The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

Tissue-specific overexpression of systemic RNA interference components limits lifespan in C. elegans.

Intertissue RNA transport recently emerged as a novel signaling mechanism. In mammals, mounting evidence suggests that small RNA transfer between cells is widespread and used in various physiological contexts. In the nematode C. elegans, a similar mechanism is conferred by the systemic RNAi pathway. Members of the Systemic RNA Interference Defective (SID) family act at different steps of cellular RNA uptake and export. The limiting step in systemic RNA interference (RNAi) is the import of extracellular RNAs via the conserved double-stranded (dsRNA)-gated dsRNA channel SID-1. To better understand the role of RNAs as intertissue signaling molecules, we modified the function of SID-1 in specific tissues of C. elegans. We observed that sid-1 loss-of-function mutants are as healthy as wild-type worms. Conversely, overexpression of sid-1 in C. elegans intestine, muscle, or neurons rendered worms short-lived. The effects of intestinal sid-1 overexpression were attenuated by silencing the components of systemic RNAi sid-1, sid-2 and sid-5, implicating systemic RNA signaling in the lifespan reduction. Accordingly, tissue-specific overexpression of sid-2 and sid-5 also reduced worm lifespan. Additionally, an RNAi screen for components of several non-coding RNA pathways revealed that silencing the miRNA biogenesis proteins PASH-1 and DCR-1 rendered the lifespan of worms with intestinal sid-1 overexpression similar to controls. Collectively, our data support the notion that systemic RNA signaling must be tightly regulated, and unbalancing that process provokes a reduction in lifespan. We termed this phenomenon Intercellular/Extracellular Systemic RNA imbalance (InExS).

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Tracking soluble and nanoparticulated titanium released in vivo from metal dental implant debris using (single-particle)-ICP-MS.

BACKGROUND: This work studies the presence of the Ti, Al and V metal ions and Ti nanoparticles released from the debris produced by the implantoplasty, a surgical procedure used in the clinic, in rat organs. METHODS: The sample preparation for total Ti determination was carefully optimized using microsampling inserts to minimize the dilution during the acid attack of the lyophilized tissues by a microwave-assisted acid digestion method. An enzymatic digestion method was optimized and applied to the different tissue samples in order to extract the titanium nanoparticles for the single-particle ICP-MS analysis. RESULTS: A statistically significant increase was found for Ti concentrations from control to experimental groups for several of the studied tissues, being and particularly significant in the case of brain and spleen. Al and V concentrations were detected in all tissues but they were not different when comparing control and experimental animals, except for V in brain. The possible presence of Ti-containing nanoparticles mobilized from the implantoplasty debris was tested using enzymatic digestions and SP-ICP-MS. The presence of Ti-containing nanoparticles was observed in all the analyzed tissues, however, differences on the Ti mass per particle were found between the blanks and the digested tissue and between control and experimental animals in some organs. CONCLUSION: The developed methodologies, both for ionic and nanoparticulated metal contents in rat organs, have shown the possible increase in the levels of Ti both as ions and nanoparticles in rats subjected to implantoplasty.

Low muscle mass in lung cancer is associated with an inflammatory and immunosuppressive tumor microenvironment.

BACKGROUND: Computed tomographies (CT) are useful for identifying muscle loss in non-small lung cancer (NSCLC) cachectic patients. However, we lack consensus on the best cutoff point for pectoralis muscle loss. We aimed to characterize NSCLC patients based on muscularity, clinical data, and the transcriptional profile from the tumor microenvironment to build a cachexia classification model. METHODS: We used machine learning to generate a muscle loss prediction model, and the tumor's cellular and transcriptional profile was characterized in patients with low muscularity. First, we measured the pectoralis muscle area (PMA) of 211 treatment-naive NSCLC patients using CT available in The Cancer Imaging Archive. The cutoffs were established using machine learning algorithms (CART and Cutoff Finder) on PMA, clinical, and survival data. We evaluated the prediction model in a validation set (36 NSCLC). Tumor RNA-Seq (GSE103584) was used to profile the transcriptome and cellular composition based on digital cytometry. RESULTS: CART demonstrated that a lower PMA was associated with a high risk of death (HR = 1.99). Cutoff Finder selected PMA cutoffs separating low-muscularity (LM) patients based on the risk of death (P-value = 0.003; discovery set). The cutoff presented 84% of success in classifying low muscle mass. The high risk of LM patients was also found in the validation set. Tumor RNA-Seq revealed 90 upregulated secretory genes in LM that potentially interact with muscle cell receptors. The LM upregulated genes enriched inflammatory biological processes. Digital cytometry revealed that LM patients presented high proportions of cytotoxic and exhausted CD8+ T cells. CONCLUSIONS: Our prediction model identified cutoffs that distinguished patients with lower PMA and survival with an inflammatory and immunosuppressive TME enriched with inflammatory factors and CD8+ T cells.

