Interest in biomarker patterns and disease has led to the development of immunoassays that evaluate multiple analytes in parallel with little sample. However, there are no current standards for multiplex configuration, validation, and quality, thus, validation by platform, population, and question of interest is recommended. We sought to determine the best blood fraction for multiplex evaluation of circulating biomarkers in postmenopausal women and to explore body composition phenotype discrimination by biomarkers.
Archived serum and plasma samples from a sample of healthy postmenopausal women with the highest (n=9) and lowest (n=11) percent lean mass, by dual-energy X-ray absorptiometry, were used to measure 90 analytes by bead-based, suspension multiplex assays. Replicates of serum and plasma were analyzed in a random selection of 4 of these individuals.
Ninety-percent of the analytes were detectable for ≥50% of samples; when limited to these well detected analytes, mean replicate correlations for serum and plasma were 0.87and 0.85 respectively. Serum had lower error rates discriminating phenotypes; 7 serum versus 2 plasma analytes discriminated extreme body phenotypes.
Serum and plasma performed similarly for the majority of the analytes. Serum demonstrated a slight advantage in predicting extreme body composition phenotypes in postmenopausal women using parallel evaluation of analytes.
biomarkers; lean mass; multi-analyte; multiplex; phenotype discrimination
Vitamin D levels and calcium intake have been associated with risk of colorectal neoplasia, and genetic variation in vitamin D-pathway genes may affect circulating vitamin D metabolite concentrations and/or risk for colorectal lesions. This study evaluated associations between polymorphic variation in the Gc-globulin (GC) and Calcium-sensing receptor (CASR) and odds for metachronous colorectal neoplasia and vitamin D metabolite concentrations.
Participants from the Ursodeoxycholic Acid (UDCA) and Wheat Bran Fiber (WBF) trials (n=1439) were analyzed using a single nucleotide polymorphism (SNP) tagging approach, with a subset (n=404) of UDCA trial participants for whom vitamin D metabolite concentrations were also available. A total of 25 GC and 35 CASR tagSNPs were evaluated using multiple statistical methods.
Principal components analyses did not reveal gene-level associations between GC or CASR and colorectal neoplasia, however, a significant gene-level association between GC and 25(OH)D concentrations (p < 0.01) was observed. At the individual SNP-level and following multiple comparisons adjustments, significant associations were observed between seven GC (rs7041, rs222035, rs842999, rs1155563, rs12512631, rs16846876, rs1746825) polymorphisms and circulating measures of 25(OH)D (adjusted p < 0.01), and CASR SNP rs1042636 and proximal colorectal neoplasia (adjusted p = 0.01).
These results demonstrate a possible association between variation in CASR and odds of colorectal neoplasia as well as the potential role of variation in GC with circulating 25(OH)D concentrations.
Additional research is warranted to determine the mechanism of GC genotype in influencing 25(OH)D concentrations and to further elucidate the role of CASR in colorectal neoplasia.
GC; CASR; polymorphism; colorectal neoplasia; vitamin D
Development of human immunodeficiency virus resistance mutations is a major cause of failure of antiretroviral treatment. We develop a recursive partitioning method to correlate high-dimensional viral sequences with repeatedly measured outcomes. The splitting criterion of this procedure is based on a class of U-type score statistics. The proposed method is flexible enough to apply to a broad range of problems involving longitudinal outcomes. Simulation studies are performed to explore the finite-sample properties of the proposed method, which is also illustrated through analysis of data collected in 3 phase II clinical trials testing the antiretroviral drug efavirenz.
Antiretroviral drugs; Longitudinal data; Recursive partitioning; Repeated measurements; Resistance mutations; Tree method
The three-dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α-helices and/or β-strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β-sheet in a protein, there has been little progress on generating native-like packing of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β-sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ2 motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands.
Protein structure prediction; super secondary structure; ab initio modeling; helix-sheet packing
UVB irradiation of epidermal keratinocytes results in the activation of the p38 MAPK pathway and subsequently activator protein-1 (AP-1) transcription factor activation and COX-2 expression. AP-1 and COX-2 have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-2 expression and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-2 expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and growth compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and non-transgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-2 expression and proliferation of UVB-irradiated cells.
ultraviolet light; dominant negative p38; non-melanoma skin cancer; COX-2; AP-1
Examine psychological functioning and beliefs about medicine in adolescents with HIV-1 on HAART in a community-based directly observed therapy (DOT) pilot feasibility study.
Youth with behaviorally-acquired HIV (n=20; 65% female; median age 21 years) with adherence problems, received once-daily DOT. Youth were assessed at baseline, week 12 (post-DOT) and week 24 (follow-up).
