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Objective To conduct a proof-of-concept study on preferential binding of polymeric IgA (pIgA) using a novel recombinant rabbit/human chimeric secretory component (cSC) and preliminary assessment of the diagnostic potential of virus-specific pIgA in discriminating acute hepatitis A, E, and C (HAV, HEV, HCV) patients and uninfected controls using an indirect enzyme-linked immunoassay. Results cSC binds > 0.06 μg/ml of purified human and mouse pIgA with negligible cross-reactivity against IgM and IgA. Virus-specific pIgA was significantly higher in serum of acute HAV (n = 6) and HEV (n = 12) patients than uninfected samples (HEV: p

Objective Mexico has one of the world’s highest rates of obesity, which is influenced by lipid-genetic and lifestyle factors. This study aimed to determine whether FABP2 (Ala54Thr) and MTTP (-493 G/T) genetic polymorphisms are associated with metabolic disorders in Mexican subjects. Methods A total of 523 subjects participated in a cross-sectional study. Genotyping for FABP2 and MTTP was performed using real-time RT-PCR. Biochemical and anthropometric data were evaluated. Results The genetically at-risk group (Thr54/-493T) was associated with significantly higher total and low-density lipoprotein cholesterol levels (difference between genetically at-risk group and wild-type group: 10.6 mg/dL and 8.94 mg/dL, respectively). Carriers within the genetically at-risk group had a significantly higher prevalence rate of hypercholesterolaemia (42.5% vs. 32.0%) and higher LDL-C levels (37.6% vs. 26.4%) than did non-carriers. Conclusions Subjects who are genetically at risk (Thr54/-493T) have higher total cholesterol levels, low-density lipoprotein cholesterol levels, and prevalence rate of hypercholesterolaemia. These findings highlight the importance of basing nutritional intervention strategies for preventing and treating chronic diseases on individual genetic characteristics.

Tuberculosis (TB) is ranked among the top 10 causes of death worldwide. New biomarker-based serodiagnostics and vaccines are unmet needs stalling disease control. Antigen 60 (A60) is a thermostable mycobacterial complex typically purified from Bacillus Calmette-Gu&eacute, rin (BCG) vaccine. A60 was historically evaluated for TB serodiagnostic and vaccine potential with variable findings. Despite containing immunogenic proteins, A60 has yet to be proteomically characterized. Here, commercial A60 was (1) trypsin-digested in-solution, analyzed by LC-MS/MS, searched against M. tuberculosis H37Rv and M. bovis BCG Uniprot databases, (2) analyzed using STRING to predict protein&ndash, protein interactions, and (3) probed with anti-TB monoclonal antibodies and patient immunoglobulin G (IgG) on Western blot to evaluate antigenicity. We detected 778 proteins in two A60 samples (440 proteins shared), including DnaK, LprG, LpqH, and GroEL1/2, reportedly present in mycobacterial extracellular vesicles (EV). Of these, 107 were also reported in EVs of M. tuberculosis, and 27 key proteins had significant protein&ndash, protein interaction, with clustering for chaperonins, ribosomal proteins, and proteins for ligand transport (LpqH and LprG). On Western blot, 7/8 TB and 1/8 non-TB sera samples had reactivity against 37&ndash, 50 kDa proteins, while LpqH, GroEL2, and PstS1 were strongly detected. In conclusion, A60 comprises numerous proteins, including EV proteins, with predicted biological interactions, which may have implications on biomarker and vaccine development.