Ion release and local effects of titanium metal particles from dental implants: An experimental study in rats.

BACKGROUND: The objective of this study was to evaluate the accumulation of ions in blood and organs caused by titanium (Ti) metal particles in a mandibular defect in rats, together with a description of the local reaction of oral tissues to this Ti alloy debris. METHODS: Twenty Sprague-Dawley rats were randomly distributed into three groups: an experimental group with a mandibular bone defect filled with metallic debris obtained by implantoplasty; a positive control group; and a negative control group. Thirty days after surgery, the rats were euthanized and perilesional tissue surrounding the mandibular defect was removed, together with the lungs, spleen, liver, and brain. Two blood samples were collected: immediately before surgery and before euthanasia. The perilesional tissue was histologically analyzed using hematoxylin-eosin staining, and Ti, aluminum, and vanadium ion concentrations in blood and organs were measured by TQ-ICP-MS. Descriptive and bivariate analyses of the data were performed. RESULTS: All rats with implanted metal debris showed metal particles and a bone fracture callus on the osseous defect. The metal particles were surrounded by a foreign body reaction characterized by the presence of histiocytes and multinucleated giant cells (MNGCs). The experimental group had a significant higher concentration of Ti ions in all studied organs except lung tissue (p < 0.05). In addition, there were more V ions in the brain in the experimental group (p = 0.008). CONCLUSIONS: Although further studies are required to confirm the clinical relevance of these results, Ti metal particles in the jaw might increase the concentration of metal ions in vital organs and induce a foreign body reaction.

Meiotic behavior, transmission and active genes of B chromosomes in the cichlid Astatotilapia latifasciata: new clues about nature, evolution and maintenance of accessory elements.

Supernumerary B chromosomes (Bs) are dispensable genetic elements widespread in eukaryotes and are poorly understood mainly in relation to mechanisms of maintenance and transmission. The cichlid Astatotilapia latifasciata can harbor Bs in a range of 0 (named B -) and 1-2 (named B +). The B in A. latifasciata is rich in several classes of repetitive DNA sequences, contains protein coding genes, and affects hosts in diverse ways, including sex-biased effects. To advance in the knowledge about the mechanisms of maintenance and transmission of B chromosomes in A. latifasciata, here, we studied the meiotic behavior in males and transmission rates of A. latifasciata B chromosome. We also analyzed structurally and functionally the predicted B chromosome copies of the cell cycle genes separin-like, tubb1-like and kif11-like. We identified in the meiotic structure relative to the B chromosome the presence of proteins associated with Synaptonemal Complex organization (SMC3, SYCP1 and SYCP3) and found that the B performs self-pairing. These data suggest that isochromosome formation was a step during B chromosome evolution and this element is in a stage of diversification of the two arms keeping the self-pairing behavior to protect the A chromosome complement of negative effects of recombination. Moreover, we observed no occurrence of B-drive and confirmed the presence of cell cycle genes copies in the B chromosome and their transcription in encephalon, muscle and gonads, which can indicates beneficial effects to hosts and contribute to B maintenance.

Decreased miR-497-5p Suppresses IL-6 Induced Atrophy in Muscle Cells.

Interleukin-6 (IL-6) is a pro-inflammatory cytokine associated with skeletal muscle wasting in cancer cachexia. The control of gene expression by microRNAs (miRNAs) in muscle wasting involves the regulation of thousands of target transcripts. However, the miRNA-target networks associated with IL6-induced muscle atrophy remain to be characterized. Here, we show that IL-6 promotes the atrophy of C2C12 myotubes and changes the expression of 20 miRNAs (5 up-regulated and 15 down-regulated). Gene Ontology analysis of predicted miRNAs targets revealed post-transcriptional regulation of genes involved in cell differentiation, apoptosis, migration, and catabolic processes. Next, we performed a meta-analysis of miRNA-published data that identified miR-497-5p, a down-regulated miRNAs induced by IL-6, also down-regulated in other muscle-wasting conditions. We used miR-497-5p mimics and inhibitors to explore the function of miR-497-5p in C2C12 myoblasts and myotubes. We found that miR-497-5p can regulate the expression of the cell cycle genes CcnD2 and CcnE1 without affecting the rate of myoblast cellular proliferation. Notably, miR-497-5p mimics induced myotube atrophy and reduced Insr expression. Treatment with miR-497-5p inhibitors did not change the diameter of the myotubes but increased the expression of its target genes Insr and Igf1r. These genes are known to regulate skeletal muscle regeneration and hypertrophy via insulin-like growth factor pathway and were up-regulated in cachectic muscle samples. Our miRNA-regulated network analysis revealed a potential role for miR-497-5p during IL6-induced muscle cell atrophy and suggests that miR-497-5p is likely involved in a compensatory mechanism of muscle atrophy in response to IL-6.