Baseline to week 12 comparisons: 55% of youth reported clinical depressive symptoms compared to 27% at week 12 with sustained improvements at week 24. Substance use: Borderline clinical range (Tscore=68), with clinical but statistically non-significant improvement (Tscore=61). Hopelessness scores reflected optimism for the future. Coping strategies showed significantly decreased Cognitive Avoidance (p=0.02), Emotional Discharge (p=0.004), and Acceptance/Resignation (“nothing I can do,” p=0.004); Positive Reappraisal and Seeking Support emerged. Aside from depressive symptoms, week 12 improvements were not sustained at week 24. DOT adherence was predicted by higher baseline depression (p=0.05), Beliefs About Medicine (p=0.006) and Perceived Threat of illness scores (p=0.03).
Youth with behaviorally-acquired HIV and adherence problems who participated in a community-based DOT intervention reported clinically improved depressive symptoms, and temporarily reduced substance use and negative coping strategies. Depressive symptoms, Beliefs About Medicine and viewing HIV as a threat predicted better DOT adherence.
HIV; adolescent; psychological functioning; directly observed therapy (DOT); adherence
Plasma concentrations of several protease inhibitors are decreased during pregnancy. Few data are available describing atazanavir exposure during pregnancy, especially when used in combination with tenofovir, whose coadministration with atazanavir results in decreased atazanavir exposure.
IMPAACT 1026s is an on-going, prospective, non-blinded study of antiretroviral pharmacokinetics in HIV-infected pregnant women that included two cohorts receiving atazanavir/ritonavir 300mg/100mg once daily during the third trimester through 6-12 weeks postpartum either with or without tenofovir.
Intensive steady-state 24-hour pharmacokinetic profiles were performed during the third trimester and at 6-12 weeks postpartum. Atazanavir was measured by reverse-phase HPLC with a detection limit of 0.13 mcg/mL. Pharmacokinetic targets were the estimated 10th percentile atazanavir AUC (29.4 mcg*hr/mL) in non-pregnant historical controls taking the standard dose (mean AUC=57 mcg*hr/mL) and a trough concentration of 0.15 mcg/mL, the concentration target used in therapeutic drug monitoring programs. Infant bilirubin concentrations were measured at 24-48 hours and 4-6 days after birth.
Atazanavir pharmacokinetic data were available for 38 women (18 without tenofovir, 20 with tenofovir. Median atazanavir AUC was reduced during the third trimester compared to postpartum for subjects not receiving tenofovir (41.9 vs 57.9 mcg*hr/mL, p=.02) and for subjects receiving tenofovir (28.8 vs. 39.6 mcg*hr/mL, p=.04). During the third trimester, AUC was below the target in 33% (6/18) of women not receiving tenofovir and 55% (11/20) of women receiving tenofovir. Trough concentration was below the target in 6% (1/18) of women not receiving tenofovir and 15% (3/20) of women receiving tenofovir. The median (range) ratio of cord blood/maternal atazanavir concentration in 29 paired samples was 0.18 (0 - 0.45). No excessive infant bilirubin concentrations were observed.
Atazanavir exposure is reduced by pregnancy and by concomitant tenofovir use. A dose increase of atazanavir/ritonavir to 400mg/100mg may be necessary in pregnant women to ensure atazanavir exposure equivalent to that seen in nonpregnant adults, especially for pregnant women who are antiretroviral-experienced and/or who are also receiving tenofovir.
atazanavir; tenofovir; pharmacokinetics; pregnancy; HIV; mother to child transmission
The Hippo pathway regulates organ size by inhibiting cell proliferation and promoting cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector of the Hippo pathway that promotes cell growth as a transcription co-activator. In human cancer, the YAP1 gene was reported as amplified and over-expressed in several tumor types.
Immunohistochemical staining of YAP1 protein was used to assess the expression of YAP1 in pancreatic tumor tissues. siRNA oligonucleotides were used to knockdown the expression of YAP1 and their effects on pancreatic cancer cells were investigated using cell proliferation, apoptosis, and anchorage-independent growth assays. The Wilcoxon signed-rank, Pearson correlation coefficient, Kendall's Tau, Spearman's Rho, and an independent two-sample t (two-tailed) test were used to determine the statistical significance of the data.
Immunohistochemistry studies in pancreatic tumor tissues revealed YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the tumor cells, whereas the adjacent normal epithelial showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell density. YAP1 total protein expression increased in the nuclear fractions in BxPC-3 and PANC-1, while it declined in HPDE6 as cell density increased. Additionally, treatment of pancreatic cancer cell lines, BxPC-3 and PANC-1, with YAP1-targeting siRNA oligonucleotides significantly reduced their proliferation in vitro. Furthermore, treatment with YAP1 siRNA oligonucleotides diminished the anchorage-independent growth on soft agar of pancreatic cancer cells, suggesting a role of YAP1 in pancreatic cancer tumorigenesis.
