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Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Background This research aimed at exploring the mechanisms of alterations of metabolites and pathways in T2D from the perspective of metabolomics and transcriptomics, as well as uncovering novel drug candidate for T2D treatment. Methods Metabolites in human plasma from 42 T2D patients and 45 non‐diabetic volunteers were detected by liquid chromatography‐mass spectrometer (LC‐MS). Microarray dataset of the transcriptome was obtained from Gene Expression Omnibus (GEO) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to conduct pathway enrichment analysis. Connectivity Map (CMap) was employed to select potential drugs for T2D therapy. In vivo assay was performed to verify above findings. The protein expression levels of ME1, ME2 and MDH1 were detected by Western blot to determine the status of NAD/NADH cofactor system. Results In our study, differentially expressed metabolites were selected out between healthy samples and T2D samples with selection criteria P value 2, including N‐acetylglutamate and Malate. Genes set enrichment analysis (GSEA) revealed that 34 pathways were significantly enriched in T2D. Based on CMap analysis and animal experiments, Atractyloside was identified as a potential novel drug for T2D treatment via targeting ME1, ME2 and MDH1 and regulating the NAD/NADH cofactor system. Conclusion The present research revealed differentially expressed metabolites and genes, as well as significantly altered pathways in T2D via an integration of metabolomics, transcriptomics and CMap analysis. It was also demonstrated that comprehensive analysis based on metabolomics and transcriptomics was an effective approach for identification and verification of metabolic biomarkers and alternated pathways.

Background Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder. Although most patients present with isolated CH, some patients present with CH and extra-thyroidal congenital malformations (ECMs), for which less is known about the underlying genetics. The aim of this study was to investigate the genetic mechanisms in patients with CH and ECMs using chromosomal microarray (CMA) and whole exome sequencing (WES). Methods Peripheral venous blood samples were collected from 16 patients with CH and ECMs. Genomic DNA was extracted from peripheral blood leukocytes. CMA and WES were performed to detect copy number and single nucleotide variants. Results CMA identified clinically significant copy number variants in 7 patients consistent with their phenotypes. For 6 of them, the genotype and phenotype suggested a syndromic diagnosis, and the remaining patient carried a pathogenic microdeletion and microduplication including GLIS3. WES analysis identified 9 different variants in 7 additional patients. The variants included 2 known mutations (c.1096C>T (p.Arg366Trp) in KCNQ1 and c.848C>A (p.Pro283Gln) in NKX2-5) and 7 novel variants: one nonsense mutation (c.4330C>T (p.Arg1444*) in ASXL3), one frameshift mutation (c.1253_1259delACTCTGG (p.Asp418fs) in TG), three missense variants (c.1472C>T (p.Thr491Ile) in TG, c.4604A>G (p.Asp1535Gly) in TG, and c.2139G>T (p.Glu713Asp) in DUOX2, and two splice site variants (c.944-1G>C and c.3693 + 1G>T) in DUOX2. Conclusions We report the first genetic study of CH patients with ECMs using CMA and WES. Overall, our detection rate for pathogenic and possibly pathogenic variants was 87.5% (14/16). We report 7 novel variants, expanding the mutational spectrum of TG, DUOX2, and ASXL3.

