Performance of HIV-1 drug resistance testing at low-level viremia and its ability to predict future virologic outcomes and viral evolution in treatment-naive individuals.

BACKGROUND: Low-level viremia (LLV; human immunodeficiency virus [HIV-1] RNA 50-999 copies/mL) occurs frequently in patients receiving antiretroviral therapy (ART), but there are few or no data available demonstrating that HIV-1 drug resistance testing at a plasma viral load (pVL) <1000 copies/mL provides potentially clinically useful information. Here, we assess the ability to perform resistance testing by genotyping at LLV and whether it is predictive of future virologic outcomes in patients beginning ART. METHODS: Resistance testing by genotyping at LLV was attempted on 4915 plasma samples from 2492 patients. A subset of previously ART-naive patients was analyzed who achieved undetectable pVL and subsequently rebounded with LLV (n = 212). A genotypic sensitivity score (GSS) was calculated based on therapy and resistance testing results by genotyping, and stratified according to number of active drugs. RESULTS: Eighty-eight percent of LLV resistance assays produced useable sequences, with higher success at higher pVL. Overall, 16 of 212 (8%) patients had pretherapy resistance. Thirty-eight of 196 (19%) patients without pretherapy resistance evolved resistance to 1 or more drug classes, primarily the nucleoside reverse transcriptase (14%) and/or nonnucleoside reverse transcriptase (9%) inhibitors. Patients with resistance at LLV (GSS <3) had a 2.1-fold higher risk of virologic failure (95% confidence interval, 1.2- to 3.7-fold) than those without resistance (P = .007). Progressively lower GSS scores at LLV were associated with a higher increase in pVL over time (P < .001). Acquisition of additional resistance mutations to a new class of antiretroviral drugs during LLV was not found in a subset of patients. CONCLUSIONS: Routine HIV-1 genotyping of LLV samples can be performed with a reasonably high success rate, and the results appear predictive of future virologic outcomes.

DSM-IV defined conduct disorder and oppositional defiant disorder: an investigation of shared liability in female twins.

BACKGROUND: DSM-IV specifies a hierarchal diagnostic structure such that an oppositional defiant disorder (ODD) diagnosis is applied only if criteria are not met for conduct disorder (CD). Genetic studies of ODD and CD support a combination of shared genetic and environmental influences but largely ignore the imposed diagnostic structure. METHOD: We examined whether ODD and CD share an underlying etiology while accounting for DSM-IV diagnostic specifications. Data from 1446 female twin pairs, aged 11-19 years, were fitted to two-stage models adhering to the DSM-IV diagnostic hierarchy. RESULTS: The models suggested that DSM-IV ODD-CD covariation is attributed largely to shared genetic influences. CONCLUSIONS: This is the first study, to our knowledge, to examine genetic and environmental overlap among these disorders while maintaining a DSM-IV hierarchical structure. The findings reflect primarily shared genetic influences and specific (i.e. uncorrelated) shared/familial environmental effects on these DSM-IV-defined behaviors. These results have implications for how best to define CD and ODD for future genetically informed analyses.

The Effectiveness of a Family-Centered Childhood Obesity Intervention at the YMCA: A Pilot Study.

OBJECTIVE: Community-based, family-centered obesity prevention/treatment initiatives have been shown to be effective in reducing body mass index (BMI) and improving healthy habits in children if implemented with high intensity and sufficient duration. Let's Go! 5-2-1-0 Program (5-2-1-0) was incorporated into family-centered, monthly physical activity classes and cooking classes over six months delivered by Young Men's Christian Association (YMCA) staff. We hypothesized that implementation of this intervention would improve 5-2-1-0 knowledge attainment, increase healthy behavior (based on 5- 2-1-0 curriculum), and improve BMI and waist circumference measurements in children. METHODS: Children attending YMCA summer camps in Rochester, MN, during 2016 were recruited via study packets mailed to their families. Height, weight, and waist circumference measurements as well as the results of the Modified Healthy Habits Survey and the 5-2-1-0 Knowledge Acquisition Survey were recorded for each participating child at baseline and 6-month follow-up. The intervention group received monthly healthy habit reminder emails, and was invited to monthly evening cooking and physical activity classes for 7 sessions over a 6-month period. RESULTS: Fifteen families in the intervention group attended classes. Of those, 13 families regularly participated in (attended at least 5 out of 7) both the monthly physical activity and cooking classes. The children in the intervention group had a significant improvement in the number of Knowledge Acquisition Survey questions answered correctly (p<0.001), while there was no improvement in the control group. As compared to children in the control group, there was no significant change in BMI or waist circumference or healthy habits in the intervention group. CONCLUSION: Our study findings indicate that our intervention resulted in improved knowledge about healthy habits, but did not significantly impact healthy habits or BMI. Potential reasons for this were the small sample size and the attenuated length and/or intensity of the intervention.

