Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the "pros and cons" of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers.

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD⁺ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.

The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P. aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.

To investigate the incidence of coronavirus disease 2019 (COVID-19) in a single-center cohort of patients with MS and to explore the contribution of their comorbidities and therapies to the outcome.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

A prevalent side-effect of simvastatin is attenuated glucose homeostasis. The underlying mechanism is unknown, but impaired lipid metabolism may provide the link. The aim of this study was to investigate whether simvastatin-treated patients had a lower capacity to oxidize lipids and reduced expression of the major proteins regulating lipid uptake, synthesis, lipolysis, and storage in skeletal muscle than matched controls.

CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1beta is primarily neurotoxic in ischemic stroke, TNF-alpha may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.

Macrophage infiltration in two subcutaneous adipose tissue depots and systemic low-grade inflammation were studied in post-obese (PO), obese (O), and control (C) subjects. Young males were recruited into PO: (n = 10, weight-loss avg. 26%, BMI: 26.6 ± 0.7, mean ±SEM kg/m2), O: (n = 10, BMI: 33.8 ± 1.0kg/m2) and C: (n = 10, BMI: 26.6 ± 0.6 kg/m2). PO and C were matched by BMI. Blood and abdominal and gluteal subcutaneous adipose tissue were obtained in the overnight fasted state. Plasma concentrations of IL-6 and CRP were higher (p < 0.05) in O than in PO and C, TNF-α was higher (p < 0.05) only in O compared to PO and IL-18 was similar between groups. The number of CD68+ macrophages was higher (p < 0.05) in the gluteal than the abdominal depot, and higher (p < 0.05) in O and PO compared to C in both depots. The content of CD163+ macrophages was similar between depots but was higher (p < 0.05) in PO compared to C and O in the gluteal depot. In post obese men with a long-term sustained weight loss, systemic low-grade inflammation was similar to non-obese controls despite a higher subcutaneous adipose tissue CD68+ macrophage content. Interestingly, the anti-inflammatory CD163+ macrophage adipose tissue content was consistently higher in post obese than obese and controls.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

Cording was the first virulence factor identified in Mycobacterium tuberculosis (Mtb). We aimed to ascertain its role in the induction of active tuberculosis (TB) in the mouse strain C3HeB/FeJ by testing the immunopathogenic capacity of the H37Rv strain. We have obtained two batches of the same strain by stopping their growth in Proskauer Beck liquid medium once the mid-log phase was reached, in the noncording Mtb (NCMtb) batch, and two days later in the cording Mtb (CMtb) batch, when cording could be detected by microscopic analysis. Mice were challenged with each batch intravenously and followed-up for 24 days. CMtb caused a significant increase in the bacillary load at an early stage post-challenge (day 17), when a granulomatous response started, generating exudative lesions characterized by neutrophilic infiltration, which promoted extracellular bacillary growth together with cording formation, as shown for the first time in vivo. In contrast, NCMtb experienced slight or no bacillary growth and lesions could barely be detected. Previous Bacillus Calmette-Guérin (BCG) vaccination or low dose aerosol (LDA) Mtb infection were able to delay the progression towards active TB after CMtb challenge. While BCG vaccination also reduced bacillary load when NCMtb was challenged, LDA did not, and its proliferative lesions experienced neutrophil infiltration. Analysis of lung cytokine and chemokine profiles points to their capacity to block the production of CXCL-1 and further amplification of IL-1β, IL-17 and neutrophilic extracellular trap formation, all of which are essential for TB progression. These data highlight the key role of cording formation in the induction of active TB.

