased risk of pancreatic cancer.

A Comparison of the Diagnostic Accuracy and Reliability of Subjective Grading and Computer-Aided Assessment of Intranodal Vascularity in Differentiating Metastatic and Reactive Cervical Lymphadenopathy.

PURPOSE: Ultrasound is a well-established imaging modality in the assessment of malignant cervical lymphadenopathy. With the use of Doppler ultrasound, intranodal vascularity can be evaluated. However, the major limitation of ultrasound is operator dependency. Therefore, this study aimed to evaluate and compare the diagnostic accuracy and reliability of the subjective grading and computer-aided approach in assessing intranodal vascularity for the differentiation of benign and malignant lymph nodes. MATERIALS AND METHODS: The present study retrospectively assessed 99 power Doppler ultrasound images of cervical lymph nodes and evaluated the degree of intranodal vascularity using qualitative subjective grading (QSG) and quantitative computer-aided (QCA) methods. The diagnostic accuracy of the two methods in distinguishing metastatic and reactive nodes and their inter- and intra-rater reliability in assessing intranodal vascularity were evaluated and compared. RESULTS: The results showed that the QCA method was more accurate than the QSG method with a significantly higher sensitivity (67.8 % and 61.9 %, respectively, p < 0.05) and specificity (73.3 % and 57.3 %, respectively, p < 0.05). Using the intranodal vascularity index as determined by the QCA approach, the optimum cut-off to differentiate metastatic and reactive cervical lymph nodes was 32 %. The QCA method showed higher inter- and intra-rater reliability than the QSG method. CONCLUSION: In the assessment of the degree of intranodal vascularity, the QCA method was more accurate and reliable than the QSG method in distinguishing metastatic and reactive lymph nodes.

[Effect of metanephric mesenchymal stem cells after renal ischemia reperfusion injury in mice].

OBJECTIVE: To study the effect of metanephric mesenchymal stem cells (MMSCs ) after renal ischemia reperfusion injury (IRI). METHODS: C57BL/6J male mice were divided into control group (n=5) and experimental group (n=30). The control group was given a sham operation, while in the experimental group, a model of renal IRI was established. The experimental group was further divided into two groups according to the material injected through the femoral vein: IRI group (injected with normal saline, 10 ml/kg) and cell therapy group (injected with normal saline containing 5×10(5) MMSCs, 10 ml/kg). Samples of blood and kidney tissues were collected from five mice from each group at 12 h, 24 h and 72 h after IRI. The serum creatinine (SCr) level was detected, and the results of kidney tissue pathological staining and Paller score of renal tubules were analyzed to assess the effect of MMSCs after renal IRI. In addition, the expression of microRNA-26a(miR-26a)in kidney tissues was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared among the groups. RESULTS: (1) In both IRI group and cell therapy group, the levels of SCr at 12 h, 24 h and 72 h after operation were all significantly higher than those of the control group, besides, the level of SCr at 24 h was significantly higher than that at 12 h and 72 h (all P<0.05). The levels of SCr at 12 h, 24 h and 72 h were of no significant differences between IRI group and cell therapy group(all P>0.05). (2)Paller scores of renal tubules at 12 h, 24 h and 72 h in both IRI group and cell therapy group were significantly higher than those in the control group, and the scores at 24 h were significantly higher than that at 72 h, while the latter were in turn higher than the scores at 12 h (all P<0.05). In the cell therapy group, Paller score of renal tubules at 24 h(57.2±6.3)was significantly lower than that in IRI group(70.8±14.8) (P<0.05). Histological examination showed renal tubular epithelial cell atrophy, swelling and protein cast in kidney tissues from IRI group at 24 h, compared with the control group and the cell therapy group at the same time.(3) In IRI group and cell therapy group, the levels of miR-26a in the kidney tissues at 12 h and 24 h were significantly lower than those of the control group (P<0.05). In the cell therapy group, the level of miR-26a in the kidney tissues at 24 h (0.416±0.139) and 72 h (1.152±0.239)were significantly higher than that in the IRI group(0.244±0.067, 0.855±0.038, both P<0.05). A negative correlation between the levels of miR-26a and SCr level were found (r=-0.5, P<0.05). CONCLUSION: MMSCs have a certain repairing effect on renal IRI, accompanied by an increase in miR-26a expression.

[Value of intravoxel incoherent motion diffusion-weighted imaging in differential diagnosis of benign and malignant hepatic lesions and blood perfusion evaluation].

