Schizophrenia-derived hiPSC brain microvascular endothelial-like cells show impairments in angiogenesis and blood-brain barrier function.

Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed to a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ. As SZ is a neurodevelopmental disease, the use of human-induced pluripotent stem cells (hiPSC) allows disease biology modeling while retaining the patient's unique genetic signature. Previously, we reported a VEGFA signaling impairment in SZ-hiPSC-derived neural lineages leading to decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective blood-brain barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distinctive expression pattern of angiogenic and BBB-associated genes. Functionally, SZP BEC showed a decreased angiogenic response in vitro and higher transpermeability than Ctrl BEC. Immunofluorescence staining revealed less expression and altered distribution of tight junction proteins in SZP BEC. Moreover, SZP BEC's conditioned media reduced barrier capacities in the brain microvascular endothelial cell line HCMEC/D3 and in an in vivo permeability assay in mice. Overall, our results describe an intrinsic failure of SZP BEC for proper barrier function. These findings are consistent with the hypothesis tracing schizophrenia origins to brain development and BBB dysfunction.

High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

Preserved Ca2+ handling and excitation-contraction coupling in muscle fibres from diet-induced obese mice.

AIMS/HYPOTHESIS: Disrupted intracellular Ca2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity- and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitation-contraction coupling process: upon plasma membrane electrical activity, the CaV1.1 voltage sensor/Ca2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca2+, which activates contraction while Ca2+ uptake by the SR ATPase Ca2+-pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. METHODS: Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. CaV1.1 Ca2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca2+ release through ryanodine receptors was simultaneously detected with the Ca2+-sensitive dye Rhod-2 in the cytosol. CaV1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca2+ release at rest was assessed with the Ca2+-sensitive dye Fluo-4. The rate of SR Ca2+ uptake was assessed from the time course of cytosolic Ca2+ recovery after the end of voltage excitation using the Ca2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100 Hz. RESULTS: The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The CaV1.1 Ca2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/µF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/µF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca2+ release of 76.0 ± 6.5 and 78.1 ± 4.4 µmol l-1 ms-1, in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca2+ removal and the spontaneous Ca2+ release activity at rest. CONCLUSIONS/INTERPRETATION: The functional properties of the main mechanisms involved in the control of muscle Ca2+ homeostasis are well preserved in muscle fibres from obese mice, at the level of both the plasma membrane and of the SR. We conclude that intracellular Ca2+ handling and excitation-contraction coupling in skeletal muscle fibres are not primary targets of obesity and type 2 diabetes. Graphical abstract.

Epidemiology and Outcomes of Kocuria Keratitis.

OBJECTIVE: To determine the characteristics of infectious keratitis caused by Kocuria spp. METHODS: Retrospective case series. Information included demographic data, medical history, risk factors associated with infectious keratitis, clinical characteristics, microbiological results and drug sensitivity, clinical course, management, and visual outcomes. RESULTS: Eight patients were included (7 females and 1 male; mean age, 66.2 years; age range, 42-84 years). All patients presented risk factors for infectious complications, such as filamentous keratitis, dry eye, blepharitis, and persistent corneal edema. In all cases, ulcers were classified as severe. The infection resolved with medical treatment in one eye only. One case was treated with amniotic membrane graft. Two patients required keratoplasty (lamellar and penetrating), and one case needed sclerokeratoplasty. In three cases, the keratitis was severe enough to require evisceration. The final visual acuity ranged from 20/25 to no light perception. CONCLUSIONS: Bacterial keratitis by Kocuria spp. is a rare infection that may have an unexpected clinical course and possible serious outcomes. This pathogen should be considered in patients with unusual clinical course. Local or systemic immune compromise in the genesis of the disease must also be taken into account, and ophthalmologists should be more suspicious in this vulnerable group of patients.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity.

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P3 production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P3 was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

ATP signaling in skeletal muscle: from fiber plasticity to regulation of metabolism.

Tetanic electrical stimulation releases adenosine triphosphate (ATP) from muscle fibers through pannexin-1 channels in a frequency-dependent manner; extracellular ATP activates signals that ultimately regulate gene expression and is able to increase glucose transport through activation of P2Y receptors, phosphatidylinositol 3-kinase, Akt, and AS160. We hypothesize that this mechanism is an important link between exercise and the regulation of muscle fiber plasticity and metabolism.

Clinical and microbiological profile of infectious keratitis in children.

BACKGROUND: Infectious keratitis is a sight-threatening condition for children. The purpose of this study was to describe the clinical profile, risk factors and microbiological profile of infectious keratitis in children. METHODS: Retrospective review of clinical records of patients under 16 years of age with history of microbial keratitis seen at a tertiary referral center. Clinical characteristics, risk factors, visual and surgical outcomes as well as the microbiological profile are analyzed. RESULTS: Forty-one eyes of 41 patients. Mean age was 8.7 years. Time between the onset of symptoms and ophthalmological examination was 12.7 days. Predisposing factors were found in 78%; ocular trauma was the most common (25%). Visual acuity equal or worse than 20/200 at admission correlated positively with a poorer visual outcome, p=0.002. Positivity of cultures was 34%. Gram-positive bacteria were isolated in 78.5%; Staphylococcus epidermidis (28.6%) was the most common microorganism. CONCLUSIONS: Our study emphasizes the importance of a prompt diagnosis and treatment of infectious corneal ulcers in children. Trauma and contact lenses were the main predisposing factors. Gram-positive organisms were isolated in the vast majority of cases and visual outcomes are usually poor.