Background Syphilis has been and still is one of the greatest global health concerns. Syphilis can seriously damage the nervous system of infected individuals including infants born to infected mother. Treatment of syphilis is simple and effective with penicillin but diagnosis is challenging, particularly in resource-constrained settings, due to the need for a laboratory-based confirmatory test. Current point of care (POC) tests for syphilis are available but cannot distinguish active infections from past treated infections with a misclassification rate of up to 50% (low specificity). We developed a prototype rapid POC test (IgA Confirm) that can differentiate active syphilis from past treated infections at the point of care. Methods We conducted a prospective diagnostic accuracy study to assess the specificity (and sensitivity) of the IgA Confirm test in identifying active syphilis infections classified by Treponema pallidum Antibobody (TPAb) and rapid plasma regain (RPR) laboratory serology. Between June-December 2018, 500 pregnant women attending Rahima Moosa Mother and Child hospital, South Africa were recruited and provided venous blood samples for syphilis testing including the IgA Confirm (index) and laboratory serology (reference) tests. Results The IgA Confirm demonstrate a sensitivity of 100% (5/5) for identifying samples with active syphilis infections (TPAb positive and RPR positive); 100% (9/9) specificity for identifying samples with past or treated infections (TPAb positive, RPR negative) and, 99.4% (484/487) specificity for samples with no evidence of syphilis (TPAb and RPR negative). Conclusion This study showed that the IgA Confirm test has the ability to identify active syphilis infection and meet the WHO Target Product Profile for syphilis confirmatory testing. Future study is needed to further evaluate diagnostic performance of the test in high prevalence setting. Disclosure No significant relationships.

Measuring CD4 counts remains an important component of HIV care. The Visitect CD4 is the first instrument-free low-cost point-of-care CD4 test with results interpreted visually after 40 min, providing a result of ≥350 CD4 cells/mm3., Measuring CD4 counts remains an important component of HIV care. The Visitect CD4 is the first instrument-free low-cost point-of-care CD4 test with results interpreted visually after 40 min, providing a result of ≥350 CD4 cells/mm3. The field performance and diagnostic accuracy of the test was assessed among HIV-infected pregnant women in South Africa. A nurse performed testing at the point-of-care using both venous and finger-prick blood, and a counselor and laboratory staff tested venous blood in the clinic laboratory (four Visitect CD4 tests/participant). Performance was compared to the mean CD4 count from duplicate flow cytometry tests on venous blood (FACSCalibur Trucount). In 2017, 156 patients were enrolled, providing a total of 624 Visitect CD4 tests (468 venous and 156 finger-prick samples). Of 624 tests, 28 (4.5%) were inconclusive. Generalized linear mixed modeling showed better performance of the test on venous blood (sensitivity = 81.7%; 95% confidence interval [CI] = 72.3 to 91.1]; specificity = 82.6%, 95% CI = 77.1 to 88.1) than on finger-prick specimens (sensitivity = 60.7%; 95% CI = 45.0 to 76.3; specificity = 89.5%, 95% CI = 83.2 to 95.8; P = 0.001). No difference in performance was detected by cadre of health worker (P = 0.113) or between point-of-care versus laboratory-based testing (P = 0.108). Adequate performance of Visitect CD4 with different operators and at the point of care, with no need of electricity or instrument, shows the potential utility of this device, especially for facilitating decentralization of CD4 testing services in rural areas.

Background Current point-of-care tests (POCT) for syphilis, based on the detection of Treponema pallidum (TP) total antibodies, have limited capacity in distinguishing between active and past/treated syphilis. We report the development and early evaluation of a new prototype POCT based on the detection of TP-IgA antibodies, a novel biomarker for active syphilis. Methods The TP-IgA POCT (index test) was developed in response to the World Health Organisation (WHO) target product profile (TPP) for a POCT for confirmatory syphilis testing. Two sub-studies were conducted consecutively using 458 pre-characterised stored plasma samples in China (sub-study one, addressing the criteria for the WHO TPP), and 503 venous blood samples collected from pregnant/postpartum women in South Africa (sub-study two, addressing potential clinical utility). Performance of the index test was assessed against standard laboratory-based serology using a combination of treponemal (TPHA) and non-treponemal (rapid plasma reagin [RPR]) tests. Findings In sub-study one, the index test demonstrated 96·1% (95%CI=91·7%-98·5%) sensitivity and 84·7% (95%CI=80·15–88·6%) specificity for identification of active syphilis (TPHA positive, RPR positive). It correctly identified 71% (107/150) samples of past-treated syphilis (TPHA positive, RPR negative). In sub-study two, the index test achieved 100% (95%CI=59%-100%) sensitivity for active syphilis and correctly identified all nine women with past syphilis. Interpretation The TP-IgA POCT has met the WHO TPP for a POCT for diagnosis of active syphilis and demonstrated its potential utility in a clinical setting. Future studies are warranted to evaluate field performance of the final manufactured test. Funding Saving Lives at Birth: Grand Challenge for Development, Thrasher Research Fund, and the Victorian Government Operational Infrastructure Scheme.

