Changes in task-based effective connectivity in language networks following rehabilitation in post-stroke patients with aphasia

In this study, we examined regions in the left and right hemisphere language network that were altered in terms of the underlying neural activation and effective connectivity subsequent to language rehabilitation. Eight persons with chronic post-stroke aphasia and eight normal controls participated in the current study. Patients received a 10 week semantic feature-based rehabilitation program to improve their skills. Therapy was provided on atypical examples of one trained category while two control categories were monitored; the categories were counterbalanced across patients. In each fMRI session, two experimental tasks were conducted: (a) picture naming and (b) semantic feature verification of trained and untrained categories. Analysis of treatment effect sizes revealed that all patients showed greater improvements on the trained category relative to untrained categories. Results from this study show remarkable patterns of consistency despite the inherent variability in lesion size and activation patterns across patients. Across patients, activation that emerged as a function of rehabilitation on the trained category included bilateral IFG, bilateral SFG, LMFG, and LPCG for picture naming; and bilateral IFG, bilateral MFG, LSFG, and bilateral MTG for semantic feature verification. Analysis of effective connectivity using Dynamic Causal Modeling (DCM) indicated that LIFG was the consistently significantly modulated region after rehabilitation across participants. These results indicate that language networks in patients with aphasia resemble normal language control networks and that this similarity is accentuated by rehabilitation.The funding for this project comes from NIDCD/NIH 1P50DC012283 and NIDCD/NIH 1K18DC011517. (1P50DC012283 - NIDCD/NIH; 1K18DC011517 - NIDCD/NIH)Published versio

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer

Ultra-rare genetic variation in common epilepsies: a case-control sequencing study

BACKGROUND:Despite progress in understanding the genetics of rare epilepsies, the more common epilepsies have proven less amenable to traditional gene-discovery analyses. We aimed to assess the contribution of ultra-rare genetic variation to common epilepsies. METHODS:We did a case-control sequencing study with exome sequence data from unrelated individuals clinically evaluated for one of the two most common epilepsy syndromes: familial genetic generalised epilepsy, or familial or sporadic non-acquired focal epilepsy. Individuals of any age were recruited between Nov 26, 2007, and Aug 2, 2013, through the multicentre Epilepsy Phenome/Genome Project and Epi4K collaborations, and samples were sequenced at the Institute for Genomic Medicine (New York, USA) between Feb 6, 2013, and Aug 18, 2015. To identify epilepsy risk signals, we tested all protein-coding genes for an excess of ultra-rare genetic variation among the cases, compared with control samples with no known epilepsy or epilepsy comorbidity sequenced through unrelated studies. FINDINGS:We separately compared the sequence data from 640 individuals with familial genetic generalised epilepsy and 525 individuals with familial non-acquired focal epilepsy to the same group of 3877 controls, and found significantly higher rates of ultra-rare deleterious variation in genes established as causative for dominant epilepsy disorders (familial genetic generalised epilepsy: odd ratio [OR] 2·3, 95% CI 1·7-3·2, p=9·1 × 10-8; familial non-acquired focal epilepsy 3·6, 2·7-4·9, p=1·1 × 10-17). Comparison of an additional cohort of 662 individuals with sporadic non-acquired focal epilepsy to controls did not identify study-wide significant signals. For the individuals with familial non-acquired focal epilepsy, we found that five known epilepsy genes ranked as the top five genes enriched for ultra-rare deleterious variation. After accounting for the control carrier rate, we estimate that these five genes contribute to the risk of epilepsy in approximately 8% of individuals with familial non-acquired focal epilepsy. Our analyses showed that no individual gene was significantly associated with familial genetic generalised epilepsy; however, known epilepsy genes had lower p values relative to the rest of the protein-coding genes (p=5·8 × 10-8) that were lower than expected from a random sampling of genes. INTERPRETATION:We identified excess ultra-rare variation in known epilepsy genes, which establishes a clear connection between the genetics of common and rare, severe epilepsies, and shows that the variants responsible for epilepsy risk are exceptionally rare in the general population. Our results suggest that the emerging paradigm of targeting of treatments to the genetic cause in rare devastating epilepsies might also extend to a proportion of common epilepsies. These findings might allow clinicians to broadly explain the cause of these syndromes to patients, and lay the foundation for possible precision treatments in the future. FUNDING:National Institute of Neurological Disorders and Stroke (NINDS), and Epilepsy Research UK

Essential amino acid and carbohydrate ingestion before resistance exercise does not enhance postexercise muscle protein synthesis