SLC25A46 (solute carrier family 25 member 46) mutations have been linked to various neurological diseases with recessive inheritance, including Leigh syndrome, optic atrophy, and lethal congenital pontocerebellar hypoplasia. SLC25A46 is expressed in the outer membrane of mitochondria, where it plays a critical role in mitochondrial dynamics. A deceased 7-month-old female infant was suspected to have Leigh syndrome. Clinical exome sequencing was non-diagnostic, but research reanalysis of the sequencing data identified two novel variants in SLC25A46: a missense (c.1039C>T, p.Arg347Cys; NM_138773, hg19) and a donor splice region variant (c.283+5G>A) in intron 1. Both variants were predicted to be damaging. Sanger sequencing of cDNA detected a single missense allele in the patient compared to control, and the SLC25A46 transcript levels were also reduced due to the splice region variant. Additionally, Western blot analysis of whole-cell lysate showed a decrease of SLC25A46 expression in proband fibroblasts, relative to control cells. Further, analysis of mitochondrial morphology revealed evidence of increased fragmentation of the mitochondrial network in proband fibroblasts, compared to control cells. Collectively, our findings suggest that these novel variants in SLC24A46, the donor splice one and the missense variant, are the cause of the neurological phenotype in this proband.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder., Author summary Childhood interstitial lung disease (chILD) is a heterogeneous group of rare disorders characterized by diffuse pulmonary infiltrates, respiratory signs and symptoms, and impaired gas exchange. These disorders are complex to diagnose and are associated with substantial morbidity and mortality. Although pathogenic variants in a number of genes have been described to cause chILD, these known genetic etiologies explain only a minority of the cases and there are additional genes yet to be identified. We have identified an 8-year-old girl with chILD, systemic hypertension, and immune abnormalities who carries a homozygous stop-gain variant in the ARHGAP42 gene. Functional studies demonstrate that this stop-gain variant leads to exon 5 skipping and reduced levels of ARHGAP42 protein. We also show enhanced RhoA expression and its activity in the patient’s lymphoblastoid cell lines. ARHGAP42 is involved in regulation of blood pressure and its deficiency causes hypertension in murine models and human adults. This is the first report to link a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in a pediatric patient. Identification of additional chILD patients carrying ARHGAP42 mutations will better define its role in chILD.

Background Gene variants have been reported to be associated with congenital hypothyroidism (CH), the purpose of this study was to analyze the mutation spectrum and prevalence of 12 known causative genes ( TSHR , PAX8 , NKX2 . 1 , NKX2 . 5 , FOXE1 , DUOX2 , TG , TPO , GLIS3 , NIS , SLC26A4 and DEHAL1 ) in CH in China. Methods Peripheral venous blood samples were collected from the patients. Genomic DNA was extracted from peripheral blood leukocytes. All exons and their exon-intron boundary sequences of the 12 known CH associated genes in 66 CH patients were screened by next-generation sequencing (NGS). Results NGS analysis of 12 known CH associated genes revealed that 32 patients (32/66, 48.5%) were detected to have at least one potentially functional variant. 21, 9, 1, 1, 1 and 1 patients were found to have potential pathogenic variants in DUOX2 , TG , PAX8 , SLC26A4 , TSHR and TPO genes, respectively. Novel variants included one DUOX2 and one TPO missense variants of unknown significance (VUS). Conclusion Our study expands the mutation spectrum of DUOX2 and TPO genes. 48.5% CH patients had at least one potential pathogenic variant. We found relatively high frequency of DUOX2 (31.8%) and TG (13.6%) mutations in our cohort.

People are taking up antioxidants in their daily diet and being exposed to a potential diesel exhaust particles (DEP)-containing environment. Thus it is important to study in vitro cellular responses when cells are exposed to DEP with or without antioxidant treatment. The investigation of DEP and resveratrol (RES) on cellular biophysical and biochemical changes is needed to better understand the mechanisms of DEP and RES in mammalian cells. A combination of two non-invasive techniques (atomic force microscopy, AFM, and Raman spectroscopy, RM) and multimodal tools were applied to evaluate the biophysical, biochemical alterations and cytokine, membrane potential and cell cycle of cells with or without RES pretreatment to different times of DEP exposure. AFM results indicated that RES protected cells from DEP-induced damage to cytoskeleton and cell architectures, and noted that RES treatments also attenuated DEP-induced alterations in cell elasticity and surface adhesion force over DEP incubation time. RM monitored the changes in characteristic Raman peak intensities of DNA and protein over the DEP exposure time for both RES and non-RES treated groups. The cytokine and chemokine changes quantified by Multiplex ELISA revealed that the inflammatory responses were enhanced with the increase in DEP exposure time and that RES enhanced the expression levels of cytokine and chemokine. This work demonstrated that significant biophysical and biochemical changes in cells might be relevant to early pathological changes induced by DEP damage. Copyright © 2016 John Wiley & Sons, Ltd.

