Explore the vast range of titles available.

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

Diabetes mellitus (DM) induces a variable degree of muscle sarcopenia, which may be related to protein degradation and to the expression of both E3 ubiquitin ligases and some specific microRNAs (miRNAs). The present study investigated the effect of diabetes and acute muscle contraction upon the TRIM63 and FBXO32 expression as well as the potential involvement of some miRNAs. Diabetes was induced by streptozotocin and studied after 30 days. Soleus muscles were harvested, stimulated to contract in vitro for twitch tension analysis (0.5 Hz), 30 min later for tetanic analysis (100 Hz), and 30 min later were frozen. TRIM63 and FBXO32 proteins were quantified by western blotting; Trim63 mRNA, Fbxo32 mRNA, miR-1-3p, miR-29a-3p, miR-29b-3p, miR-133a-3p, and miR-133b-3p were quantified by qPCR. Diabetes induced sarcopenia by decreasing ( P  < 0.05) muscle weight/tibia length index, maximum tetanic contraction and relaxation rates, and absolute twitch and tetanic forces ( P  < 0.05). Diabetes decreased ( P  < 0.05) the Trim63 and Fbxo32 mRNAs (30%) and respective proteins (60%), and increased ( P  < 0.01) the miR-29b-3p (2.5-fold). In muscle from diabetic rats, acute contractile stimulus increased TRIM63 protein, miR-1-3p, miR-29a-3p, and miR-133a/b-3p, but decreased miR-29b-3p ( P  < 0.05). Independent of the metabolic condition, after muscle contraction, both TRIM63 and FBXO32 proteins correlated significantly with miR-1-3p, miR-29a/b-3p, and miR-133a/b-3p. All diabetes-induced regulations were reversed by insulin treatment. Concluding, the results depict that muscle wasting in long-term insulinopenic condition may not be accompanied by increased proteolysis, pointing out the protein synthesis as an important modulator of muscle sarcopenia in DM.

The therapeutic potential of mesenchymal stem cell-conditioned medium (MSC-CM) has been reported with various types of disease models. Here, we examine the therapeutic effect of umbilical cord MSC-CM (UCMSC-CM) on muscle-related disease, using a dexamethasone (Dex)-induced muscle atrophy in vitro model. The expressions of muscle atrophy-related proteins (MuRF-1 and MAFbx) and muscle-specific proteins (desmin and myogenin) were evaluated by Western blot analysis. The level of production of reactive oxygen species (ROS) was determined using a 2',7'-dichlorofluorescein diacetate (DCFDA) dye assay. The expression of antioxidant enzymes (copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPx-1), and catalase (CAT)) was verified by reverse transcription polymerase chain reaction (RT-PCR). When L6 cells were exposed to Dex, the expression of muscle atrophy-related proteins was increased by 50–70%, and the expression of muscle-specific proteins was in turn decreased by 23–40%. Conversely, when the L6 cells were co-treated with UCMSC-CM and Dex, the expression of muscle atrophy-related proteins was reduced in a UCMSC-CM dose-dependent manner and the expression of muscle-specific proteins was restored to near-normal levels. Moreover, ROS generation was effectively suppressed and the expression of antioxidant enzymes was recovered to a normal degree. These data imply that UCMSC-CM clearly has the potential to prevent muscle atrophy. Thus, our present study offers fundamental data on the potential treatment of musclerelated disease using UCMSC-CM.

Cysteine string protein (CSP) is a chaperone of the Dnaj/Hsp40 family of proteins and is essential for synaptic maintenance. Mutations in the human gene encoding CSP, DNAJC5, cause adult neuronal ceroid lipofucinosis (ANCL) which is characterised by progressive dementia, movement disorders, seizures and premature death. CSP null models in mice, flies and worms have been shown to also exhibit similar neurodegenerative phenotypes. Here we have explored the mechanisms underlying ANCL disease progression using Caenorhaditis elegans mutant strains of dnj-14 , the worm orthologue of DNAJC5. Transcriptional profiling of these mutants compared to control strains revealed a broad down-regulation of ubiquitin proteasome system (UPS)-related genes, in particular, components of multimeric RING E3 ubiquitin ligases including F-Box, SKR and BTB proteins. These data were supported by the observation that dnj-14 mutant worm strains expressing a GFP-tagged ubiquitin fusion degradation substrate exhibited decreased ubiquitylated protein degradation. The results indicate that disruption of an essential synaptic chaperone leads to changes in expression levels of UPS-related proteins which has a knock-on effect on overall protein degradation in C. elegans. The specific over-representation of E3 ubiquitin ligase components revealed in our study, suggests that proteins and complexes upstream of the proteasome itself may be beneficial therapeutic targets.

