A novel trafficking signal within the HLA-C cytoplasmic tail allows regulated expression upon differentiation of macrophages

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.     

Nonlinear smooth orthogonal decomposition of kinematic features of sawing reconstructs muscle fatigue evolution as indicated by electromyography

Tracking or predicting physiological fatigue is important for developing more robust training protocols and better energy supplements and/or reducing muscle injuries. Current methodologies are usually impractical and/or invasive and may not be realizable outside of laboratory settings. It was recently demonstrated that smooth orthogonal decomposition (SOD) of phase space warping (PSW) features of motion kinematics can identify fatigue in individual muscle groups. We hypothesize that a nonlinear extension of SOD will identify more optimal fatigue coordinates and provide a lower-dimensional reconstruction of local fatigue dynamics than the linear SOD. Both linear and nonlinear SODs were applied to PSW features estimated from measured kinematics to reconstruct muscle fatigue dynamics in subjects performing a sawing motion. Ten healthy young right-handed subjects pushed a weighted handle back and forth until voluntary exhaustion. Three sets of joint kinematic angles were measured from the right upper extremity in addition to surface electromyography (EMG) recordings. The SOD coordinates of kinematic PSW features were compared against independently measured fatigue markers (i.e., mean and median EMG spectrum frequencies of individual muscle groups). This comparison was based on a least-squares linear fit of a fixed number of the dominant SOD coordinates to the appropriate local fatigue markers. Between subject variability showed that at most four to five nonlinear SOD coordinates were needed to reconstruct fatigue in local muscle groups, while on average 15 coordinates were needed for the linear SOD. Thus, the nonlinear coordinates provided a one-order-of-magnitude improvement over the linear ones.     

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.     

Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 ± 8 and 50 ± 7%, respectively, in skeletal muscle of α2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.The ubiquitously expressed serine/threonine AMP-activated protein kinase (AMPK)² is an αβγ heterotrimer postulated to play a key role in the response to energetic stress (1, 2), because of its sensitivity to increased cellular AMP levels (3). Pharmacological activation of AMPK (primarily via the AMP analogue ZMP) increases catabolic processes such as GLUT4 translocation (4, 5), glucose uptake (6, 7), long chain fatty acid (LCFA) uptake (8), and substrate oxidation (6). Concomitantly, pharmacological activation of AMPK inhibits anabolic processes, and in skeletal muscle genetic reduction of the catalytic AMPKα2 subunit eliminates these pharmacological effects (9–12). Thus, AMPK has been proposed to act as a metabolic master switch (2, 13, 14). Physiologically, exercise at intensities sufficient to increase free cytosolic AMP (AMP_free) levels is a potent stimulus of AMPK, preferentially activating AMPKα2 in skeletal muscle (15–17). The metabolic profile of skeletal muscle during moderate to high intensity exercise is remarkably similar to skeletal muscle in which AMPK has been pharmacologically activated (i.e. increases in catabolic processes). This is consistent with the hypothesis that AMPK activation is required for the metabolic response to increased cellular stress. Given this, it is surprising that the direct role(s) of skeletal muscle AMPK during exercise under physiological in vivo conditions is unknown.A number of studies have tried to attribute causality to the AMPK and metabolic responses to exercise using transgenic models. In mouse models in which AMPKα2 protein expression and/or activity has been impaired, contractions performed in isolated skeletal muscle in vitro, ex vivo, or in situ have demonstrated that skeletal muscle glucose uptake (MGU) is normal (9, 10), partially impaired (11, 18), or ablated (19). Furthermore, ex vivo skeletal muscle LCFA uptake and oxidation in response to contraction appears to be AMPK-independent (20, 21). A key limitation of these studies is that the experimental models were not physiological. Under in vivo conditions, mice expressing a kinase-dead (18) or inactive (22) AMPKα2 subunit in cardiac and skeletal muscle have impaired voluntary and maximal physical activity, respectively, indicative of a physiological role for AMPK during exercise. In this context, obese non-diabetic and diabetic individuals have impaired skeletal muscle AMPK activation during moderate intensity exercise (23) as well as during the post-exercise period (24), yet the contribution of this impairment to the disease state is unclear. Thus, in vivo studies are essential to define the role of AMPK in skeletal muscle during exercise.Physical exercise of a moderate intensity is an effective adjunct treatment for chronic metabolic diseases such as obesity and type 2 diabetes (25). Given the importance of elucidating the molecular mechanism(s) regulating skeletal muscle substrate metabolism during exercise and the putative role of AMPK as a critical mediator in this process, we tested the hypothesis that AMPKα2 is functionally linked to substrate metabolism in vivo.     