Energy (and Reactive Oxygen Species Generation) Saving Distribution of Mitochondria for the Activation of ATP Production in Skeletal Muscle.

Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived reactive oxygen species (ROS) are beneficial, and the amount and location of these ROS is important to avoid muscle damage associated with oxidative stress. We discuss here some of the evidence that involves ROS production associated with skeletal muscle contraction and the potential oxidative stress associated with muscle contraction. We also discuss the potential role of H2O2 produced after NOX activation in the regulation of glucose transport in skeletal muscle. Finally, we propose a model based on evidence for the role of different populations of mitochondria in skeletal muscle in the regulation of ATP production upon exercise. The subsarcolemmal population of mitochondria has the enzymatic and metabolic components to establish a high mitochondrial membrane potential when fissioned at rest but lacks the capacity to produce ATP. Calcium entry into the mitochondria will further increase the metabolic input. Upon exercise, subsarcolemmal mitochondria will fuse to intermyofibrillar mitochondria and will transfer the mitochondria membrane potential to them. These mitochondria are rich in ATP synthase and will subsequentially produce the ATP needed for muscle contraction in long-term exercise. These events will optimize energy use and minimize mitochondria ROS production.

Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K/Akt/mTOR signaling pathway.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a "myokine", synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as "Excitation-Transcription coupling". eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.

Pannexin-1 and CaV1.1 show reciprocal interaction during excitation-contraction and excitation-transcription coupling in skeletal muscle.

One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of CaV1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also CaV1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.

ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle.

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca(2+) homeostasis and muscle physiology.

Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle.

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 µM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP.

Gli3 is a zinc finger transcription factor proteolytically processed into a truncated repressor lacking C-terminal activation domains. Gli3 processing is stimulated by protein kinase A (PKA) and inhibited by Hedgehog signaling, a major signaling pathway in vertebrate development and disease. We show here that multisite glycogen synthase kinase 3beta (GSK3beta) phosphorylation and ubiquitination by SCFbetaTrCP are required for Gli3 processing. We identified multiple betaTrCP-binding sites related to the DSGX2-4S motif in Gli3, which are intertwined with PKA and GSK3beta sites, and SCFbetaTrCP target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3beta and CK1 that leads to direct betaTrCP binding and ubiquitination by SCFbetaTrCP. Binding of betaTrCP to Gli3 N- and C-terminal domains lacking DSGX2-4S-related motifs was also observed, which could reflect indirect interaction via other components of Hedgehog signaling, such as the tumor suppressor Sufu. Gli3 therefore joins a small set of transcription factors whose processing is regulated by the ubiquitin-proteasome pathway. Our study sheds light on the role of PKA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling.

Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse.

Skeletal muscle is described as an endocrine organ, constitutively or intermittently secreting bioactive molecules. The signaling pathways by which these molecules mediate changes in skeletal muscle and regulate interorgan crosstalk are only partly understood. Lactate is widely described as a signaling molecule in different cells, but the role of lactate as a signaling molecule in mature skeletal muscle has not been fully unveiled. The aim of this study was to determine the role of lactate on activation of signaling pathways in adult mouse skeletal muscle. Male mice were injected intraperitoneally with lactate or saline, and tissues were dissected after 40 min. Phosphorylation levels of relevant proteins in muscle were assessed by Western blotting. After lactate administration, we found an increase in p-ERK1/2Thr202/Tyr204 (3.5-fold; P = 0.004) and p-p70S6KThr389 (1.9-fold; P = 0.01) in quadriceps; and an increase in p-rpS6Ser235/236 in both quadriceps (6.3-fold; P = 0.01) and EDL (2.3-fold; P = 0.01), without changes in soleus. There was a tendency toward an increase in p-AMPKThr172 (1.7-fold; P = 0.08), with a significant increase in p-ACCSer79 (1.5-fold; P = 0.04) in soleus, without changes in quadriceps and EDL. These results support the hypothesis that lactate plays a role in the molecular signaling related to hypertrophy and to oxidative metabolism on adult skeletal muscle and suggest that this activation depends on the skeletal muscle type. The mechanisms that underlie the effect of lactate in mature skeletal muscles remain to be established.

Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice.

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion.

Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100 nmol/L) for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.

Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle.