Denervation causes muscle atrophy and incapacity in humans. Although electrical stimulation (ES) and stretching (St) are commonly used in rehabilitation, it is still unclear whether they stimulate or impair muscle recovery and reinnervation. The purpose of this study was to evaluate the effects of ES and St, alone and combined (ES + St), on the expression of genes that regulate muscle mass (MyoD, Runx1, atrogin-1, MuRF1 and myostatin), on muscle fibre cross-sectional area and excitability, and on the expression of the neural cell adhesion molecule (N-CAM) in denervated rat muscle. ES, St and ES + St reduced the accumulation of MyoD, atrogin-1 and MuRF1 and maintained Runx1 and myostatin expressions at normal levels in denervated muscles. None of the physical interventions prevented muscle fibre atrophy or N-CAM expression in denervated muscles. In conclusion, although ES, St and ES + St changed gene expression, they were insufficient to avoid muscle fibre atrophy due to denervation.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a major advance in treating NSCLC with EGFR-activating mutations. However, acquired resistance, due partially to secondary mutations limits their use. Here we report that NSCLC cells with acquired resistance to gefitinib or osimertinib (AZD9291) exhibit EMT features, with a decrease in E-cadherin, and increases in vimentin and stemness, without possessing any EGFR secondary mutations. Knockdown of E-cadherin in parental cells increased gefitinib resistance and stemness, while knockdown of vimentin in resistant cells resulted in opposite effects. Src activation and Hakai upregulation were found in gefitinib-resistant cells. Knockdown of Hakai elevated E-cadherin expression, attenuated stemness, and resensitized the cells to gefitinib. Clinical cancer specimens with acquired gefitinib resistance also showed a decrease in E-cadherin and an increase in Hakai expression. The dual HDAC and HMGR inhibitor JMF3086 inhibited the Src/Hakai and Hakai/E-cadherin interaction to reverse E-cadherin expression, and attenuated vimentin and stemness to restore gefitinib sensitivity. The EMT features of AZD9291-resistant H1975 cells were related to the upregulation of Zeb1. Both gefitinib and AZD9291 sensitivity was restored by JMF3086 through reversing EMT. Our study not only revealed a common mechanism of EMT in both gefitinib and AZD9291 resistance beyond EGFR mutations per se, but also provides a new strategy to overcome it.

Wu and colleagues report that under hypoxic conditions the LATS2 kinase is targeted for degradation by the SIAH2 ubiquitin ligase, leading to inhibition of the Hippo kinase cascade and activation of YAP, which promotes tumour growth. The Hippo signalling pathway plays important roles in animal development, physiology and tumorigenesis 1 , 2 , 3 . Understanding how the activity of this pathway is regulated by the cellular microenvironment remains a major challenge. Here we elucidate a molecular mechanism by which hypoxia deactivates Hippo signalling. We demonstrate that the E3 ubiquitin ligase SIAH2 stimulates YAP by destabilizing LATS2, a critical component of the Hippo pathway, in response to hypoxia. Loss of SIAH2 suppresses tumorigenesis in a LATS2-dependent manner in a xenograft mouse model. We further show that YAP complexes with HIF1α and is essential for HIF1α stability and function in tumours in vivo . LATS2 is downregulated in human breast tumours and negatively correlates with SIAH2 expression levels, indicating that the SIAH2–LATS2 pathway may have a role in human cancer. Our data uncover oxygen availability as a microenvironment signal for the Hippo pathway and have implications for understanding the regulation of Hippo signalling in tumorigenesis.

Purpose Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival. Methods We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations. Results High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival. Conclusion Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases—a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l −1 in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance. 4-hydroxycoumarin (4HC), a precursor for anticoagulant drugs such as warfarin, has a major role in the treatment of thromboembolic diseases. Here, the authors present an artificial biosynthetic pathway for 4HC production in E. coli and demonstrate its potential for large-scale microbial production.

Aberrant protein aggregation and mitochondrial dysfunction have each been linked to aging and a number of age-onset neurodegenerative disorders, including Parkinson disease. Loss-of-function mutations in parkin , an E3 ubiquitin ligase that functions to promote the ubiquitin–proteasome system of protein degradation and also in mitochondrial quality control, have been implicated in heritable forms of Parkinson disease. The question of whether parkin can modulate aging or positively impact longevity, however, has not been addressed. Here, we show that ubiquitous or neuron-specific up-regulation of Parkin, in adult Drosophila melanogaster , increases both mean and maximum lifespan without reducing reproductive output, physical activity, or food intake. Long-lived Parkin-overexpressing flies display an increase in K48-linked polyubiquitin and reduced levels of protein aggregation during aging. Recent evidence suggests that Parkin interacts with the mitochondrial fission/fusion machinery to mediate the turnover of dysfunctional mitochondria. However, the relationships between parkin gene activity, mitochondrial dynamics, and aging have not been explored. We show that the mitochondrial fusion-promoting factor Drosophila Mitofusin, a Parkin substrate, increases in abundance during aging. Parkin overexpression results in reduced Drosophila Mitofusin levels in aging flies, with concomitant changes in mitochondrial morphology and an increase in mitochondrial activity. Together, these findings reveal roles for Parkin in modulating organismal aging and provide insight into the molecular mechanisms linking aging to neurodegeneration.