Prediction of SARS-CoV Interaction with Host Proteins during Lung Aging Reveals a Potential Role for TRIB3 in COVID-19.

COVID-19 is prevalent in the elderly. Old individuals are more likely to develop pneumonia and respiratory failure due to alveolar damage, suggesting that lung senescence may increase the susceptibility to SARS-CoV-2 infection and replication. Considering that human coronavirus (HCoVs; SARS-CoV-2 and SARS-CoV) require host cellular factors for infection and replication, we analyzed Genotype-Tissue Expression (GTEx) data to test whether lung aging is associated with transcriptional changes in human protein-coding genes that potentially interact with these viruses. We found decreased expression of the gene tribbles homolog 3 (TRIB3) during aging in male individuals, and its protein was predicted to interact with HCoVs nucleocapsid protein and RNA-dependent RNA polymerase. Using publicly available lung single-cell data, we found TRIB3 expressed mainly in alveolar epithelial cells that express SARS-CoV-2 receptor ACE2. Functional enrichment analysis of age-related genes, in common with SARS-CoV-induced perturbations, revealed genes associated with the mitotic cell cycle and surfactant metabolism. Given that TRIB3 was previously reported to decrease virus infection and replication, the decreased expression of TRIB3 in aged lungs may help explain why older male patients are related to more severe cases of the COVID-19. Thus, drugs that stimulate TRIB3 expression should be evaluated as a potential therapy for the disease.

An Integrated Meta-Analysis of Secretome and Proteome Identify Potential Biomarkers of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is extremely aggressive, has an unfavorable prognosis, and there are no biomarkers for early detection of the disease or identification of individuals at high risk for morbidity or mortality. The cellular and molecular complexity of PDAC leads to inconsistences in clinical validations of many proteins that have been evaluated as prognostic biomarkers of the disease. The tumor secretome, a potential source of biomarkers in PDAC, plays a crucial role in cell proliferation and metastasis, as well as in resistance to treatments, which together contribute to a worse clinical outcome. The massive amount of proteomic data from pancreatic cancer that has been generated from previous studies can be integrated and explored to uncover secreted proteins relevant to the diagnosis and prognosis of the disease. The present study aimed to perform an integrated meta-analysis of PDAC proteome and secretome public data to identify potential biomarkers of the disease. Our meta-analysis combined mass spectrometry data obtained from two systematic reviews of the pancreatic cancer literature, which independently selected 20 studies of the secretome and 35 of the proteome. Next, we predicted the secreted proteins using seven in silico tools or databases, which identified 39 secreted proteins shared between the secretome and proteome data. Notably, the expression of 31 genes of these secretome-related proteins was upregulated in PDAC samples from The Cancer Genome Atlas (TCGA) when compared to control samples from TCGA and The Genotype-Tissue Expression (GTEx). The prognostic value of these 39 secreted proteins in predicting survival outcome was confirmed using gene expression data from four PDAC datasets (validation set). The gene expression of these secreted proteins was able to distinguish high- and low-survival patients in nine additional tumor types from TCGA, demonstrating that deregulation of these secreted proteins may also contribute to the prognosis in multiple cancers types. Finally, we compared the prognostic value of the identified secreted proteins in PDAC biomarkers studies from the literature. This analysis revealed that our gene signature performed equally well or better than the signatures from these previous studies. In conclusion, our integrated meta-analysis of PDAC proteome and secretome identified 39 secreted proteins as potential biomarkers, and the tumor gene expression profile of these proteins in patients with PDAC is associated with worse overall survival.

The expression landscape of cachexia-inducing factors in human cancers.