Phase transitions in a programmable quantum spin glass simulator.

Understanding magnetic phases in quantum mechanical systems is one of the essential goals in condensed matter physics, and the advent of prototype quantum simulation hardware has provided new tools for experimentally probing such systems. We report on the experimental realization of a quantum simulation of interacting Ising spins on three-dimensional cubic lattices up to dimensions 8 × 8 × 8 on a D-Wave processor (D-Wave Systems, Burnaby, Canada). The ability to control and read out the state of individual spins provides direct access to several order parameters, which we used to determine the lattice's magnetic phases as well as critical disorder and one of its universal exponents. By tuning the degree of disorder and effective transverse magnetic field, we observed phase transitions between a paramagnetic, an antiferromagnetic, and a spin-glass phase.

A feasible approach to all-electronic digital labeling and readout for cell identification.

We present two critical innovations that enable a unique, purely electronic approach to microfluidic whole-cell analysis, focusing on the problem of cell identification and sorting. We used fully-scalable lithographic techniques to microfabricate digital barcodes, providing a means for low-cost, large volume production. We have demonstrated molecular functionalization of the barcodes, using biotin-streptavidin, as well as human CD4 antibody, and we have successfully linked the barcodes to polystyrene beads using the biotin-streptavidin complex. This functionalization allows unique barcodes to be attached to specific cell types, based on phenotype. We have also implemented an electronic barcode readout scheme, using a radio frequency microsensor integrated in an elastomeric microfluidic channel, that can read individual barcodes at rates in excess of 1000 labels s(-1). The barcodes are biologically compatible, and coupled with the electronic sensing technology, provide a route to compact, inexpensive, disposable cell identification, sorting and purification.

Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution.

BACKGROUND: Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS: The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS: The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

Effect of diet and Lactobacillus acidophilus supplements on human fecal bacterial enzymes.

The effect of diet and Lactobacillus acidophilus supplements on fecal microflora enzyme activity was studied in humans. The bacterial enzymes that were investigated are known to catalyze reactions that may result in formation of proximal carcinogens. Compared to vegetarians, omnivores eating a "Western-type" diet had higher levels of beta-glucuronidase, nitroreductase, azoreductase, and steroid 7-alpha-dehydroxylase in their fecal microflora. Removal of red meat or addition of fiber in the form of bran or wheat germ to the diet of omnivores for 30 days had no effect on beta-glucuronidase, nitroreductase, or azoreductase activity. However, removal of red meat or addition of fiber reduced fecal steroid 7-alpha-dehydroxylase activity. The addition of viable Lactobacillus acidophilus supplements to the diet of omnivores significantly decreased fecal bacterial beta-glucuronidase and nitroreductase activities. Thirty days after Lactobacillus supplements were curtailed, fecal enzyme levels returned to normal base-line activities. These findings suggested that the metabolic activity of the fecal microflora was influenced by diet and could be altered by Lactobacillus supplements and to a lesser extent by dietary fiber.

Effect of diet on excretion of estrogens in pre- and postmenopausal women.

Fecal, urinary, and plasma estrogens and plasma androgens were studied in healthy pre- and postmenopausal vegetarian and omnivorous women. Dietary histories of the subjects revealed that omnivores consumed a higher percentage of total protein and fat from animal sources. The total 72-hr fecal excretion as measured by dry weight was higher for vegetarians. Preliminary results indicate that vegetarian women excrete 2 to 3 times more estrogens in feces than do omnivores and that omnivores have about 50% higher mean plasma level of unconjugated estrone and estradiol than vegetarians. Estriol-3-glucuronide, a compound that is formed upon reabsorption of free estriol from the intestine, is found in lower concentrations in the urine of vegetarians. These data suggest that in vegetarians a greater amount of the biliary estrogens escape reabsorption and are excreted with the feces. The differences in estrogen metabolism may explain the lower incidence of breast cancer in vegetarian women.