Diesel exhaust particles (DEPs) are major air pollutants that lead to numerous human disorders, especially pulmonary diseases, partly through the induction of oxidative stress. Resveratrol is a polyphenol that ameliorates the production of reactive oxygen species (ROS) and delays aging-related processes. Herein we studied the cytoprotective effect of resveratrol on DEP-exposed human lung cells in a factorial experimental design. This work investigates biophysical features including cellular compositions and biomechanical properties, which were measured at the single-cell level using confocal Raman microspectroscopy (RM) and atomic force microscopy (AFM), respectively. Principal component analysis (PCA), hierarchical cluster analysis (HCA) and partial least square regression (PLS) analysis were applied to analyze Raman spectra with and without resveratrol protection. The health status of individual cells could be effectively predicted using an index derived from characteristic Raman spectral peak (e.g., 1006 cm−1) based on PLS model. AFM measurements indicated that cellular adhesion force was greatly reduced, while Young’s modulus was highly elevated in resveratrol treated DEP-exposed cells. Anti-oxidant resveratrol reduced DEP-induced ROS production and suppressed releases of several cytokines and chemokines. These findings suggest resveratrol may enhance resistance of human lung cells (e.g., SAEC) to air pollutants (e.g. DEPs).

Multidrug resistance (MDR) refers to the cross-resistance of cancer cells to one drug, accompanied by other drugs with different mechanisms and structures, which is one of the main obstacles of clinical chemotherapy. Overexpression of P-glycoprotein (P-gp) was an extensively studied cause of MDR. Therefore, inhibiting P-gp have become an important strategy to reverse MDR. In this study, two series of triazole-tetrahydroisoquinoline-core P-gp inhibitors were designed and synthesized. Among them, compound I-5 had a remarkable reversal activity of MDR activity and the preliminary mechanism study was also carried out. All the results proved that compound I-5 was considered as a promising P-gp-mediated MDR reversal candidate.

Mutations in striated preferentially expressed protein kinase (SPEG), a member of the myosin light chain kinase protein family, are associated with centronuclear myopathy (CNM), cardiomyopathy, or a combination of both. Burgeoning evidence suggests that SPEG plays critical roles in the development, maintenance, and function of skeletal and cardiac muscles. Here we review the genotype-phenotype relationships and the molecular mechanisms of SPEG-related diseases. This review will focus on the progress made toward characterizing SPEG and its interacting partners, and its multifaceted functions in muscle regeneration, triad development and maintenance, and excitation-contraction coupling. We will also discuss future directions that are yet to be investigated including understanding of its tissue-specific roles, finding additional interacting proteins and their relationships. Understanding the basic mechanisms by which SPEG regulates muscle development and function will provide critical insights into these essential processes and help identify therapeutic targets in SPEG-related disorders.

Proteolysis-targeting chimera (PROTAC) could selectively degrade target protein and may become a promising strategy for treating estrogen receptor α (ERα) positive breast cancers. Here, we designed penetrated peptide-based PROTACs by constructing an N-terminal lactam cyclic to improve proteolytic stability and cell penetration. We used a lactam cyclic peptide as ERα binding ligand, 6-aminocaproic acid as a linker, and a hydroxylated pentapeptide structure for recruiting E3 ligase to obtain heterobifunctional compounds. The resulting optimized compound I-6 selectively recruited ERα to the E3 ligase complex for promoting the degradation of ERα. Compound I-6 possessed strong effect on MCF-7 cell toxicity (IC50 ∼9.7 μM) and significantly enhanced activities in inducing ERα degradation. Meanwhile, I-6 performed much stronger potency in inhibition of tumors growth than tamoxifen. This work is a successful template to construct PROTACs based on cell-permeable peptides, which could extend the chemical space of PROTACs.

Objectives Defects in the human GLI-similar 3 (GLIS3) gene are reported to be a rare cause of congenital hypothyroidism (CH) and neonatal diabetes. The aim of this study was to examine the prevalence of GLIS3 mutation among CH patients in the Guangxi Zhuang Autonomous Region of China and to define the relationships between GLIS3 genotypes and clinical phenotypes. Methods Blood samples were collected from 592 patients with CH in Guangxi Zhuang Autonomous Region, China, and genomic DNA was extracted from peripheral blood leukocytes. All exons of the GLIS3 gene with their exon-intron boundaries were screened by next-generation sequencing (NGS) and CNVplex®. Chromosomal microarray analysis (CMA) was performed to detect the existence of the adjacent gene deletion. Results NGS and CNVplex® analysis of GLIS3 in 592 CH patients revealed two different variations in two individuals (2/592, 0.3%). Patient 1 was the paternal allele of 9p24.3p23 heterozygous deletion including the whole GLIS3 gene, and patient 2 was heterozygous for c.2159G > A (p.R720Q) GLIS3 variant combined with compound heterozygous DUOX2 mutations (p.R683L/p.L1343F). Conclusions Our study indicated that the prevalence of GLIS3 variations was 0.3% among studied Chinese CH patients. Multiple variations in one or more CH associated genes can be found in one patient. We found a novel GLIS3 variation c.2159G > A (p.R720Q), thereby expanding the variation spectrum of the gene.