BACKGROUND: Cachexia is a multifactorial syndrome highly associated with specific tumour types, but the causes of variation in cachexia prevalence and severity are unknown. While circulating plasma mediators (soluble cachectic factors) derived from tumours have been implicated with the pathogenesis of the syndrome, these associations were generally based on plasma concentration rather than tissue-specific gene expression levels. Here, we hypothesized that tumour gene expression profiling of cachexia-inducing factors (CIFs) in human cancers with different prevalence of cachexia could reveal potential cancer-specific cachexia mediators and biomarkers of clinical outcome. METHODS: First, we combined uniformly processed RNA sequencing data from The Cancer Genome Atlas and Genotype-Tissue Expression databases to characterize the expression profile of secretome genes in 12 cancer types (4651 samples) compared with their matched normal tissues (2737 samples). We systematically investigated the transcriptomic data to assess the tumour expression profile of 25 known CIFs and their predictive values for patient survival. We used the Xena Functional Genomics tool to analyse the gene expression of CIFs according to neoplastic cellularity in pancreatic adenocarcinoma, which is known to present the highest prevalence of cachexia. RESULTS: A comprehensive characterization of the expression profiling of secreted genes in different human cancers revealed pathways and mediators with a potential role in cachexia within the tumour microenvironment. Cytokine-related and chemokine-related pathways were enriched in tumour types frequently associated with the syndrome. CIFs presented a tumour-specific expression profile, in which the number of upregulated genes was correlated with the cachexia prevalence (r2 : 0.80; P value: 0.002) and weight loss (r2 : 0.81; P value: 0.002). The distinct gene expression profile, according to tumour type, was significantly associated with prognosis (P value ≤ 1.96 E-06). In pancreatic adenocarcinoma, the upregulated CIF genes were associated with tumours presenting low neoplastic cellularity and high leucocyte fraction and not with tumour grade. CONCLUSIONS: Our results present a biological dimension of tumour-secreted elements that are potentially useful to explain why specific cancer types are more likely to develop cachexia. The tumour-specific profile of CIFs may help the future development of better targeted therapies to treat cancer types highly associated with the syndrome.

An inexpensive microscopy system for microfluidic studies in budding yeast.

Recently, microfluidic technologies have been developed to allow higher throughput collection of yeast replicative lifespan data. Adoption of these devices has been limited, in part, due to the high cost of the motorized microscopy instrumentation from mainline manufacturers. Inspired by recent development of open source microscopy hardware and software, we developed minimal-cost hardware attachments to provide long-term focus stabilization for lower-cost microscopes and open source software to manage concurrent time-lapse image acquisition from multiple microscopes. We hope that these tools will help spur the wider adoption of microfluidic technologies for the study of aging in yeast.

Tumor Transcriptome Reveals High Expression of IL-8 in Non-Small Cell Lung Cancer Patients with Low Pectoralis Muscle Area and Reduced Survival.

Cachexia is a syndrome characterized by an ongoing loss of skeletal muscle mass associated with poor patient prognosis in non-small cell lung cancer (NSCLC). However, prognostic cachexia biomarkers in NSCLC are unknown. Here, we analyzed computed tomography (CT) images and tumor transcriptome data to identify potentially secreted cachexia biomarkers (PSCB) in NSCLC patients with low-muscularity. We integrated radiomics features (pectoralis muscle, sternum, and tenth thoracic (T10) vertebra) from CT of 89 NSCLC patients, which allowed us to identify an index for screening muscularity. Next, a tumor transcriptomic-based secretome analysis from these patients (discovery set) was evaluated to identify potential cachexia biomarkers in patients with low-muscularity. The prognostic value of these biomarkers for predicting recurrence and survival outcome was confirmed using expression data from eight lung cancer datasets (validation set). Finally, C2C12 myoblasts differentiated into myotubes were used to evaluate the ability of the selected biomarker, interleukin (IL)-8, in inducing muscle cell atrophy. We identified 75 over-expressed transcripts in patients with low-muscularity, which included IL-6, CSF3, and IL-8. Also, we identified NCAM1, CNTN1, SCG2, CADM1, IL-8, NPTX1, and APOD as PSCB in the tumor secretome. These PSCB were capable of distinguishing worse and better prognosis (recurrence and survival) in NSCLC patients. IL-8 was confirmed as a predictor of worse prognosis in all validation sets. In vitro assays revealed that IL-8 promoted C2C12 myotube atrophy. Tumors from low-muscularity patients presented a set of upregulated genes encoding for secreted proteins, including pro-inflammatory cytokines that predict worse overall survival in NSCLC. Among these upregulated genes, IL-8 expression in NSCLC tissues was associated with worse prognosis, and the recombinant IL-8 was capable of triggering atrophy in C2C12 myotubes.

The Pathway to Cancer Cachexia: MicroRNA-Regulated Networks in Muscle Wasting Based on Integrative Meta-Analysis.