Ingestion of an essential amino acid-carbohydrate (EAA + CHO) solution following resistance exercise enhances muscle protein synthesis during postexercise recovery. It is unclear whether EAA + CHO ingestion before resistance exercise can improve direct measures of postexercise muscle protein synthesis (fractional synthetic rate; FSR). We hypothesized that EAA + CHO ingestion before a bout of resistance exercise would prevent the exercise-induced decrease in muscle FSR and would result in an enhanced rate of muscle FSR during postexercise recovery. We studied 22 young healthy subjects before, during, and for 2 h following a bout of high-intensity leg resistance exercise. The fasting control group (n = 11) did not ingest nutrients, and the EAA + CHO group (n = 11) ingested a solution of EAA + CHO 1 h before beginning the exercise bout. Stable isotopic methods were used in combination with muscle biopsies to determine FSR. Immunoblotting procedures were utilized to assess cell signaling proteins associated with the regulation of FSR. We found that muscle FSR increased in the EAA + CHO group immediately following EAA + CHO ingestion (P < 0.05), returned to basal values during exercise, and remained unchanged at 1 h postexercise. Muscle FSR decreased in the fasting group during exercise and increased at 1 h postexercise (P < 0.05). However, the 2 h postexercise FSR increased by ∼50% in both groups with no differences between groups (P > 0.05). Eukaryotic elongation factor 2 phosphorylation was reduced in both groups at 2 h postexercise (EAA + CHO: 39 ± 7%; fasting: 47 ± 9%; P < 0.05). We conclude that EAA + CHO ingestion before resistance exercise does not enhance postexercise FSR compared with exercise without nutrients

Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery

Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKα2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased (∼35%) during postexercise recovery (P ≤ 0.05). Glucose uptake increased during exercise and postexercise recovery (P ≤ 0.05). Akt phosphorylation was increased at 1 h and AMPKα2 activity increased at 2 h postexercise (P ≤ 0.05). Phospho(Ser/Thr)-Akt substrate (PAS) phosphorylation (often used as a marker for AS160) was unchanged immediately postexercise and increased at 1 h (P ≤ 0.05) and 2 h postexercise (P = 0.07). The PAS antibody is not always specific for AS160/TBC1D4 and can detect proteins at a similar molecular weight. Therefore, we immunoprecipitated AS160/TBC1D4 and then blotted with the PAS antibody, which confirmed that PAS phosphorylation is occurring on AS160/TBC1D4. There was also a positive correlation between PAS phosphorylation and leg glucose uptake during recovery (P < 0.05). We conclude that resistance exercise increases AS160/TBC1D4 phosphorylation in association with an increase in leg glucose uptake during postexercise recovery

An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1–3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) α-subunit (Ser52)]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser240/244) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h (P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2α phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA (P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery

Muscle protein breakdown has a minor role in the protein anabolic response to essential amino acid and carbohydrate intake following resistance exercise

Muscle protein breakdown (MPB) is increased following resistance exercise, but ingestion of carbohydrate during postexercise recovery can decrease MPB with no effect on muscle protein synthesis (MPS). We sought to determine whether a combination of essential amino acids (EAA) with low carbohydrate or high carbohydrate could effectively reduce MPB following resistance exercise and improve muscle protein net balance (NB). We hypothesized that higher levels of carbohydrate and resulting increases in circulating insulin would inhibit MPB and associated signaling, resulting in augmented NB. Thirteen male subjects were assigned to one of two groups receiving equivalent amounts of EAA (∼20 g) but differing carbohydrate levels (low = 30, high = 90 g). Groups ingested nutrients 1 h after an acute bout of leg resistance exercise. Leg phenylalanine kinetics (e.g., MPB, MPS, NB), signaling proteins, and mRNA expression were assessed on successive muscle biopsies using stable isotopic techniques, immunoblotting, and real-time quantitative PCR, respectively. MPB tended to decrease (P < 0.1) and MPS increased (P < 0.05) similarly in both groups following nutrient ingestion. No group differences were observed, but muscle ring finger 1 (MuRF1) protein content and MuRF1 mRNA expression increased following resistance exercise and remained elevated following nutrient ingestion, while autophagy marker (light-chain 3B-II) decreased after nutrient ingestion (P < 0.05). Forkhead box-O3a phosphorylation, total muscle atrophy F-box (MAFbx) protein, and MAFbx and caspase-3 mRNA expression were unchanged. We conclude that the enhanced muscle protein anabolic response detected when EAA+carbohydrate are ingested postresistance exercise is primarily due to an increase in MPS with minor changes in MPB, regardless of carbohydrate dose or circulating insulin level