Traditional approaches to characterize stem cell differentiation are time-consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) - both considered as non-invasive techniques - are applied to detect the biochemical and biophysical properties of trophoblast derived stem-like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed. Monitoring trophoblast cells differentiation.

Ofloxacin has been widely used as a form of quinolone antibiotics. However, it has the potential to exert biological effects on aquatic organisms and cause surface water pollution. It is necessary to find an efficient way to remove ofloxacin. This study reports on the degradation of ofloxacin in solution using TiO2 nanotubes (TiO2 NTs) as photocatalyst. The TiO2 NTs were synthesized through anodization. The morphology, elemental composition and state, crystalline phase, and photocatalytic activity of this photocatalyst were characterized by a variety of surface analysis techniques. The obtained TiO2 NTs were applied to ofloxacin degradation by photoelectrocatalysis. The degradation efficiency was assessed by in situ monitoring the UV-vis absorbance spectrum of ofloxacin solution during the degradation process. The effects of initial pH, bias potential, and initial concentration of ofloxacin were investigated systematically. Moreover, the toxicity of ofloxacin during the photoelectrocatalytic degradation process was evaluated using the growth inhibition test with Microcystis aeruginosa. The TiO2 NT-based photoelectrocatalytic method provided a high degradation rate for ofloxacin removal.

In this study, confocal Raman spectroscopy, atomic force microscope (AFM) and multiplex ELISA were applied to analyze the biophysical responses (biomechanics and biospectroscopy) of normal human primary small airway epithelial cells (SAECs) and human lung carcinoma epithelial A549 cells to in vitro short term DEP exposure (up to 2 h). Raman spectra revealed the specific cellular biomolecular changes in cells induced by DEP compared to unexposed control cells. Principal component analysis was successfully applied to analyze spectral differences between control and treated groups from multiple individual cells, and indicated that cell nuclei are more sensitive than other cell locations. AFM measurements indicated that 2 h of DEP exposure induced a significant decrease in cell elasticity and a dramatic change in membrane surface adhesion force. Cytokine and chemokine production measured by multiplex ELISA demonstrated DEP-induced inflammatory responses in both cell types.

The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses cancer metastasis without affecting the growth of the primary tumor. Previous work has shown that it decreases the expression of protein mediators involved in chemoresistance. This study measured the biomechanical and biochemical changes in BRMS1 expression and the responses of BRMS1 to drug treatments on cancer cells in vitro. The results show that BRMS1 expression affects biomechanical properties by decreasing the Young's modulus and adhesion force of breast cancer cells after doxorubicin (DOX) exposure. Raman spectral bands corresponding to DNA/RNA, lipids and proteins were similar for all cells after DOX treatment. The expression of cytokines were similar for cancer cells after DOX exposure, although BRMS1 expression had different effects on the secretion of cytokines for breast cancer cells. The absence of significant changes on apoptosis, reactive oxygen species (ROS) expression and cell viability after BRMS1 expression shows that BRMS1 has little effect on cellular chemoresistance. Analyzing cancer protein expression is critical in evaluating therapeutics. Our study may provide evidence of the benefit of metastatic suppressor expression before chemotherapy.

Porcine trophoblast-derived stem-like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast-derived cells, cultured in vitro in a defined and non-serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Young's modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix-related genes were significantly impacted by medium type (non-serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy-both considered as label-free, non-invasive techniques-can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton-related genes exhibit similar pattern to that of Young's modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton-related genes and cellular stiffness.