Lateral flow assays (LFAs) are the mainstay of rapid point-of-care diagnostics, with the potential to enable early case management and transform the epidemiology of infectious disease. However, most LFAs only detect single biomarkers. Recognizing the complex nature of human disease, overlapping symptoms and states of co-infections, there is increasing demand for multiplexed systems that can detect multiple biomarkers simultaneously. Due to innate limitations in the design of traditional membrane-based LFAs, multiplexing is arguably limited to a small number of biomarkers. Here, we summarize the need for multiplexed LFA, key technical and operational challenges for multiplexing, inherent in the design and production of multiplexed LFAs, as well as emerging enabling technologies that may be able to address these challenges. We further identify important areas for research in efforts towards developing multiplexed LFAs for more impactful diagnosis of infectious diseases.

Influence of host factors, including human immunodeficiency virus (HIV) co-infection, on the distribution and diagnostic potential of previously evaluated biomarkers of pulmonary tuberculosis (PTB), such as anti-antigen 60 (A60) immunoglobulin (Ig) G, anti-A60 IgA, and C-reactive protein (CRP), remain unclear. Anti-A60 IgG, anti-A60 IgA, and CRP in PTB and non-PTB patient sera (n = 404, including smear-positive/negative, culture-positive (SPCP/SNCP) and HIV+ve/−ve) were measured by enzyme-linked immunoassay and statistically analysed. In multinomial logistic regression, expectoration, chest pain, wasting, and culture count positively associated with CRP (p < 0.001), while smear count positively associated with anti-A60 IgG (p = 0.090). Expectoration and enlarged lymph nodes negatively associated with anti-A60 IgA (p = 0.018). Biomarker distribution and diagnostic potential varied significantly by symptoms and bacilli burden, and across different PTB subpopulations. CRP was correlated poorly with anti-A60 antibodies, while anti-A60 IgA and IgG were correlated in non-tuberculosis (TB) and SPCP patients (p < 0.001). When combined, anti-A60 IgG and CRP best discriminated SPCP/HIV−ve from non-TB (AUC: 0.838, 95% CI: 0.783–0.894), while anti-A60 IgA and CRP performed best in discriminating HIV+ve PTB from non-TB (AUC: 0.687, 95% CI: 0.598–0.777). Combined CRP and anti-A60 antibodies had significantly reduced accuracy in SNCP and SNCP/HIV+ve compared to SPCP/HIV−ve subpopulations. The complex relationships between host factors and biomarkers suggest their limited utility, especially in SNCP/HIV+ve subpopulations, highlighting the importance of examining host response and immune biomarkers across relevant patient subpopulations.

Emerging research has shown that subtle factors during pregnancy and gestation can influence long-term health in offspring. In an attempt to be proactive, we set out to explore whether a nonpharmacological intervention, perinatal exercise, might improve offspring health. Female mice were separated into sedentary or exercise cohorts, with the exercise cohort having voluntary access to a running wheel prior to mating and during pregnancy and nursing. Offspring were weaned, and analyses were performed on the mature offspring that did not have access to running wheels during any portion of their lives. Perinatal exercise caused improved glucose disposal following an oral glucose challenge in both female and male adult offspring ( P < 0.05 for both). Blood glucose concentrations were reduced to lower values in response to an intraperitoneal insulin tolerance test for both female and male adult offspring of parents with access to running wheels ( P < 0.05 and P < 0.01, respectively). Male offspring from exercised dams showed increased percent lean mass and decreased fat mass percent compared with male offspring from sedentary dams ( P < 0.01 for both), but these parameters were unchanged in female offspring. These data suggest that short-term maternal voluntary exercise prior to and during healthy pregnancy and nursing can enhance long-term glucose homeostasis in offspring.

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

The aims of the study were to assess whether Treponema pallidum-specific IgM may provide a useful marker of infectious syphilis in human immunodeficiency virus (HIV)-infected patients, and to compare the performance of a prototype IgM-rapid point-of-care test (PoCT) with a standard IgM-enzyme immunoassay (EIA). Twenty samples from HIV-infected patients with untreated syphilis ( n = 4 primary syphilis, n = 11 secondary and n = 5 early latent) and 51 follow-up samples at three, six or 12 months after treatment were tested for the presence of IgM with the Mercia-EIA (Microgen Bioproducts Ltd, Camberley, UK) and a prototype PoCT (Select Vaccines Ltd, Melbourne, Australia). Although sample numbers were small, IgM detection by EIA appears to be a reliable marker for untreated syphilis in HIV-infected patients with primary (4/4 IgM-positive) or secondary syphilis (10/11 IgM-positive, 1/11 equivocal). After treatment, IgM was no longer detected after three months in the majority of patients (87%) and was either negative or equivocal in all patients after six and 12 months. The overall sensitivity of the IgM-PoCT was 82% and varied with clinical stage, being highest in secondary (10/10 EIA positives) but lower in primary (2/4 EIA positives) and early latent syphilis (2/3 EIA positives). Overall specificity was 95%. Rapid detection of IgM would enable clinicians to distinguish between past-treated and infectious syphilis and allow for diagnosis and treatment in a single visit.

Ca2+ desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (ΔK210) in cardiac troponin T (cTnT). ΔK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Quantitative analysis of cardiac myofibrils isolated from ΔK210 hearts identified a decrease in basal phosphorylation of cTnI (46%), cTnT (29%) and MyBP-C (31%) compared with wild type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr203 (28%) and decreased phosphorylation of cTnI-Ser23/24 (41%) in mutant myocardium. In vitro kinase assays indicate that ΔK210 increases phosphorylation propensity of cTnT-Thr203 three fold without changing cTnI-Ser23/24 phosphorylation. Molecular modeling of cTnT-ΔK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr203, which enhances PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-ΔK210 has enhanced binding to cTnI compared with cTnT-wt, and may impair Ca2+ sensing/transmission leading to myofilament desensitization. Collectively, our observations suggest that cardiomyopathy-causing ΔK210 has far-reaching effects influencing posttranslational modifications of key sarcomeric proteins, and potentially cTnI-cTnT interaction.

Objective The purpose of this study is to describe the focus and comprehensiveness of domains measured in e-cigarette research. Methods A portfolio analysis of National Institutes of Health grants focusing on e-cigarette research and funded between the fiscal years 2007 and 2015 was conducted. Grant proposals were retrieved using a government database and coded using the Host–Agent–Vector-Environment (HAVE) model as a framework to characterise the measures proposed. Eighty-one projects met the criteria for inclusion in the analysis. Results The primary HAVE focus most commonly found was Host (73%), followed by Agent (21%), Vector (6%) and Environment (0%). Intrapersonal measures and use trajectories were the most common measures in studies that include Host measures (n=59 and n=51, respectively). Product composition was the most common area of measurement in Agent studies (n=24), whereas Marketing (n=21) was the most common (n=21) area of Vector measurement. When Environment measures were examined as secondary measures in studies, they primarily focused on measuring Peer, Occupation and Social Networks (n=18). Although all studies mentioned research on e-cigarettes, most (n=52; 64%) did not specify the type of e-cigarette device or liquid solution under study. Conclusions This analysis revealed a heavy focus on Host measures (73%) and a lack of focus on Environment measures. The predominant focus on Host measures may have the unintended effect of limiting the evidence base for tobacco control and regulatory science. Further, a lack of specificity about the e-cigarette product under study will make comparing results across studies and using the outcomes to inform tobacco policy difficult.

Cardiac troponin T (cTnT) is a phosphoprotein that modulates cardiac muscle contraction through its extensive and diverse interactions with neighboring thin filament proteins. Its N-terminal half is the "glue" that anchors the troponin complex to tropomyosin-actin. Until now, studies aimed at investigating the role of the N-terminal tail region have not considered the effects of phosphorylation. To understand better the regulatory role of the N-terminal tail region of phosphorylated cTnT, we investigated the functional effects of N-terminal deletion (amino acids 1-91) and phosphorylation on Ca(2+) dependence of myofilament isometric force production, isometric ATPase rate, and thin filament sliding speed. Chemomechanical profiles were assessed in detergent permeabilized fiber preparations where the native troponin (cTn) was exchanged with recombinant cTn engineered to contain modified cTnT (truncated, phosphorylated) in the presence of wild-type cTnI and cTnC. Removal of the cTnT N-terminal amino acids 1-91 (cTnT-del) enhances myofilament responsiveness to nonsaturating Ca(2+) levels (the physiological range in cardiac myocytes). However, at saturating Ca(2+) levels, there is a reduction in isometric tension and ATPase rate. On one hand, phosphorylation of cTnT-del attenuates the sensitizing effect induced by truncation of the N-terminal tail, "resetting" myofilament Ca(2+) responsiveness back to control levels. On the other hand, it impairs isometric tension development and ATPase rate. Interestingly, phosphorylation of cTnT (cTnT-P) differentially regulates tension cost (an index of cross-bridge cycling rate): increased by cTn-del-P and decreased by intact cTn-wt-P. Like the isometric fiber data, sliding speed of thin filaments regulated by cTn-del is more sensitive to Ca(2+) compared with cTn-wt. Phosphorylation of cTnT (whether cTnT-del or -wt) depresses sliding speed and is associated with Ca(2+) desensitization of thin filament sliding speed.

Activation of the endogenous alpha1-adrenergic receptor (AR) associated with human aortic smooth muscle cells resulted in a dose- and time-dependent increase in the levels of mitochondrial reactive oxygen species (ROS). ROS increases were apparent within 10 min and maximal after 45 min. Prolonged activation (4 h) of the alpha1-AR resulted in smooth muscle cell apoptosis. Both the increase in ROS and apoptotic cell death were blocked by the nonselective alpha1-AR antagonist prazosin as well as the selective alpha1D-AR antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7, 9-dione (BMY 7378). Increases in ROS and apoptosis produced by alpha1-AR activation were also blocked by the p38 mitogen-activated protein kinase inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190) and the NAPDH oxidase inhibitor apocynin. The extracellular signal-regulated kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) or the c-Jun NH2-terminal kinase inhibitor 1, 9-pyrazoloanthrone anthra(1, 9-cd)pyrazol-6(2H)-one (SP 600125) was without effect on increases in ROS levels or apoptosis. Pifithrin-alpha, an inhibitor of the tumor suppressor protein p53, had no effect on ROS generation but did block alpha1D-AR-induced apoptosis. Activation of the alpha1D-AR resulted in translocation of p53 to the mitochondria. The mitochondrial translocation of p53 was blocked by prazosin, BMY 7378, apocynin, SB 202190, and pifithrin-alpha. Apoptosis was also blocked by small interfering RNA directed against p53. These data show that the alpha1D-AR is coupled to the generation of mitochondrial ROS by a pathway involving p38 and NADPH oxidase. Sustained activation of the alpha1D-AR results in smooth muscle cell apoptosis in a pathway that involves the tumor suppressor protein p53 and the mitochondrial translocation of p53. The data also provide evidence of a linkage between the alpha1D-AR and p53.

© 2015 Elsevier Inc. The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of this post-translational modification in HIV-1 infection is not fully understood. Detailed biological studies are hindered in part by the difficulty in accessing homogeneous sulfopeptides and sulfoproteins through biological expression and established synthetic techniques. Herein we describe an efficient approach to the synthesis of sulfopeptides bearing discrete sulfation patterns through the divergent, site-selective incorporation of sTyr residues on solid support. By employing three orthogonally protected Tyr building blocks and a solid-phase sulfation protocol, we demonstrate the synthesis of a library of target N-terminal CCR5(2-22) sulfoforms bearing discrete and differential sulfation at Tyr10, Tyr14, and Tyr15, from a single resin-bound intermediate. We demonstrate the importance of distinct sites of Tyr sulfation in binding gp120 through a competitive binding assay between the synthetic CCR5 sulfopeptides and an anti-gp120 monoclonal antibody. These studies revealed a critical role of sulfation at Tyr14 for binding and a possible additional role for sulfation at Tyr10. N-terminal CCR5 variants bearing a sTyr residue at position 14 were also found to complement viral entry into cells expressing an N-terminally truncated CCR5 receptor.

The U.S. dietary supplement market increased 7.5% in 2012 compared to 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine...

The U.S. dietary supplement market increased by 7.5% in 2012 compared with 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine their health effects, safety, and efficacy. A portfolio analysis was performed across the NIH and the Office of Dietary Supplements (ODS) for fiscal years (FYs) 2009–2011 by using the databases Human Nutrition Research Information Management (HNRIM) and Computer Access to Research on Dietary Supplements (CARDS). The results indicated that total NIH dietary supplement–related funding for FYs 2009–2011 was $855 million ($295 million in 2009, $311 million in 2010, and $249 million in 2011). The institutes and centers with the highest investment in dietary supplement research were as follows: the National Heart, Lung, and Blood Institute ($135 million); the National Cancer Institute ($188 million); the National Center for Complementary and Alternative Medicine ($99 million); the National Institute of Diabetes and Digestive and Kidney Diseases ($68 million); the National Institute of Environmental Health Sciences ($58 million); and the ODS ($32 million). The dietary supplement ingredients receiving the most funding were botanicals (22%), vitamins (20%), lipids (14%), and minerals and trace elements (10%). The top 3 outcome research areas were cancer (61% of total dietary supplement investment), cardiovascular disease (47%), and women’s reproductive health (38%). In FYs 2009, 2010, and 2011, the ODS provided 3.5%, 3.6%, and 4.1%, respectively, of the NIH investment in dietary supplement research. ODS funding focused on cellular, enzymatic, or molecular mechanisms (64% of total ODS funding). This portfolio analysis demonstrates that the NIH has committed substantial funding to dietary supplement research in an effort to expand the scientific knowledge base on the efficacy and safety of dietary supplements.

Point-of-care tests (POCTs) play an important role in bridging the gap between centralized laboratory diagnostics and peripheral healthcare service providers. Particularly in infectious diseases such as HIV/AIDS and TB where early detection is imperative to improve disease outcome, uptake of an accurate test that is simple, rapid and robust can significantly alter the epidemiology and control of the disease. However, a good POCT can only serve its full potential when adopted in a comprehensive programmatic context linking patients to on-site case management. Immunochromatographic lateral flow devices for detection of antibody or antigen currently dominate available POCTs, and development of such devices has relied on the discovery and optimization of definitive biomarkers suitable for such platforms. In the future, however, there will be an increasing need to develop cost-effective POCTs that address biomarkers that are well established in laboratory settings but are not currently amenable to point-of-care, such as molecular tests for drug resistance in TB and viral load in HIV and viral hepatitis.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles. Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C⁷. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 μg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5 mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 μM) is added to start state 3. Oligomycin (1 μM), an ATPase synthase blocker, is used to estimate state. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 μM) is added to measurestate, or uncoupled respiration. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥ 4 is considered as evidence of a viable mitochondria preparation. In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

The mitochondrial content of skeletal muscles is proportional to activity level, with the assumption that intrinsic mitochondrial function is the same in all muscles. This may not hold true for all muscles. For example, the diaphragm is a constantly active muscle; it is possible that its mitochondria are intrinsically different compared with other muscles. This study tested the hypothesis that mitochondrial respiration rates are greater in the diaphragm compared with triceps surae (TS, a limb muscle). We isolated mitochondria from diaphragm and TS of adult male Sprague Dawley rats. Mitochondrial respiration was measured by polarography. The contents of respiratory complexes, uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3), and voltage-dependent anion channel 1 (VDAC1) were determined by immunoblotting. Complex IV activity was measured by spectrophotometry. Mitochondrial respiration states 3 (substrate and ADP driven) and 5 (uncoupled) were 27 ± 8% and 24 ± 10%, respectively, lower in diaphragm than in TS ( P < 0.05 for both comparisons). However, the contents of respiratory complexes III, IV, and V, UCP1, and VDAC1 were higher in diaphragm mitochondria (23 ± 6, 30 ± 8, 25 ± 8, 36 ± 15, and 18 ± 8% respectively, P ≤ 0.04 for all comparisons). Complex IV activity was 64 ± 16% higher in diaphragm mitochondria ( P ≤ 0.01). Mitochondrial UCP2 and UCP3 content and complex I activity were not different between TS and diaphragm. These data indicate that diaphragm mitochondria respire at lower rates, despite a higher content of respiratory complexes. The results invalidate our initial hypothesis and indicate that mitochondrial content is not the only determinant of aerobic capacity in the diaphragm. We propose that UCP1 and VDAC1 play a role in regulating diaphragm aerobic capacity.

The availability of rapid, point-of-care (POC) tests has significantly expanded the capacity of both developed and resource-constrained countries (RCCs) to diagnose HIV, with immunochromatographic tests most commonly used in these settings. This has been especially important in programs for prevention of mother-to-child transmission, in both RCCs and the developed world. However, suitable POC tests are not yet commercially available for diagnosis of neonatal HIV, where persistence of maternal antibody in the infant precludes the use of current antibody tests during the first 12 to 18 months. In addition, measurement of CD4+ T cells, CD4%, and HIV viral load still relies on sophisticated laboratory infrastructure, constraining the delivery of appropriate care to many HIV-infected patients. Continued effort is required in the development and validation of additional POC tests to support HIV patient care, and in quality assurance in manufacturing and in test performance in the field to ensure appropriate use of existing and new POC tests.

People living at high altitude appear to have lower blood glucose levels and decreased incidence of diabetes. Faster glucose uptake and increased insulin sensitivity are likely explanations for these findings: skeletal muscle is the largest glucose sink in the body, and its adaptation to the hypoxia of altitude may influence glucose uptake and insulin sensitivity. This study tested the hypothesis that chronic normobaric hypoxia increases insulin-stimulated glucose uptake in soleus muscles and decreases plasma glucose levels. Adult male C57BL/6J mice were kept in normoxia [fraction of inspired O2 = 21% (Control)] or normobaric hypoxia [fraction of inspired O2 = 10% (Hypoxia)] for 4 wk. Then blood glucose and insulin levels, in vitro muscle glucose uptake, and indexes of insulin signaling were measured. Chronic hypoxia lowered blood glucose and plasma insulin [glucose: 14.3 ± 0.65 mM in Control vs. 9.9 ± 0.83 mM in Hypoxia ( P < 0.001); insulin: 1.2 ± 0.2 ng/ml in Control vs. 0.7 ± 0.1 ng/ml in Hypoxia ( P < 0.05)] and increased insulin sensitivity determined by homeostatic model assessment 2 [21.5 ± 3.8 in Control vs. 39.3 ± 5.7 in Hypoxia ( P < 0.03)]. There was no significant difference in basal glucose uptake in vitro in soleus muscle (1.59 ± 0.24 and 1.71 ± 0.15 μmol·g−1·h−1 in Control and Hypoxia, respectively). However, insulin-stimulated glucose uptake was 30% higher in the soleus after 4 wk of hypoxia than Control (6.24 ± 0.23 vs. 4.87 ± 0.37 μmol·g−1·h−1, P < 0.02). Muscle glycogen content was not significantly different between the two groups. Levels of glucose transporters 4 and 1, phosphoinositide 3-kinase, glycogen synthase kinase 3, protein kinase B/Akt, and AMP-activated protein kinase were not affected by chronic hypoxia. Akt phosphorylation following insulin stimulation in soleus muscle was significantly (25%) higher in Hypoxia than Control ( P < 0.05). Neither glycogen synthase kinase 3 nor AMP-activated protein kinase phosphorylation changed after 4 wk of hypoxia. These results demonstrate that the adaptation of skeletal muscles to chronic hypoxia includes increased insulin-stimulated glucose uptake.