Semantic networks of interests in online non-suicidal self-injury communities.

People who engage in non-suicidal self-injury (NSSI) often conceal their practices, which limits examination and understanding of their engagement. The goal of this research is to utilize data from public online social networks (namely, LiveJournal, a major blogging social networking site) to observe the NSSI population in a naturally occurring setting. Specifically, the focus of this paper is the interests publicly declared by LiveJournal users. In the course of study, we collected the self-declared interests of 25,000 users who are members of or participate in 139 NSSI-related communities. We constructed a family of semantic networks of interests based on their similarity. The semantic networks are structured and contain several dense clusters-semantic domains-that include NSSI-specific interests (such as self-injury and razor), references to music performers (such as evanescence), and general daily life and creativity related interests (such as poetry and friendship). Assuming users are genuine in their declarations, the clusters reveal distinct patterns of interest and may signal keys to NSSI.

An organo-functionalized metal-oxide cluster, [VO6{(OCH2CH2)2N(CH2CH2OH)}6], with Anderson-like structure.

A new polyoxovanadium cluster compound, [VO6{(OCH2CH2)2N(CH2CH2OH)}6]·0.5CH3CN, was synthesized and characterized by single-crystal X-ray diffraction analysis, FTIR and UV-vis spectroscopy, and TGA. The cluster is composed of a fully reduced cyclic {V6N6O18} framework, which adopts an Anderson-like structure and is comprised of a ring of six edge-sharing {VO5N} octahedra incorporating six {(OCH2CH2)2N(CH2CH2OH)} ligands. Two (OCH2CH2-) arms of each of the six triethanolamine ligands are directly incorporated into the oxometalate core and the third {-CH2CH2OH} arm remains pendant. In the condensed phase, the clusters form discrete hcp layers through inter-cluster hydrogen bonding. These layers stack through soft chemical interactions to form a 3D network structure. The neutral cluster, [VO6{(OCH2CH2)2N(CH2CH2OH)}6], is the isopolyoxovanadium analogue to the cationic clusters contained in a series of heteropolyoxovanadium compounds previously introduced by our laboratory, e.g., [LiVO6{(OCH2CH2)2N(CH2CH2OH)}6]+; its existence shows that a heteroatom is not required to form or stabilize the common organofunctionalized vanadium oxide framework: [VO6{(OCH2CH2)2N(CH2CH2OH)}6]. To the best of our knowledge, the isopolyoxovanadium and heteropolyoxovanadium clusters represent the first reported isopoly-heteropoly analogues in the polyoxometalate field. We compare the TGA profile, FTIR and UV-vis spectra of the new compound with two of its cationic heteropoly analogues.

A single untimed plasma drug concentration measurement during low-level HIV viremia predicts virologic failure.

Suboptimal untimed plasma drug levels (UDL) have been associated with lower rates of virologic suppression and the emergence of drug resistance. Our aim was to evaluate whether UDL among patients with low-level viremia (LLV) while receiving highly active antiretroviral therapy (HAART) can predict subsequent virologic failure (plasma viral load ≥1000 copies/mL) and emergence of resistance. The first documented LLV episode of 328 consenting patients was analysed in terms of drug levels, viral load and resistance, which were monitored while patients were on a consistent HAART regimen. UDL of protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), were categorized as 'therapeutic' or 'subtherapeutic' based on predefined target trough concentrations. Drug resistance genotype was assessed using the Stanford algorithm. Time to virologic failure was evaluated by Kaplan-Meier analysis and Cox proportional hazards regression. We found 78 of 328 patients (24%) with subtherapeutic drug levels at time of first detectable LLV, while 19% harboured drug-resistant virus. Both subtherapeutic UDL and drug resistance independently increased the risk of subsequent virologic failure (p <0.001 and p 0.04, respectively). In a multivariable model, variables associated with LLV and virologic failure included subtherapeutic UDL, elevated plasma viral load, and drug resistance. Patients with subtherapeutic UDL accumulated further drug resistance faster during follow-up (p 0.03). Together, resistance and UDL variables can explain a higher proportion of virologic failure than either measure alone. Our results support further prospective evaluation of UDL in the management of low-level viremia.