Cancer cachexia is a multifactorial syndrome that leads to significant weight loss. Cachexia affects 50%-80% of cancer patients, depending on the tumor type, and is associated with 20%-40% of cancer patient deaths. Besides the efforts to identify molecular mechanisms of skeletal muscle atrophy-a key feature in cancer cachexia-no effective therapy for the syndrome is currently available. MicroRNAs are regulators of gene expression, with therapeutic potential in several muscle wasting disorders. We performed a meta-analysis of previously published gene expression data to reveal new potential microRNA-mRNA networks associated with muscle atrophy in cancer cachexia. We retrieved 52 differentially expressed genes in nine studies of muscle tissue from patients and rodent models of cancer cachexia. Next, we predicted microRNAs targeting these differentially expressed genes. We also include global microRNA expression data surveyed in atrophying skeletal muscles from previous studies as background information. We identified deregulated genes involved in the regulation of apoptosis, muscle hypertrophy, catabolism, and acute phase response. We further predicted new microRNA-mRNA interactions, such as miR-27a/Foxo1, miR-27a/Mef2c, miR-27b/Cxcl12, miR-27b/Mef2c, miR-140/Cxcl12, miR-199a/Cav1, and miR-199a/Junb, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting MSTN, CXCL12, and CAMK2B, which may be considered for the development of novel therapeutic strategies for cancer cachexia. Our study has broadened the knowledge of microRNA-regulated networks that are likely associated with muscle atrophy in cancer cachexia, pointing to their involvement as potential targets for novel therapeutic strategies.

Environmental monitoring of water resources around a municipal landfill of the Rio Grande do Sul state, Brazil.

In Brazil, landfills are commonly used as a method for the final disposal of waste that is compliant with the legislation. This technique, however, presents a risk to surface water and groundwater resources, owing to the leakage of metals, anions, and organic compounds. The geochemical monitoring of water resources is therefore extremely important, since the leachate can compromise the quality and use of surface water and groundwater close to landfills. In this paper, the results of analyses of metals, anions, ammonia, and physicochemical parameters were used to identify possible contamination of surface water and groundwater in a landfill area. A statistical multivariate approach was used. The values found for alkali metals, nitrate, and chloride indicate contamination in the regional groundwater and, moreover, surface waters also show variation when compared to the other background points, mainly for ammonia. Thus, the results of this study evidence the landfill leachate influence on the quality of groundwater and surface water in the study area.

Toxic and micronutrient elements in organic, brown and polished rice in Brazil.

Concentration levels of As, Cd, Hg, Pb, Tl, Sn, Sb Co, Cu, Mn, Se, Zn, Cr, Ni and Mo in different types of rice cultivated in irrigated fields in Brazil were evaluated. Arsenic, Cd, Pb, Zn, Mn and Cu were found in higher concentrations in brown rice samples, suggesting the prevalence of these elements in the bran. Meanwhile, lower concentrations of Pb, Mo, Cr, Se and Co were found in parboiled rice. Organic rice did not differ of cultivated conventionally rice. Thallium, Hg and Sb were not detected in any rice sample whose limits of detection were 0.7 µg kg(-1), 2.5 µg kg(-1) and 8 µg kg(-1), respectively. The concentrations of the investigated elements were compared with those reported for polished rice and brown rice from other countries, unveiling concentrations in general at the same level for rice produced at non-contaminated sites.

Sputtering deposition of magnetic Ni nanoparticles directly onto an enzyme surface: a novel method to obtain a magnetic biocatalyst.

A simple one-step method based on the sputtering deposition of Ni nanoparticles (NP) has been developed for the production of magnetic biocatalysts, avoiding the complications and drawbacks of methods based on chemical functionalisation or coating of magnetic NP. This new technique provided high levels of recovery, reusability and catalytic activity for the lipase-Ni biocatalyst.

Direct analysis of methylated trivalent arsenicals in mouse liver by hydride generation-cryotrapping-atomic absorption spectrometry.

Growing evidence suggest that the methylated trivalent metabolites of inorganic arsenic (iAs), methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), contribute to adverse effects of iAs exposure. However, the lack of suitable methods has hindered the quantitative analysis of MAs(III) and DMAs(III) in complex biological matrices. Here, we show that hydride generation-cryotrapping-atomic absorption spectrometry can quantify both MAs(III) and DMAs(III) in livers of mice exposed to iAs. No sample extraction is required, thus limiting MAs(III) or DMAs(III) oxidation prior to analysis. The limits of detection are below 6 ng As/g of tissue, making this method suitable even for studies examining low exposures to iAs.