Single cell analysis reveals the involvement of the long non-coding RNA Pvt1 in the modulation of muscle atrophy and mitochondrial network

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell and tissue specificity and subcellular compartmentalization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. How lncRNAs are expressed in different myofibers, participate in metabolism regulation and muscle atrophy or how they are compartmentalized within a single myofiber is still unknown. We compiled a comprehensive catalog of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, and demonstrating that many lncRNAs can be involved in the biological processes de-regulated during muscle atrophy. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity

Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle

Summary: Skeletal muscle is composed of different myofiber types that preferentially use glucose or lipids for ATP production. How fuel preference is regulated in these post-mitotic cells is largely unknown, making this issue a key question in the fields of muscle and whole-body metabolism. Here, we show that microRNAs (miRNAs) play a role in defining myofiber metabolic profiles. mRNA and miRNA signatures of all myofiber types obtained at the single-cell level unveiled fiber-specific regulatory networks and identified two master miRNAs that coordinately control myofiber fuel preference and mitochondrial morphology. Our work provides a complete and integrated mouse myofiber type-specific catalog of gene and miRNA expression and establishes miR-27a-3p and miR-142-3p as regulators of lipid use in skeletal muscle. : Chemello et al. characterize coding mRNAs and non-coding microRNAs expressed by myofibers of hindlimb mouse muscles, identifying complex interactions between these molecules that modulate mitochondrial functions and muscle metabolism. They demonstrate that specific short non-coding RNAs influence the contractile fiber composition of skeletal muscles by modulating muscle metabolism. Keywords: single myofiber, skeletal muscle metabolism, mitochondria, miRNAs, lipid

Postmenopausal estrogen and progestin effects on the serum proteome

Background: Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated. Methods: Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool. Results: In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P less than 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates less than 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates less than 0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways. Conclusions: Serum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects

Investigation of two Fermi-LAT gamma-ray blazars coincident with high-energy neutrinos detected by IceCube

After the identification of the gamma-ray blazar TXS 0506+056 as the first compelling IceCube neutrino source candidate, we perform a systematic analysis of all high-energy neutrino events satisfying the IceCube realtime trigger criteria. We find one additional known gamma-ray source, the blazar GB6 J1040+0617, in spatial coincidence with a neutrino in this sample. The chance probability of this coincidence is 30% after trial correction. For the first time, we present a systematic study of the gamma-ray flux, spectral and optical variability, and multi-wavelength behavior of GB6 J1040+0617 and compare it to TXS 0506+056. We find that TXS 0506+056 shows strong flux variability in the Fermi-LAT gamma-ray band, being in an active state around the arrival of IceCube-170922A, but in a low state during the archival IceCube neutrino flare in 2014/15. In both cases the spectral shape is statistically compatible ($\leq 2\sigma$) with the average spectrum showing no indication of a significant relative increase of a high-energy component. While the association of GB6 J1040+0617 with the neutrino is consistent with background expectations, the source appears to be a plausible neutrino source candidate based on its energetics and multi-wavelength features, namely a bright optical flare and modestly increased gamma-ray activity. Finding one or two neutrinos originating from gamma-ray blazars in the given sample of high-energy neutrinos is consistent with previously derived limits of neutrino emission from gamma-ray blazars, indicating the sources of the majority of cosmic high-energy neutrinos remain unknown.Comment: 22 pages, 11 figures, 2 Table

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Identification of specific non-coding RNAs involved in skeletal muscle metabolism: a single cell approach.