Modifying the Bank Erosion Hazard Index (BEHI) Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio

Understanding the source of pollution in a stream is vital to preserving, restoring, and maintaining the stream’s function and habitat it provides. Sediments from highly eroding streambanks are a major source of pollution in a stream system and have the potential to jeopardize habitat, infrastructure, and stream function. Watershed management practices throughout the Cleveland Metroparks attempt to locate and inventory the source and rate the risk of potential streambank erosion to assist in formulating effect stream, riparian, and habitat management recommendations. The Bank Erosion Hazard Index (BEHI), developed by David Rosgen of Wildland Hydrology is a fluvial geomorphic assessment procedure used to evaluate the susceptibility of potential streambank erosion based on a combination of several variables that are sensitive to various processes of erosion. This protocol can be time consuming, difficult for non-professionals, and confined to specific geomorphic regions. To address these constraints and assist in maintaining consistency and reducing user bias, modifications to this protocol include a “Pre-Screening Questionnaire”, elimination of the Study Bank-Height Ratio metric including the bankfull determination, and an adjusted scoring system. This modified protocol was used to assess several high priority streams within the Cleveland Metroparks. The original BEHI protocol was also used to confirm the results of the modified BEHI protocol. After using the modified assessment in the field, and comparing it to the original BEHI method, the two were found to produce comparable BEHI ratings of the streambanks, while significantly reducing the amount of time and resources needed to complete the modified protocol.     

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non‐adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

A survey of integral α-helical membrane proteins

Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families.     

Interactions of T cells with fibroblast-like synoviocytes: role of the B7 family costimulatory ligand B7-H3

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-alpha, IFN-gamma, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.     

Developing landscape habitat models for rare amphibians with small geographic ranges: a case study of Siskiyou Mountains salamanders in the western USA

To advance the development of conservation planning for rare species with small geographic ranges, we determined habitat associations of Siskiyou Mountains salamanders (Plethodon stormi) and developed habitat suitability models at fine (10 ha), medium (40 ha), and broad (202 ha) spatial scales using available Geographic Information Systems data and logistic regression analysis with an information theoretic approach. Across spatial scales, there was very little support for models with structural habitat features, such as tree canopy cover and conifer diameter. Model-averaged 95% confidence intervals for regression coefficients and associated odds ratios indicated that the occurrence of Siskiyou Mountains salamanders was positively associated with rocky soils and Pacific madrone (Abutus menziesii) and negatively associated with elevation and white fir (Abies concolor); these associations were consistent across 3 spatial scales. The occurrence of this species also was positively associated with hardwood density at the medium spatial scale. Odds ratios projected that a 10% decrease in white fir abundance would increase the odds of salamander occurrence 3.02–4.47 times, depending on spatial scale. We selected the model with rocky soils, white fir, and Oregon white oak (Quercus garryana) as the best model across 3 spatial scales and created habitat suitability maps for Siskiyou Mountains salamanders by projecting habitat suitability scores across the landscape. Our habitat suitability models and maps are applicable to selection of priority conservation areas for Siskiyou Mountains salamanders, and our approach can be easily adapted to conservation of other rare species in any geographical location.