YAP1 is overexpressed in pancreatic cancer tissues and potentially plays an important role in the clonogenicity and growth of pancreatic cancer cells.
Chemopreventive and antitumor properties of perillyl alcohol (POH) studied preclinically indicate that topical POH inhibits both ultraviolet B (UVB)-induced murine skin carcinogenesis (squamous cell tumor models) and DMBA-induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous Phase 1 clinical trial in participants with normal-appearing skin demonstrated that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here we performed a three month, double-blind, randomized, placebo-controlled Phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower dose of POH compared to placebo (p = 0.1), but this was not observed in the high dose group. However, in the high dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed using karyometric analysis (p< 0.01). There was no statistical significance demonstrated in the lower dose group. No changes were observed in p53 expression, cellular proliferation (by PCNA expression), or apoptosis in either treatment group compared to placebo. These results suggest that while our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary.
Chemoprevention; topical perillyl alcohol; karyometry; skin cancer; histopathologic score
Reduced lopinavir concentrations have been demonstrated with use of the capsule formulation during the third trimester of pregnancy. This study determined lopinavir exposure with an increased dose of the new tablet formulation during the third trimester.
International Maternal Pediatric Adolescent AIDS Clinical Trials 1026s is a prospective nonblinded pharmacokinetic study in HIV-infected pregnant women, including a cohort receiving 2 lopinavir/ritonavir tablets (400 mg/100 mg) twice daily during the second trimester, 3 tablets (600 mg/150 mg) twice daily during the third trimester, and 2 tablets (400 mg/100 mg) twice daily postdelivery through 2 weeks postpartum.
Steady-state 12-hour pharmacokinetic profiles were performed during pregnancy and at 2 weeks postpartum. Lopinavir and ritonavir were measured by reverse-phase high-performance liquid chromatography (detection limit, 0.09 mcg/mL).
Thirty-three women were studied. Median lopinavir AUC for the second trimester (n = 11), third trimester (n = 33), and postpartum (n = 27) were 72, 96, and 133 mcg·hr/mL, respectively. Median minimum lopinavir concentrations were 3.4, 4.9, and 6.9 mcg/mL.
The higher lopinavir/ritonavir tablet dose (600 mg/150 mg) provided exposure during the third trimester similar to the average AUC (98 mcg·hr·mL−1) in nonpregnant adults taking 400 mg/100 mg twice daily. The higher dose should be used during the second and third trimesters of pregnancy. Postpartum dosing can be reduced to standard dosing before 2 weeks postpartum.
HIV; lopinavir; mother-to-child transmission; pharmacokinetics; pregnancy
Adherence to medications is critical to optimizing HIV care and is a major challenge in youth. The utility of directly observed therapy (DOT) to improve adherence in youth with HIV remains undefined and prompted this pilot study. Four U.S. sites were selected for this 24-week cooperative group study to assess feasibility and to identify the logistics of providing DOT to HIV-infected youth with demonstrated adherence problems. Once-a-day DOT was provided by DOT facilitators at the participant's choice of a community-based location and DOT tapered over 12 weeks to self-administered therapy based on ongoing adherence assessments. Twenty participants, median age 21 years and median CD4 227 cells/μl, were enrolled. Participants chose their homes for 82% of DOT visits. Compliance with recommended DOT visits was (median) 91%, 91%, and 83% at weeks 4, 8, and 12, respectively. Six participants completed >90% of the study-specified DOT visits and successfully progressed to self-administered therapy (DOT success); only half sustained >90% medication adherence 12 weeks after discontinuing DOT. Participants considered DOT successes were more likely to have higher baseline depression scores (p = 0.046). Via exit surveys participants reported that meeting with the facilitator was easy, DOT increased their motivation to take medications, they felt sad when DOT ended, and 100% would recommend DOT to a friend. In conclusion, this study shows that while community-based DOT is safe, feasible, and as per participant feedback, acceptable to youth, DOT is not for all and the benefits appear short-lived. Depressed youth appear to be one subgroup that would benefit from this intervention. Study findings should help inform the design of larger community-based DOT intervention studies in youth.
The three-dimensional structure of a protein is organized around the packing of its secondary structure elements. While much is known about the packing geometry observed between α-helices and between β-sheets, there has been little progress on characterizing helix-sheet interactions. We present an analysis of the conformation of αβ2 motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen-bonded. The geometry of the αβ2 motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β-sheet. Sidechain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ2 motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native-like conformations among a set of non-native conformations in well-known decoy datasets. The information on the geometry of αβ2 motifs as well as the related statistical potentials has applications in the field of protein structure prediction.