Skeletal muscle is a heterogeneous tissue composed of different cell types with myofibers that are the smallest complete contractile system that influence muscle contraction velocity and metabolism. Myofibers, can be classified in three main categories: slow oxidative (also known as type I), fast-oxidative (or type IIA) and fast-glycolytic (or type IIB). Recently, the non-coding RNAs have emerged as a new RNA class with regulatory functions in several biological processes. Non-coding RNAs (ncRNAs) are transcripts with little or no coding capacity, divisible in two classes: microRNAs (miRNAs; shorter than 200nt) and long non-coding RNAs (lncRNAs; longer than 200nt). To uncover the functions of ncRNAs in muscle physiopathology, we profiled both miRNAs and lncRNAs in single myofibers, evidencing the importance of single cell approaches. miRNAs. We discovered two circuits that connect myofiber metabolic traits with mir-27a-3p and mir-142-3p. We evidenced that miR-27a-3p was highly expressed in oxidative myofibers while miR-142-3p was low expressed, suggesting a complementary mechanism in the regulation of myofiber metabolism. In fact, we demonstrated a) the involvement of miR-27a-3p in the inhibition of the synthesis of Phosphoglucomutase 2 (Pgm2) and acid α-glucosidase (Gaa) enzymes, both involved in glycogenolysis and b) the control of the Fndc5-Irisin pathway by miR-142-3p that instead modulate lipids content within myofibers. lncRNAs. Since there are no data available on lncRNAs expressed by myofibers, we defined them according to their expression in skeletal muscle fibers. We showed that lncRNAs are fiber type specific, with a peculiar subcellular localization and are sensible to muscle atrophy. We showed that the lncRNA Plasmacytoma variant translocation 1 (Pvt1) is activated early during muscle atrophy and it is more expressed in fast myofibers. These observations were the starting points for the pathway dissection throughout which Pvt1 acts in skeletal muscle. In fact, atrophy causes metabolic changes and myofiber switching, both inter-related myofiber characteristics. We demonstrated the impact of Pvt1 on mitochondrial respiration and morphology, its regulatory activity on autophagy and apoptosis, and its consequent ability to influence myofiber size. Pvt1 regulates mitochondria physiology though its capacity in modulating c-Myc, which in turn, regulates Bcl2, Bax/Bak, Mfn1, and Becn1, impinging mitochondrial dynamics and finally muscle fiber dimension. The story is then made difficult by the fact that Pvt1 locus codify also for miRNAs. We showed that three miRNAs encoded by Pvt1 locus (miR-1207-3p, miR-1207-5p and miR-1208) seems influence mitochondrial dynamics. We speculate that these miRNAs could target the lncRNA Pvt1 revealing a new regulatory loop of miRNAs and lncRNAs that may govern skeletal muscle homeostasis. In skeletal muscle research, the evolution of new single cell approaches allows the identification of signatures that specify functional and metabolic behavior of myofibers, otherwise impossible to detect analyzing the whole muscle. Our works contribute to better explain not only the importance of single cell studies, but also how ncRNAs participate in the regulation of skeletal muscle functions

Mutations in Assembly Factors Required for the Biogenesis of Mitochondrial Respiratory Chain

The mitochondrial respiratory chain, which provides to the cells most of their ATP requirement, is composed of five multisubunit complexes. Its biogenesis is a multi-step process characterized by the sequential formation of intermediate assemblies composed of subunits encoded by two distinct genomes, mitochondrial or nuclear DNA. This process is assisted by a diverse set of ancillary proteins of nuclear origin called assembly factors that are not part of the final complexes and exert different functions. Mutations in several genes encoding these proteins have been identified in patients affected by mitochondrial diseases, exceeding those found in genes encoding structural subunits for some complexes. The hallmark of these disorders, which are often multisystemic and mainly affect high energy demanding organs, is the broad genetic and clinical heterogeneity, making their diagnosis problematic. The number of assembly factors associated with human diseases is rapidly increasing, owing to the employment of next generation sequencing methods in the diagnostic workflow. Therapy for these conditions is mostly based on supportive care, emphasizing the need to elucidate their pathological mechanisms to find novel treatments

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts

A multidisciplinary study unveils the nature of a Roman ink of the I century AD

A multi-instrumental approach combining highly sensitive Synchrotron Radiation-based techniques was used to provide information on the real composition of a dry black ink powder found in a bronze inkwell of the first century AD. The presence of Pb, Cu and Fe in the powder, revealed by XRF and ICP-OES data, leads to raise several hypotheses on their origin. The inkpot and its lid were also investigated by Hand-Held XRF, revealing a bronze alloy (Cu-Sn) with a certain amount of Fe and Pb. The lid was found to be particularly enriched in lead. XRPD, XAS and FTIR measurements showed a substantial presence of silicates and common clay minerals in the ink along with cerussite and malachite, Pb and Cu bearing-carbonates, respectively. These evidences support the hypothesis of an important contamination of the ink sample by the burial environment (soil) and the presence of degradation products of the bronze inkpot. The combined use of IR, Raman, and GC-MS evidenced that the black ink is mainly composed of amorphous carbon deriving from the combustion of organic material mixed with a natural binding agent, Arabic gum