The α₇β₁-integrin increases muscle hypertrophy following multiple bouts of eccentric exercise

Mechanical stimuli increase skeletal muscle growth in a mammalian target of rapamycin (mTOR)- and p70(S6K)-dependent manner. It has been proposed that costameric proteins at Z bands may sense and transfer tension to these initiators of protein translation, but few candidates have been identified. The purpose of this study was to determine whether a role exists for the α(7)-integrin in the activation of hypertrophic signaling and growth following eccentric exercise training. Five-week-old, wild-type (WT) and α(7)BX2-integrin transgenic (α(7)Tg) mice were randomly assigned to one of two groups: 1) sedentary (SED), or 2) exercise training (EX). Exercise training consisted of downhill running 3 sessions/wk for 4 wk (-20°, 17 m/min, 30 min). Downhill running was used to induce physiological mechanical strain. Twenty-four hours following the final training session, maximal isometric hindlimb plantar flexor force was measured. Gastrocnemius-soleus complexes were collected for further analysis of signaling changes, which included AKT, mTOR and p70(S6K), and muscle growth. Despite increased p70(S6K) activity in WT/EX, no significant changes in cross-sectional area or force were observed in WT/EX compared with WT/SED. AKT, mTOR, and p70(S6K) activation was higher, and whole muscle hypertrophy, relative muscle weight, myofibrillar protein, and force were significantly elevated in α(7)Tg/EX compared with α(7)Tg/SED. A marked increase in average myofiber cross-sectional area was observed in α(7)Tg/EX compared with all groups. Our findings demonstrate that the α(7)β(1)-integrin sensitizes skeletal muscle to mechanical strain and subsequent growth. Thus the α(7)β(1)-integrin may represent a novel molecular therapy for the treatment of disuse muscle atrophy.     

Optical reversal of halothane-induced immobility in C. elegans

Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [ [1] and [2] ]. Genetic studies in Caenorhabditis elegans [ [3] and [4] ], Drosophila [ [3] and [5] ], and mice [ [6] , [7] , [8] and [9] ] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K⁺ [ [10] , [11] , [12] and [13] ] or permeable to Na⁺ [14] are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane-induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of nonspecific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.     

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear–mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.     

High butyric acid amounts induce oxidative stress,alter calcium homeostasis,and cause neurite retraction in nerve growth factor-treated PC12 cells

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC12 cells to varying BA concentrations (0.5, 1.0, 5.0 mM). We measured total heme, H₂O₂, catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0 mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5 mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.     

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy,and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.     

Oral TNFα Modulation Alters Neutrophil Infiltration,Improves Cognition and Diminishes Tau and Amyloid Pathology in the 3xTgAD Mouse Model

Cytokines such as TNFα can polarize microglia/macrophages into different neuroinflammatory types. Skewing of the phenotype towards a cytotoxic state is thought to impair phagocytosis and has been described in Alzheimer’s Disease (AD). Neuroinflammation can be perpetuated by a cycle of increasing cytokine production and maintenance of a polarized activation state that contributes to AD progression. In this study, 3xTgAD mice, age 6 months, were treated orally with 3 doses of the TNFα modulating compound isoindolin-1,3 dithione (IDT) for 10 months. We demonstrate that IDT is a TNFα modulating compound both in vitro and in vivo. Following long-term IDT administration, mice were assessed for learning & memory and tissue and serum were collected for analysis. Results demonstrate that IDT is safe for long-term treatment and significantly improves learning and memory in the 3xTgAD mouse model. IDT significantly reduced paired helical filament tau and fibrillar amyloid accumulation. Flow cytometry of brain cell populations revealed that IDT increased the infiltrating neutrophil population while reducing TNFα expression in this population. IDT is a safe and effective TNFα and innate immune system modulator. Thus small molecule, orally bioavailable modulators are promising therapeutics for Alzheimer’s disease.     

Fish, marine n-3 polyunsaturated fatty acids and coronary heart disease: a minireview with focus on clinical trial data

In this paper, we will briefly deal with the background for the possible effects of long-chain marine n-3 (also called omega-3) polyunsaturated fatty acids (PUFA) in coronary heart disease (CHD) and then focus on findings from clinical trials in humans. We will not deal with effects of alpha-linolenic acid, the non-marine type of n-3 PUFA derived from plant oils.     

Cytokinin dehydrogenase differentially regulates cytokinin and indirectly affects hydrogen peroxide accumulation in tomato leaf

Cytokinin dehydrogenase (CKX) catalyzes the irreversible degradation of cytokinins (CKs). CKs play a role in the regulation of hydrogen peroxide (H(2)O(2)) accumulation while H(2)O(2) is involved in chlorophyll degradation. Here, we elucidated how CKX differentially regulates representative CK levels and indirectly influence H(2)O(2) accumulation in tomato leaves. We induced drought, salt and iron-deficiency stresses in tomato plants and found that chlorosis-inducing stresses (salt and iron-deficiency) induced lower chlorophyll levels as compared to drought stress and control. Protein gel blot analysis detected two CKXs in tomato leaves: a 35 kDa protein (CKX35) found in chlorotic leaves associated with a reduction in representative CK levels and high H(2)O(2) concentrations, and a 37 kDa protein (CKX37) found in green leaves associated with representative CKs and H(2)O(2) at normal levels. In summary, CKX isoforms in the tomato leaf appear to have distinct roles in differentially regulating CK levels and indirectly influencing H(2)O(2) accumulation.     

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.     

Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays

Protein microarray technologies are rapidly expanding to fulfill current needs of proteome discovery for disease management. Nanostructured materials have been shown to present interesting features when used in biological settings: nanostructured titanium oxide film (ns-TiOx), synthesized by supersonic cluster beam deposition (SCBD), has recently emerged as a biocompatible substrate in different biological assays. The ns-TiOx surface is characterized by a morphology at the nanoscale that can be tuned to modulate specific biomolecule–material interactions. Here we present a systematic characterization of ns-TiOx coatings as protein binding surfaces, comparing their performances with those of most common commercial substrates in protein and antibody microarray assays. Through a robust statistical evaluation of repeatability in terms of coefficient of variation (CV) analysis, we demonstrate that ns-TiOx can be used as reliable substrate for biochips in analytical protein microarray application.