Objective: To investigate the value of intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) in the differential diagnosis and blood perfusion evaluation of benign and malignant hepatic lesions. Methods: A retrospective analysis was performed for 86 patients (96 lesions) with pathologically or clinically confirmed hepatic lesions or hepatic lesions diagnosed based on follow-up results, among whom 48 had malignant lesions (53 lesions) and 38 had benign lesions (43 lesions). The patients underwent conventional magnetic resonance (MR) plain scan, contrast-enhanced scan, and diffusion-weighted imaging (DWI) with different b values (b = 0, 50, 100, 150, 200, 400, 600, 800, 1 000, and 1 200 s/mm2) to determine the parameters of the double exponential model for intravoxel incoherent motion (IVIM): fast diffusion coefficient Dfast, slow diffusion coefficient Dslow, and percentage of fast-diffusion constituent F value. The patients were divided into groups according to the blood supply to lesions on conventional MR plain scan and contrast-enhanced scan, and there were 47 lesions in abundant blood supply group and 49 in poor blood supply group. The data for analysis were Dfast, Dslow, and F values of benign/malignant lesion groups and abundant/poor blood supply groups. The independent samples t-test was used for statistical analysis; the independent samples non-parametric test Mann-Whitney U test was used for the comparison of F value; the receiver operating characteristic (ROC) curve was used to evaluate the value of above parameters in the differentiation of benign and malignant lesions and blood supply evaluation. Results: Compared with the malignant lesion group, the benign lesion group had significantly higher Dslow, and F values (P< 0.001 orP= 0.001) and a higher Dfast value (P= 0.053). Compared with the poor blood supply group, the abundant blood supply group had significantly higher Dfast and F values (P< 0.001 orP= 0.001) and a higher Dslow value (P= 0.185). According to the ROC curve, the cut-off values of Dslow, Dfast, and F values in the diagnosis of benign/malignant hepatic lesions and evaluation of abundant/poor blood supply were 1.18×10-3mm2/s, 27.20×10-3mm2/s, 20.25%, 1.17×10-3mm2/s, 20.30×10-3mm2/s, and 17.80%, respectively, with sensitivities, specificities, accuracy, and areas under the ROC curve of 90.69%/92.45%/91.66%/0.938, 46.51%/73.58%/61.45%/0.589, 74.41%/50.94%/62.50%/0.653, 59.57%/57.14%/58.33%/0.559, 55.32%/63.26%/59.37%/0.618, and 93.61%/89.79%/90.62%/0.961, respectively. Conclusion: The parameter of the double exponential model for IVIM, Dslow value, has a certain value in the differential diagnosis of benign and malignant hepatic lesions, and F value can show blood perfusion in benign and malignant hepatic lesions without the need for contrast-enhanced scan, which provides a reference for the qualitative diagnosis of liver tumor.

[Rho kinase inhibitor Y27632 promotes survival of human induced pluripotent stem cells during differentiation into functional midbrain dopaminergic progenitor cells in vitro].

OBJECTIVE: To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells (hiPSCs-NECs) into functional midbrain dopaminergic progenitor cells (DAPs). METHODS: HiPSCs were cultured in mTeSRTM medium containing DMH1 (10 µmol/L), SB431542 (10 µmol/L), SHH (200 ng/mL), FGF8 (100 ng/mL), purmorphamine (2 µmol/L), CHIR99021 (3 µmol/L), and N2 (1%) for 12 days to induce their differentiation into primitive neuroepithelial cells (NECs). The hiPSCs-NECs were digested with collagenase Ⅳ and then cultured in neurobasal medium supplemented with 1% N2, 2% B27-A, BDNF (10 ng/mL), GDNF (10 ng/mL), AA, TGF-ß, cAMP, and 1% GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632, and the culture medium was changed the next day to remove Y27632. Continuous induction was performed until day 28 to obtain DAPs. RESULTS: Human iPSCs expressed the pluripotency markers OCT4, SOX2, Nanog, and SSEA1 and were positive for alkaline phosphatase staining. The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2, nestin, and PAX6. In digested hiPSCs-NECs, the addition of 5 µmol/L Y27632 significantly promoted survival of the adherent cells, increased cell viability and the proportion of S-phase cells (P < 0.01), and reduced the rate of apoptotic cells (P < 0.05). On day 28 of induction, the obtained cells highly expressed the specific markers of DAPS (TH, FOXA2, NURR1, and Tuj1). CONCLUSION: Treatment with Y27632 (5 µmol/L) for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation, which indirectly enhances the efficiency of cell differentiation.

miRNA-132 orchestrates chromatin remodeling and translational control of the circadian clock.

Mammalian circadian rhythms are synchronized to the external time by daily resetting of the suprachiasmatic nucleus (SCN) in response to light. As the master circadian pacemaker, the SCN coordinates the timing of diverse cellular oscillators in multiple tissues. Aberrant regulation of clock timing is linked to numerous human conditions, including cancer, cardiovascular disease, obesity, various neurological disorders and the hereditary disorder familial advanced sleep phase syndrome. Additionally, mechanisms that underlie clock resetting factor into the sleep and physiological disturbances experienced by night-shift workers and travelers with jet lag. The Ca(2+)/cAMP response element-binding protein-regulated microRNA, miR-132, is induced by light within the SCN and attenuates its capacity to reset, or entrain, the clock. However, the specific targets that are regulated by miR-132 and underlie its effects on clock entrainment remained elusive until now. Here, we show that genes involved in chromatin remodeling (Mecp2, Ep300, Jarid1a) and translational control (Btg2, Paip2a) are direct targets of miR-132 in the mouse SCN. Coordinated regulation of these targets underlies miR-132-dependent modulation of Period gene expression and clock entrainment: the mPer1 and mPer2 promoters are bound to and transcriptionally activated by MeCP2, whereas PAIP2A and BTG2 suppress the translation of the PERIOD proteins by enhancing mRNA decay. We propose that miR-132 is selectively enriched for chromatin- and translation-associated target genes and is an orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment. These findings will further our understanding of mechanisms governing clock entrainment and its involvement in human diseases.

Uncovering the proteome response of the master circadian clock to light using an AutoProteome system.

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 µg and 200 µg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.

Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

First detection of multiple knockdown resistance (kdr)-like mutations in voltage-gated sodium channel using three new genotyping methods in Anopheles sinensis from Guangxi Province, China.

To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.