Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients’ condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages

A primer on machine learning techniques for genomic applications

High throughput sequencing technologies have enabled the study of complex biological aspects at single nucleotide resolution, opening the big data era. The analysis of large volumes of heterogeneous “omic” data, however, requires novel and efficient computational algorithms based on the paradigm of Artificial Intelligence. In the present review, we introduce and describe the most common machine learning methodologies, and lately deep learning, applied to a variety of genomics tasks, trying to emphasize capabilities, strengths and limitations through a simple and intuitive language. We highlight the power of the machine learning approach in handling big data by means of a real life example, and underline how described methods could be relevant in all cases in which large amounts of multimodal genomic data are available

Oxytocin/Osteocalcin/IL-6 and NGF/BDNF mRNA Levels in Response to Cold Stress Challenge in Mice: Possible Oxytonic Brain-Bone-Muscle-Interaction

Oxytocin (Oxt), osteocalcin (Ost), and NGF/BDNF have a role in bone homeostasis, reproduction, and cognition. Oxt/Ost is required for muscle repair. We investigated gene response of muscle and the inter-organ communication following cold stress (CS). The mRNA quantity of Ngf, Ost, Oxt, Bdnf, p75ntr, Ntrk1, Gprc6a, Oxtr, Ntrk2, UCP1, and Il-6 genes in bone, brain, soleus (SOL), and tibialis anterior (TA) muscles from adult mice following CS were investigated. The myosin heavy-chain Mhc2b, Mhc1, Mhc2x, and Mhc2a gene expression were investigated. Mice were maintained at T = 23°C or 4°C for 6 h and 5-days (5d). CS mice did not show signs of muscle degeneration. An upregulation of Ucp1 and Ngf genes by 2 and 1.5 folds, respectively, in TA after 6 h CS and Ntrk1 by 4 and 22 folds in SOL muscle after 6 h and 5d CS, respectively, was observed; while after 6 h CS p75Ntr was downregulated in either muscle. Bdnf was unaffected, while after 5d CS Ntrk2 was upregulated in TA. Ost was downregulated in SOL by 0.9-folds at 5d. Following 5d CS, Oxtr and Il-6 genes were upregulated, respectively, by 1 and 1.5 folds in SOL. A downregulation of Mhc2b, respectively, by 0.96 and 0.88-folds after 6 h and 5d CS in SOL and Mhc2a was also downregulated by 0.88-fold after 5d CS in TA. Mhc1 and Mhc2x were not affected. Changes in the expression levels of genes in TA and SOL muscles, bone, and brain following CS were regulated by IL6 and Oxt. CS potentiates the slow-twitch phenotype of SOL which is in line with the metabolic need of this muscle, and the potentiation of the slow-twitch phenotype in TA. Oxt and IL6 coordinate a phenotype-dependent tonic effect of slow-twitch muscle and Oxt regulates the inter-organ interaction between brain and SOL muscle. Muscle tropism is maintained by NGF signaling following CS

EVIDENCE OF DIFFERENTIAL EXPRESSION RELATED TO THYROSINE HYDROXILASE GENES IN CHICK (GALLUS GALLUS) BRAIN STEM DURING EMBRYO DEVELOPMENT

By affecting efficiency of DOPA synthesis, tyrosine hydroxylase (TH) is the effective modulator of the limiting step of the biosynthetic pathways of catecholamines (dopamine, noradrenaline and adrenaline), highly conserved in vertebrate species. Catecholamines are involved in many physiological functions in the central and peripheral nervous systems as well as in the endocrine system, thus regulation of TH expression and activity are crucial for neuronal and hormonal functions that involve the entire dopaminergic, noradrenergic and adrenergic systems. THrelated expression is due to two non-allelic genes, called TH1 and TH2, reported in almost all vertebrates except placental mammalian, which have lost TH2 gene during evolution. THrelated genes have crucial ontogenetic roles, being linked to pathological onsets during embryo development. Here, we show the expression analysis of TH-related transcripts in brain stem of gallus gallus, as a key model of vertebrate central nervous system development. By real time RT-PCR assays, we assessed that TH1 and TH2 mRNAs show progressive increase during embryo development (from 8 to 21 days post fertilization) with differential trend. Moreover, a substantially different regulatory switch of expression was shown for the two genes when passing to the adult developmental phase. According to what stated in teleost fish, different expression patterns suggest different mechanism of transcriptional regulation related to potentially differing roles during development for TH1 and TH2 genes: based on our comparative results, TH1 mRNA expression in gallus gallus brain increases gradually during development reaching significantly high post-embryonic levels, whereas the TH2 mRNA seems to be more specifically linked to embryogenesis of vertebrate brain stem

Changes in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation

The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fast-twitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered

Increased sarcolemma chloride conductance as one of the mechanisms of action of carbonic anhydrase inhibitors in muscle excitability disorders

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 μM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 μM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50–100 μM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 μM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders

Detection of ribonucleotides embedded in DNA by Nanopore sequencing

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes

Growth hormone secretagogues hexarelin and JMV2894 protect skeletal muscle from mitochondrial damages in a rat model of cisplatin-induced cachexia

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia