Nutrient supplementation post ambulation in persons with incomplete spinal cord injuries: a randomized, double-blinded, placebo-controlled case series

OBJECTIVE: To examine effects of protein-carbohydrate intake on ambulation performance in persons with incomplete spinal cord injury (SCI). DESIGN: Double-blinded treatment with washout and placebo crossover. SETTING: Academic medical center. PARTICIPANTS: Three subjects aged 34 to 43 years with incomplete SCI at C5-T4. INTERVENTIONS: Subjects walked to fatigue on 5 consecutive days. On fatigue, participants consumed 48g of vanilla-flavored whey and 1g/kg of body weight of carbohydrate (CH(2)O). Weekend rest followed, and the process was repeated. A 2-week washout was interposed and the process repeated using 48g of vanilla-flavored soy. MAIN OUTCOME MEASURES: Oxygen consumed (Vo(2); in L/min), carbon dioxide evolved (Vco(2)), respiratory exchange ratio (RER: Vco(2)/Vo(2)), time (in minutes), and distance walked (in meters) were recorded. Caloric expenditure was computed as Vo(2) by time by 21kJ/L (5kcal/L) of oxygen consumed. Data were averaged across the final 2 ambulation sessions for each testing condition. RESULTS: Despite slow ambulation velocities (range, .11-.34m/s), RERs near or above unity reflected reliance on CH(2)O fuel substrates. Average ambulation time to fatigue was 17.8% longer; distance walked 37.9% longer, and energy expenditure 12.2% greater with the whey and CH(2)O supplement than with the soy drink. CONCLUSIONS: Whey and CH(2)O ingestion after fatiguing ambulation enhanced ensuing ambulation by increasing ambulation distance, time, and caloric expenditure in persons with incomplete SCI.     

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e⁻⁷⁰) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.The five-carbon molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are the fundamental building blocks for isoprenoid compounds. Beginning with DMAPP, a series of polyprenyl diphosphates with C₁₀ (geranyl diphosphate, GPP), C₁₅ (farnesyl diphosphate, FPP), C₂₀ (geranylgeranyl diphosphate, GGPP), C₂₅ (farnesylgeranyl diphosphate, FGPP), and higher molecular weight isoprenoid chains are synthesized by polyprenyl transferases (PTSs). With only a few exceptions, PTSs provide substrates for all but a few branch point enzymes for the biosynthesis of the more than 55,000 known isoprenoid metabolites, including monoterpenes, sesquiterpenes, diterpenes, sterols, carotenoids, ubiquinones, and prenylated proteins and peptides fulfilling essential roles in cells (Fig. S1) (1, 2).There are two distinct classes of PTSs, E-PTS forming trans bonds and Z-PTS forming cis bonds throughout chain elongation. The carbon skeletons of the great majority of isoprenoid metabolites are derived from products of E-PTSs, which share a common protein fold and two functionally important Asp-rich (DDXXD) motifs (1, 3, 4). E-PTSs synthesize linear allylic diphosphates ranging from C₁₀ to C₅₀, where functional assignment of these enzymes is linked to the chain length of their respective predominant products under nonforced conditions. As revealed by a variety of high-quality crystal structures, the enzymes typically are homodimers (5–7). In each monomer, the allylic binding region (S1) in the active site contains three Mg²⁺ ions ligated by two pairs of aspartates from both Asp-rich regions, which in turn holds the diphosphate of the allylic substrate in place, as illustrated with DMAPP in Fig. 1. The hydrocarbon tail of DMAPP extends into a pocket that accommodates the growing polyisoprenoid chain. The IPP binding region (S2) positions C4 of IPP near C1 of the allylic substrate. The isoprenoid moieties of the substrates are joined by a dissociative electrophilic alkylation initiated by cleavage of the carbon–oxygen bond of the allylic diphosphate to form a carbocation, which then attacks the C3–C4 double bond of allylic IPP to form a tertiary carbocationic intermediate that loses a proton from C2 to give the allylic product (8). The sequential addition of IPP to the growing chain proceeds through release of diphosphate from S1, rearrangement of the product from S2 to S1, and binding of another IPP in S2, followed by the same reaction as before (Fig. 1A). The polyisoprenoid chain grows into the elongation cavity flanked by helices D, F, G, and H of the protein near the dimer interface (Fig. 1 B and C) (7, 9). Following this elongation mechanism, these enzymes are named either according to their final product (e.g., farnesyl diphosphate synthase) or according to the longest ligand being transferred to the final product (in this case, geranyl transferase).Open in a separate windowFig. 1.Crystal structure of GGPP synthase (PDB ID 1RQI). (A) Active site S1 with DMAPP, Mg²⁺ ions, and Asp-rich motifs and active site S2 with IPP are highlighted. The electrophilic attack of the C₁ atom of DMAPP against the double bond of IPP after cleavage of diphosphate is indicated by the black arrow. (B and C) Side view (B) and top view (C) of the bioactive dimer with the active site and elongation cavity displayed. Helices D–H are identified by capital letters.Considerable effort has been made to investigate how chain elongation is terminated, establishing that steric hindrance of the growing chain in the elongation cavity is the main factor (1, 3, 9, 10). However, because the number of predicted sequences in the superfamily is so large, structural and enzymatic studies can be performed on only a small fraction of the sequences with likely E-PTS activity. We identified 5,839 such sequences at the initiation of this study in May 2011 (see below). At that time, only 46 individual sequences from 35 UniProt entries were functionally annotated based on published biochemical essays and thus available in the Gene Ontology Annotation (UniProt-GOA) database (11), and 61 had been structurally characterized with crystal structures available in the Protein Data Bank (PDB) database (12). Moreover, the rate of genome sequencing continues to increase exponentially, and even with on-going advances in high-throughput structural biology and in vitro screening methods, the gap between the number of known sequences and the number characterized experimentally will continue to grow. For this reason, reliable methods of inferring function of uncharacterized sequences are urgently needed. The highest-throughput and most widely applied approaches transfer functional annotations from characterized proteins to closely related proteins, because closely related proteins frequently are iso-functional. However, there is no simple sequence-based criterion that can be used universally to define when sequences are similar enough for the inference of iso-functionality to be made confidently (13). In the case of E-PTS enzymes, it has been demonstrated experimentally that chain-length specificity can be changed dramatically by a small number of mutations of key residues lining the elongation cavity (9, 14). Thus, in principle, even very closely related sequences could have different chain-length specificities. Furthermore, the previously characterized E-PTS enzymes were insufficient to allow inferences about the function of many members of this protein family, using any reasonable sequence-similarity cutoff, in part because many of the characterized enzymes were closely related to each other, leaving large regions of “sequence space” completely uncharacterized.We now describe a large-scale study that integrates available genomics data, in vitro experiments, X-ray crystallography, and computational approaches on a large set of representative members of the E-PTS subgroup to assign and, more importantly, to predict function for uncharacterized sequences. We show that computational tools can play a critical role in functional assignment. We use bioinformatics analysis and sequence clustering for target selection and place a particular emphasis on the use of structural information to make functional inferences. To this end, we determined 27 crystal structures and used these structures (in addition to available structures in the PDB) to create comparative models for 61 PTS enzymes for which structural information is lacking. Structures and models then were used to make predictions of chain-length specificity, using a ligand-docking method that evaluates the steric complementarity between various polyprenyl products and the elongation cavity. This structure-based approach to predicting function is validated through blind predictions on 74 PTS enzymes as well as on a subsequently obtained crystal structure in complex with a long-chain polyprenyl product.     

Evaluating a two-step approach to sexual risk reduction in a publicly-funded STI clinic: rationale, design, and baseline data from the Health Improvement Project-Rochester (HIP-R)

BackgroundSexually transmitted infection (STI) clinics provide an opportune setting for HIV prevention efforts. This randomized controlled trial evaluated a unique, two-step approach to sexual risk reduction at a publicly-funded STI clinic.MethodsDuring an initial visit, patients completed an audio-computer assisted self-interview (ACASI), were randomized to and received one of two brief interventions, obtained medical care, and completed a post-assessment. Next, two-thirds of the patients were assigned to attend an intensive sexual risk reduction workshop. At 3, 6, and 12 months, patients completed additional ACASIs and provided urine specimens to assess behavior change and incident STIs.ResultsDuring a 28-month interval, 5613 patients were screened, 2691 were eligible, and 1483 consented to participate and were randomized; the modal reason for declining was lack of time (82%). Consenting patients included 688 women and 795 men; 64% of participants were African-American. The sample was low-income, with 57% reporting an annual income of less than $15,000; most participants (62%) had a high school education or less, and 51% were unemployed. Sexual risk behavior was common, as indicated by multiple sexual partners (mean = 32.8, lifetime; mean = 2.8, past 3 months), unprotected sex (mean = 17.3 episodes, past 3 months), and prior STIs (mean = 3.3, lifetime; 23% at baseline). Bivariate analyses confirmed our prediction that HIV-related motivation and behavioral skills would be related to current sexual risk behavior. All patients received a brief intervention; patient satisfaction ratings were uniformly high for both interventions (all means ≥ 3.7 on 4-point scales). Fifty-six percent of invited patients attended the intensive workshop, and attendance did not differ as a function of brief intervention. Patient satisfaction ratings were also uniformly positive for the workshop interventions (all means ≥ 3.6). Return to follow-up assessments exceeded 70%.ConclusionsResults demonstrate that implementing an HIV preventive program in a busy, public clinic is feasible and well-accepted by patients. Ongoing evaluation will determine if the interventions reduce sexual risk behavior and lower incident STIs.     