Protein structure; super secondary structure; statistical potential; amino acid propensity
To obtain input from adolescents with HIV-1 infection to inform the design of a community-based modified directly observed therapy (MDOT) antiretroviral adherence intervention.
Pediatric AIDS Clinical Trials Group (PACTG) protocol 1036A conducted three focus groups with 17 adolescents aged 17 to 22 years (10 females, 65% African-American) from three geographically distinct US PACTG sites. Focus group sessions were scripted, audio-taped, and transcribed verbatim. A coding dictionary was developed and validated; Ethnograph v5.08 was used to summarize coded data across and within the three sites. Prevalent themes were identified via frequencies and are reported as percents.
Adolescents specified: the MDOT provider should be familiar to the participant and empathic; the MDOT location should be mutually agreed upon, flexible, and private; and participant and provider communication should be bidirectional, preferably by phone. Ideally the MDOT program should be continued until adolescents independently demonstrate adherence and include a weaning phase as a test of skill-acquisition. The most commonly endorsed barrier to the proposed program was MDOT would be an invasion of privacy. Initially, following introduction to the purpose of the focus group, all but one adolescent expressed MDOT could benefit someone other than themselves; however, at conclusion of the focus group discussion, a significant shift in openness to the intervention occurred whereby 11 participants indicated they would consider participation in a MDOT program if offered.
Focus group feedback clarified the feasibility, logistics, and patient concerns about the design and implementation of a proposed MDOT intervention for adolescents with HIV-1 infection who struggle with medication adherence
Adolescents; HIV; modified directly observed therapy (MDOT); focus group; adherence
The goal of HAART is to promote reconstitution of CD4+ T cells and other immune responses. We evaluated the extent and the kinetics of immune reconstitution in HIV-infected children over 144 weeks of successful HAART.
Thirty-seven children receiving their first HAART regimen had plasma HIV RNA; T cells and subpopulations; T-cell rearrangement excision circles (TREC) DNA; candida, HIVCD4 and HIVCD8 enzyme-linked immunospot measured at regular intervals.
Plasma HIV RNA became undetectable in 81% of patients at 24 weeks and remained undetectable in 77% at 144 weeks. In contrast, CD4+% continuously increased. Distribution of T-cell subpopulations changed rapidly during the first 48 weeks of HAART and more slowly thereafter. At 144 weeks, total, naive and activated CD4+% and naive CD8+% of HIV-infected children were not significantly different from those of healthy age-matched controls, whereas total and activated CD8+% remained elevated. CD4+ and CD8+ TREC content increased only during the first 48 weeks of HAART. They positively correlated with each other and with total CD4+%, naive CD4+% and naive CD8+%. Candida and HIVCD4 enzyme-linked immunospot increased over time reaching peak values at 48 weeks and 144 weeks, respectively. HIVCD8 enzyme-linked immunospot decreased in magnitude over 144 weeks of HAART but retained its breadth. Baseline CD4+% positively correlated with CD4+% and with functional immune reconstitution at week 144, whereas baseline TREC correlated with TREC at week 144.
HIV-infected children acquired normal distribution of CD4+ T cells and other subpopulations and recovered CD4-mediated HIV immunity after 144 weeks of HAART.
candida; cell-mediated immunity; children; highly active antiretroviral therapy; HIV; T-cell rearrangement excision circle; T-cell subpopulations
The association between baseline drug resistance mutations and subsequent increase in viral failure has not been established for HIV-infected children. We evaluated drug resistance mutations at 39 codon sites (21 protease inhibitor (PI) resistant codons and 18 nucleoside reverse transcriptase inhibitor (NRTI) resistant codons) for 92 clinically stable NRTI-experienced, PI-naive HIV-infected children 2 to 17 years of age who were initiating new therapy with ritonavir plus zidovudine (ZDV) and lamivudine or plus stavudine. The association between baseline drug resistance mutations and subsequent viral failure after 12 and 24 weeks of highly active antiretroviral therapy (HAART) was studied.
There were few primary PI associated mutations in this PI-naïve population, but 84% had NRTI mutations – codons 215 (66%), 41 (42%), 67 (37%), 210 (33%) and 70 (32%). None of the specific baseline drug resistance mutations were associated with a higher rate of virologic failure after 12 or 24 weeks of HAART. Median week 12 viral load decreased as the total number of NRTI mutations at baseline increased (P = 0.006). Specifically, a higher level of baseline ZDV resistance mutation was associated with a decrease in viral failure after 12 weeks on a ZDV-containing HAART regimen (P = 0.017).
No increase was seen in the rate of viral failure after HAART associated with the presence of resistance mutations at baseline. This paradoxical result may be due to adherence, replicative capacity, or ZDV hypersusceptibility to the new regimen.