The family of adrenergic receptors (ARs; the α1-, α2-, and β-ARs) are key regulators of the sympathetic division of the autonomic nervous system, involved in both central and peripheral cardiovascular function. Here, we review our current understanding of the cellular localization and trafficking properties of the α1-, α2-, and β-ARs. We then examine recent evidence indicating that the cellular localization of these receptors and their excursion into intracellular compartments play an underappreciated role in the activation of both G protein and novel non-G protein-dependent cellular signaling.

The cellular localization, agonist-mediated internalization, and desensitization properties of the alpha(1)-adrenoceptor (alpha(1)-AR) subtypes conjugated with green fluorescent protein (alpha(1)-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The alpha(1B)-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the alpha(1B)-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid alpha(1B)-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the alpha(1B)-AR-mediated activation of ERK1/2 phosphorylation. Alpha(1A)-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population alpha(1A)-AR/GFPs was slower than that seen for the alpha(1B)-AR. Agonist exposure also resulted in desensitization of the alpha(1A)-AR-mediated increase in ERK1/2 phosphorylation. The alpha(1D)-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of alpha(1D)-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with beta-arrestin 1 did not increase the rate or extent of agonist-stimulated alpha(1A)- or alpha(1B)-AR/GFP internalization. However, a dominant-negative form of the beta-arrestin 1, beta-arrestin 1 (319-418), blocked agonist-mediated internalization of both the alpha(1A)- and alpha(1B)-ARs. These data show that transfected alpha(1)-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the alpha(1)-ARs, and that agonist-mediated alpha(1)-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the alpha(1)-ARs.

Extraocular muscles are the effector arm of the ocular motor system and are involved in all voluntary and reflexive eye movements.1,2 The functional demands of extraocular muscles are different from those imposed on other skeletal muscles, leading to unique metabolic adaptations as demonstrated by gene expression profiling and by biochemical and functional studies.3–8 For example, extraocular muscles seem to have relatively low adenosine triphosphate (ATP)–buffering capacity, evidenced by decreased reliance on glycogenolysis and less creatine kinase activity.9–12 Moreover, extraocular muscles can reduce lactate to pyruvate for entry into the Krebs cycle to sustain their high activity demand.9,11,13 Another potential mechanism for enhancing ATP production in the extraocular muscles is enhanced glucose uptake by the muscle fibers, priming glycolysis and then mitochondrial energy pathways. In limb skeletal muscles, glucose enters muscle fibers by way of specific membrane transporters (GLUT1 and GLUT4) in a process influenced by insulin and contractile activity to match metabolic supply to demand.14 GLUT1 transporters mediate insulin- and activity-independent basal glucose uptake. On binding to its membrane receptors, insulin triggers a signaling cascade that activates protein kinase B (AKT), which in turn initiates the translocation of GLUT4-containing vesicles to the membrane, increasing glucose uptake over the basal rate.15,16 At the same time, glycogen synthase kinase 3 (GSK3) is activated and phosphorylates glycogen synthase, stimulating glycogen production.17 Contractile activity has a similar effect on glucose uptake: it induces the translocation of a separate pool of GLUT4-containing vesicles to the membrane through activation of the adenosine monophosphate (AMP)–activated kinase (AMPK) pathway.18,19 Given their functional characteristics, extraocular muscles may have very high basal glucose uptake rates, negating the need for further regulation by insulin or contractile activity. Therefore, we tested the hypothesis that glucose uptake into extraocular muscles is not influenced by insulin or contractile activity as in other skeletal muscles.

The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity.The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR.Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.

Efficient and specific phosphorylation of PKA substrates, elicited in response to beta-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A kinase anchoring protein), cardiac troponin T (cTnT). Using the yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cardiac troponin (cTn) exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.