Crystallization of thioesterase II from Escherichia coli.

X-ray quality single crystals of the Escherichia coli thioesterase II have been obtained. The protein used for crystallization was overexpressed in parent organism. The crystals are orthorhombic, space group C222(1) with axial lengths a = 99.0 A, b = 121.1 A, c = 166.6 A. A complete homotetramer (120,000 Da) of four polypeptide chains, each 286 residues long, occupies the asymmetric unit. The diffraction pattern extends to approximately 2.4 A resolution using CuK alpha radiation from a rotating anode source. The elucidation of the three-dimensional structure of this unusual bacterial thioesterase will provide basis for the analysis of its unique catalytic mechanism and substrate specificity.

Crystallization of human beta-hexosaminidase B.

beta-Hexosaminidase is a lysosomal hydrolase that is important in the metabolism of sphingoglycolipids. beta-Hexosaminidase B and beta-hexosaminidase A are the major isozymes in normal human tissue. beta-Hexosaminidase B is a homodimer of beta subunits, and beta-hexosaminidase A is a heterodimer composed of an alpha and a beta subunit. Crystals of beta-hexosaminidase B (M(r) 112,000) have been grown using the handling drop technique. They are elongated hexagonal prisms with maximum dimensions of 0.2 mm x 0.2 mm x 0.7 mm. The space group is P6(1)22 (or enantiomorph); the unit cell dimensions are a = b = 114.2 A, c = 402.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one dimer per asymmetric unit. Crystals diffract to 3.2 A resolution.

Crystallization and preliminary crystallographic data of a Streptomyces scabies extracellular esterase.

X-ray quality single crystals of an extracellular esterase from pathogenic Streptomyces scabies were obtained by the hanging drop method. The crystals are monoclinic (space group C2, a = 161.1 A, b = 51.2 A, c = 124.2 A, beta = 100.6 degrees) with two molecules related by a noncrystallographic dyad in the asymmetric unit, with a solvent content of approximately 64%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The study has been initiated in order to elucidate the mechanism of this unusual non-serine-dependent esterase, and to gain better understanding of the molecular basis of the pathogenesis of the scab disease.

Expression, purification and crystallization of the Vibrio harveyi acyltransferase.

We have obtained X-ray quality single crystals of Vibrio harveyi acyltransferase. The protein was obtained from V. harveyi by a gene mobilization expression system. The crystals are monoclinic (space group P2(1), a = 89.9 A, b = 83.6 A, c = 47.1 A, beta = 97.3 degrees) with two molecules related by a pronounced non-crystallographic dyad in the asymmetric unit, with a solvent content of approximately 50%. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube CuK alpha radiation. The elucidation of the three-dimensional structure of this enzyme, believed to contain a proteinase-like catalytic triad, which resembles in many ways other eukaryotic fatty acid chain terminating enzymes, may have important consequences for our understanding of the molecular basis of the final stages of the synthesis of fatty acids.

Crystallization of canine cardiac calsequestrin.

Calsequestrin is the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum membranes. Two X-ray quality crystal forms of canine cardiac calsequestrin were obtained by the hanging drop method using KCl as a precipitant. One form is monoclinic (space group P2(1), a = 73.4 A, b = 104.4 A, c = 60.2 A, beta = 120.4 degrees) with two molecules in the asymmetric unit and a solvent content of approximately 40%. The second form is trigonal (P3(1)21 or P3(2)21, a = b = 99.3 A, c = 89.8 A) with a single molecule in the asymmetric unit and 55% solvent content. Cross rotation function calculations show that despite the different space groups the packing of the molecules in both crystals is likely to be similar suggesting the existence of a stable dimer. The monoclinic crystals diffract beyond 3 A using a laboratory rotating anode source, while under the same conditions the trigonal crystals diffract only to approximately 4.5 A. This is the first report of successful preparation of X-ray quality crystals of a high capacity Ca2+ binding protein.

Crystallization of the malonyl coenzyme A-acyl carrier protein transacylase from Escherichia coli.

The malonyl coenzyme A-acyl carrier protein transacylase, a single polypeptide chain of 358 amino acid residues and a molecular mass of 32 kDa, is a key component of the fatty acid synthase multienzyme complex. The elucidation of its three-dimensional structure will help in the understanding of the molecular basis of the biosynthesis of fatty acids, as well as of polyketides and related biologically active molecules. Three X-ray-quality crystal forms of the Escherichia coli fabD gene product encoding for malonyl coenzyme A-acyl carrier protein transacylase have been obtained using the hanging-drop method and ammonium sulfate as precipitant. Two are tetragonal and each contains two molecules in the asymmetric unit (form I: space group P4(3(1))2(1)2 with a = b = 83.9 A, c = 166.5 A and form II: space group P4 with a = b = 132.64 A, c = 38.85 A), whereas the third form belongs to the hexagonal system and contains one molecule in the asymmetric unit (space group P6(1(5)) with a = b = 68.52 A, c = 117.71 A). In each case, the diffraction pattern extends to approximately 2.0 A resolution using CuK alpha radiation from a rotating anode source.

The structures of caspases-1, -3, -7 and -8 reveal the basis for substrate and inhibitor selectivity.

BACKGROUND: Peptide inhibitors of caspases have helped define the role of these cysteine proteases in biology. Structural and biochemical characterization of the caspase enzymes may contribute to the development of new drugs for the treatment of caspase-mediated inflammation and apoptosis. RESULTS: The crystal structure of the previously unpublished caspase-7 (Csp7; 2.35 A) bound to the reversible tetrapeptide aldehyde inhibitor acetyl-Asp-Glu-Val-Asp-CHO is compared with crystal structures of caspases-1 (2.3 A), -3 (2.2 A), and -8 (2.65 A) bound to the same inhibitor. Csp7 is a close homolog of caspase-3 (Csp3), and these two caspases possess some quarternary structural characteristics that support their unique role among the caspase family. However, although Csp3 and Csp7 are quite similar overall, they were found to have a significantly different substitution pattern of amino acids in and around the S4-binding site. CONCLUSIONS: These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.

Implementing Child-focused Activity Meter Utilization into the Elementary School Classroom Setting Using a Collaborative Community-based Approach.

INTRODUCTION: The prevalence of pediatric obesity has increased over the past 3 decades and is a pressing public health program. New technology advancements that can encourage more physical in children are needed. The Zamzee program is an activity meter linked to a motivational website designed for children 8-14 years of age. The objective of the study was to use a collaborative approach between a medical center, the private sector and local school staff to assess the feasibility of using the Zamzee Program in the school-based setting to improve physical activity levels in children. METHODS: This was a pilot 8-week observational study offered to all children in one fifth grade classroom. Body mass index (BMI), the amount of physical activity by 3-day recall survey, and satisfaction with usability of the Zamzee Program were measured pre- and post-study. RESULTS: Out of 11 children who enrolled in the study, 7 completed all study activities. In those who completed the study, the median (interquartile range) total activity time by survey increased by 17 (1042) minutes and the BMI percentile change was 0 (8). Both children and their caregivers found the Zamzee Activity Meter (6/7) and website (6/7) "very easy" or "easy" to use. CONCLUSION: The Zamzee Program was found to be usable but did not significantly improve physical activity levels or BMI. Collaborative obesity intervention projects involving medical centers, the private sector and local schools are feasible but the effectiveness needs to be evaluated in larger-scale studies.

Effect of maraviroc on non-R5 tropic HIV-1: refined analysis of subjects from the phase IIb study A4001029.

We characterized maraviroc susceptibility of dual/mixed tropic viruses from subjects enrolled onto phase IIb study A4001029. Maraviroc baseline plasma samples from 13 multidrug-experienced subjects were sequenced and the HIV-1-env gene cloned into pNL4.3Δenv to obtain recombinant viruses. The V3 region was sequenced by the Sanger method and ultradeep sequencing. By analysing subjects having a weighted optimized background therapy susceptibility (wOBT) score of <1, 3/7 subjects were characterized by good in vivo and in vitro response to maraviroc therapy. Molecular docking simulations allowed us to rationalize the maraviroc susceptibility of dual/mixed tropic viruses. A subset of subjects with dual/mixed tropic viruses responded to maraviroc. Further investigations are warranted of CCR5 antagonists in subjects carrying dual/mixed tropic virus that explore the feasible use of maraviroc in subjects that is potentially larger than those infected with a pure R5 virus.