BACKGROUND: Electrical activity regulates the expression of skeletal muscle genes by a process known as "excitation-transcription" (E-T) coupling. We have demonstrated that release of adenosine 5'-triphosphate (ATP) during depolarization activates membrane P2X/P2Y receptors, being the fundamental mediators between electrical stimulation, slow intracellular calcium transients, and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin 1 channels (Panx1, ATP release conduit), nucleotide receptors, and other signaling molecules. The goal of this study was to assess protein-protein interactions within the E-T machinery and to look for novel constituents in order to characterize the signaling complex. METHODS: Newborn derived myotubes, adult fibers, or triad fractions from rat or mouse skeletal muscles were used. Co-immunoprecipitation, 2D blue native SDS/PAGE, confocal microscopy z-axis reconstruction, and proximity ligation assays were combined to assess the physical proximity of the putative complex interactors. An L6 cell line overexpressing Panx1 (L6-Panx1) was developed to study the influence of some of the complex interactors in modulation of gene expression. RESULTS: Panx1, DHPR, P2Y2 receptor (P2Y2R), and dystrophin co-immunoprecipitated in the different preparations assessed. 2D blue native SDS/PAGE showed that DHPR, Panx1, P2Y2R and caveolin-3 (Cav3) belong to the same multiprotein complex. We observed co-localization and protein-protein proximity between DHPR, Panx1, P2Y2R, and Cav3 in adult fibers and in the L6-Panx1 cell line. We found a very restricted location of Panx1 and Cav3 in a putative T-tubule zone near the sarcolemma, while DHPR was highly expressed all along the transverse (T)-tubule. By Panx1 overexpression, extracellular ATP levels were increased both at rest and after electrical stimulation. Basal mRNA levels of the early gene cfos and the oxidative metabolism markers citrate synthase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were significantly increased by Panx1 overexpression. Interleukin 6 expression evoked by 20-Hz electrical stimulation (270 pulses, 0.3 ms each) was also significantly upregulated in L6-Panx1 cells. CONCLUSIONS: We propose the existence of a relevant multiprotein complex that coordinates events involved in E-T coupling. Unveiling the molecular actors involved in the regulation of gene expression will contribute to the understanding and treatment of skeletal muscle disorders due to wrong-expressed proteins, as well as to improve skeletal muscle performance.

Adaptación de una metodología de especificaciones en la calificación y retroalimentación en la enseñanza de cursos de ciencias básicas en una carrera de la salud para mejorar los aprendizajes y la motivación / Adaptation of a specifications gradings and feedback methodology for basic sciences courses teaching in a health science career to improving learning and motivation

Introducción: la enseñanza de cursos de ciencias básicas en carreras de la salud es un desafío por no estar directa e inmediatamente rela-cionada con el ámbito profesional. Por otra parte, las condiciones de estrés que ha impuesto el trabajo a distancia requiere de metodologías motivantes, y, que a su vez permitan una evaluación significativa. Objetivos: reportar las adaptaciones metodológicas y los resultados de una adaptación local de la metodología de especificaciones de las calificaciones y retroalimentación del trabajo. Métodos: se aplica una metodología de formación basada en la retroalimentación en el curso de Física para estudiantes de Tecnología Médica (N=106) durante un semestre. Las calificaciones promedio de los estudiantes fueron comparadas con las obtenidas en años anteriores. Para evaluar el desempeño docente se realizaron 2 encuestas a los estudiantes. El cumplimiento de los logros de aprendizaje se midió mediante auto-evaluación (escala likert 1 a 5) al inicio y término de cada uno de los cuatro capítulos. Resultados: las reprobaciones y eliminaciones de estudiantes en el curso fueron menores a años anteriores, siendo las notas significativamente mayores subiendo desde 4,89 a 6,29 (escala de 1 a 7, p<0,001). Los estudiantes se mostraron en un 95% satisfechos con el desempeño docente y finalmente, la auto-evaluación de logros de aprendizaje mostró un aumento promedio de 1 punto. Conclusiones: la metodología de evaluación basada en especificaciones adaptada a dos entregas y con evaluaciones en una escala no-binaria mejoró el rendimiento, los logros de los aprendizajes esperados y la motivación de los estudiantes.

Background: Teaching basic science courses in health careers is a challenge because these courses are not directly linked to professional practice. On the other hand, the stressful conditions imposed by distance work require motivating methodologies and a meaningful evaluation. Objectives: To report the methodological adaptations and the results of a local adaptation of the specifications grading and feedback methodology. Methods: A training methodology based on feedback is applied in the Physics course for Medical Technology students (N = 106) during one semester. We compared the students' average grades to those obtained in previous years with the same topics. To evaluate the teaching performance, we conducted two student surveys. We measure compliance with learning achievements by self-assessment (Likert scale 1 to 5) at the beginning and end of each of the four chapters. Results: Failures and eliminations of students in the course were lower than previous years, with significantly higher grades from 4.89 to 6.29 (p <0.001). The students were 95% satisfied with the teaching performance, and finally, the self-evaluation of learning achievements showed an average increase of 1 point. Conclusion: The evaluation methodology based on specifications adapted to two deliveries and evaluations on a non-binary scale improved the performance, expected learning achievements, and students' motivation.