Most studies, aimed at determining the incidence and transmission of SARS-CoV-2 in children and teenagers, have been developed in school settings. Our study conducted surveillance and inferred attack rates focusing on the practice of sports. Prospective and observational study of those attending the sports facilities of Fútbol Club Barcelona (FCB), in Barcelona, Spain, throughout the 2020-2021 season. Participants were young players (from five different sports) and adult workers, who belonged to stable teams (shared routines and were involved in same quarantine rules). Biweekly health questionnaires and SARS-CoV-2 screening were conducted. From the 234 participants included, 70 (30%) both lived and trained in the FCB facilities (Recruitment Pathway 1;RP1) and 164 (70%) lived at their own household and just came to the facilities to train (RP2). During the study, 38 positive cases were identified; none had severe symptoms or needed hospitalization. The overall weekly incidence in the cohorts did not differ compared to the one expected in the community, except for 2 weeks when an outbreak occurred. The attack rate (AR) was three times higher for the participants from RP1, in comparison to those from RP2 (p < 0.01). A Basketball team showed a significant higher AR.  Conclusion: Physical activities in stable teams are not related to an increased risk of transmission of SARS-CoV-2, since there were the same observed cases than expected in the community. The risk is higher in indoor sports (Basketball vs. Football), and in closed cohort living settings (RP1 vs. RP2). The fulfilment of preventive measures is essential. What is Known: • Despite the low numerical impact caused in paediatric hospitalizations during COVID-19 pandemic, the social impact has been maximum. • The transmission potential in children and teenagers is limited, and it had been widely demonstrated in school settings. What is New: • Group physical activities in children and teenagers are not also related to an increased risk of transmission of SARS-CoV-2, when preventive measures, such as washing hands, and screening protocols are applied. • Routine and semi-professional sports activities seem safe environments to promote during this pandemic.

The evolution of a tuberculosis (TB) infection toward active disease is driven by a combination of factors mostly related to the host response. The equilibrium between control of the bacillary load and the pathology generated is crucial as regards preventing the growth and proliferation of TB lesions. In addition, some experimental evidence suggests an important role of both local endogenous reinfection and the coalescence of neighboring lesions. Herein we propose a mathematical model that captures the essence of these factors by defining three hypotheses: (i) lesions grow logistically due to the inflammatory reaction; (ii) new lesions can appear as a result of extracellular bacilli or infected macrophages that escape from older lesions; and (iii) lesions can merge when they are close enough. This model was implemented in Matlab to simulate the dynamics of several lesions in a 3D space. It was also fitted to available microscopy data from infected C3HeB/FeJ mice, an animal model of active TB that reacts against Mycobacterium tuberculosis with an exaggerated inflammatory response. The results of the simulations show the dynamics observed experimentally, namely an initial increase in the number of lesions followed by fluctuations, and an exponential increase in the mean area of the lesions. In addition, further analysis of experimental and simulation results show a strong coincidence of the area distributions of lesions at day 21, thereby highlighting the consistency of the model. Three simulation series removing each one of the hypothesis corroborate their essential role in the dynamics observed. These results demonstrate that three local factors, namely an exaggerated inflammatory response, an endogenous reinfection, and a coalescence of lesions, are needed in order to progress toward active TB. The failure of one of these factors stops induction of the disease. This mathematical model may be used as a basis for developing strategies to stop the progression of infection toward disease in human lungs.

Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set.

Aging is associated with impaired mitochondrial function, whereas exercise training enhances mitochondrial content and function in part through activation of PGC-1α. Mitochondria form dynamic networks regulated by fission and fusion with profound effects on mitochondrial functions, yet the effects of aging and exercise training on mitochondrial network structure remain unclear. This study examined the effects of aging and exercise training on mitochondrial network structure using confocal microscopy on mitochondria-specific stains in single muscle fibers from PGC-1α KO and WT mice. Hyperfragmentation of mitochondrial networks was observed in aged relative to young animals while exercise training normalized mitochondrial network structure in WT, but not in PGC-1α KO. Mitochondrial fission protein content (FIS1 and DRP1) relative to mitochondrial content was increased with aging in both WT and PGC-1α KO mice, while exercise training lowered mitochondrial fission protein content relative to mitochondrial content only in WT. Mitochondrial fusion protein content (MFN1/2 and OPA1) was unaffected by aging and lifelong exercise training in both PGC-1α KO and WT mice. The present results provide evidence that exercise training rescues aging-induced mitochondrial fragmentation in skeletal muscle by suppressing mitochondrial fission protein expression in a PGC-1α dependent manner.