Treatment of pruritus of primary biliary cirrhosis with rifampin

Pruritus can be a debilitating symptom in patients with chronic cholestasis. Based on previous reports of its efficacy, we evaluated the impact of rifampin on the pruritus associated with primary biliary cirrhosis. Fourteen patients were included in a randomized, crossover study. After a 15-day washout period, subjects were followed for three weeks. During the first and third week, patients received 600 mg of rifampin or placebo; no treatment was administered during the second week. Pruritus was subjectively scored on a scale from 0 to 100. With rifampin, pruritus disappeared in 11 patients and partially improved in three; with placebo, only two had a partial response (P<0.001). Six patients with a prior poor or no response to cholestyramine improved with rifampin. No changes in biochemical tests or side effects were observed during this period. We conclude that short-term administration of rifampin relieves pruritus in primary biliary cirrhosis. When administered over a period of eight months in an open study, the relief of pruritus was maintained, while one individual developed an allergic reaction. Rifampin appears to be a safe drug in the management of the pruritus of primary biliary cirrhosis.     

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355-365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355-365 region signal normally but lack phosphorylation sites. When D71A- and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization.     

Development of an immunofiltration-based antigen-detection assay for rapid diagnosis of Ebola virus infection

Ebola virus (EBOV) has caused outbreaks of severe viral hemorrhagic fever in regions of Central Africa where medical facilities are ill equipped and diagnostic capabilities are limited. To obtain a reliable test that can be implemented easily under these conditions, monoclonal antibodies to the EBOV matrix protein (VP40), which previously had been found to work in a conventional enzyme-linked immunosorbent assay, were used to develop an immunofiltration assay for the detection of EBOV antigen in chemically inactivated clinical specimens. The assay was evaluated by use of defined virus stocks and specimens from experimentally infected animals. Its field application was tested during an outbreak of Ebola hemorrhagic fever in 2003. Although the original goal was to develop an assay that would detect all EBOV species, only the Zaire and Sudan species were detected in practice. The assay represents a first-generation rapid field test for the detection of EBOV antigen that can be performed in 30 min without electrical power or expensive or sensitive equipment.     

Interindividual Differences in Microbial Counts and Biochemical-Associated Variables in the Feces of Healthy Spanish Adults

The aim of this study was to examine, over a period of 1 year, interindividual variations in the most prominent and representative of the cultivatable microbial populations in the feces of eight healthy Spanish persons. A number of biochemical variables (enzyme activities and ammonium and short-chain fatty acid [SCFA] concentrations) thought to be influenced by the GIT microbiota were also analyzed. Total cultivatable microbial counts ranged from 10¹⁰ to 10¹¹ cfu/g of feces. The largest populations were obligate anaerobes belonging to the Clostridiumclusters, followed by species of bifidobacteria and bacteroides. Coliforms and lactobacilli were found at a more intermediate level (10⁵–10⁹ cfu/g). The predominant anaerobe populations remained quite constant over time, but all other microbial groups showed significant interindividual differences. Enzyme profiles were individual-dependent, but within subjects, moderate to high intersample variations over time were recorded for some activities. Fecal ammonium concentration was the most unpredictable variable; this fluctuated widely between individuals and samples. Acetic acid was the most abundant SCFA in the feces, followed by butyric and propionic acids. SCFA concentrations also varied according to the individual; some subjects showed specific profiles in terms of SCFA composition or concentration. The fecal microbial and biochemical parameters studied seemed to be individual-dependent. Most variables were rather stable over time, while others (e.g., ammonium concentration) varied widely.