Structural basis for sequence specific DNA binding and protein dimerization of HOXA13.

The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of genes during embryonic morphogenesis. Here we present the NMR structure of HOXA13 homeodomain (A13DBD) bound to an 11-mer DNA duplex. A13DBD forms a dimer that binds to DNA with a dissociation constant of 7.5 nM. The A13DBD/DNA complex has a molar mass of 35 kDa consistent with two molecules of DNA bound at both ends of the A13DBD dimer. A13DBD contains an N-terminal arm (residues 324 - 329) that binds in the DNA minor groove, and a C-terminal helix (residues 362 - 382) that contacts the ATAA nucleotide sequence in the major groove. The N370 side-chain forms hydrogen bonds with the purine base of A5* (base paired with T5). Side-chain methyl groups of V373 form hydrophobic contacts with the pyrimidine methyl groups of T5, T6* and T7*, responsible for recognition of TAA in the DNA core. I366 makes similar methyl contacts with T3* and T4*. Mutants (I366A, N370A and V373G) all have decreased DNA binding and transcriptional activity. Exposed protein residues (R337, K343, and F344) make intermolecular contacts at the protein dimer interface. The mutation F344A weakens protein dimerization and lowers transcriptional activity by 76%. We conclude that the non-conserved residue, V373 is critical for structurally recognizing TAA in the major groove, and that HOXA13 dimerization is required to activate transcription of target genes

Suppression of Met signaling by the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG)

Met is a prognostic indicator of colorectal cancer patient survival. Therapies that target Met may therefore have beneficial outcomes in the clinic. Recently, EGCG was reported to suppress Met activation, although the mechanisms were not elucidated. HCT116 and HT29 human colon cancer cells were used to examine the relationships between Met activation, EGCG treatment, and H₂O₂ generation. At concentrations of 0.5, 1 and 5 μM, EGCG suppressed the activation of Met induced by its ligand, hepatocyte growth factor (HGF). Concentrations of 10 μM EGCG and below generated low amounts of H₂O₂ (5 μM) were required to directly increase the phosphorylation of Met. Moreover, suppression of Met activation by EGCG occurred in the presence or absence of catalase, suggesting that such effects were not an "artifact" of H₂O₂ generated from EGCG in cell culture media. Molecular docking and enzyme kinetic analyses suggested that EGCG is a competitive inhibitor, binding to the kinase domain of Met with a Ki of 3.3 μM EGCG. The downstream effect of EGCG mediated suppression of the Met receptor included decreased signaling to members of the MAPK and PI3KK signaling pathways. Cell proliferation and migration was also significantly inhibited by EGCG. Overall, the data presented in this dissertation support that EGCG is able to suppress HGF-induced Met signaling. These findings demonstrate that EGCG might be a beneficial therapeutic agent in the colon, inhibiting Met signaling and helping to attenuate tumor metastasis

The Mass Function of Super Giant Molecular Complexes and Implications for Forming Young Massive Star Clusters in the Antennae (NGC 4038/39)

We have used previously published observations of the CO emission from the Antennae (NGC 4038/39) to study the detailed properties of the super giant molecular complexes with the goal of understanding the formation of young massive star clusters. Over a mass range from 5E6 to 9E8 solar masses, the molecular complexes follow a power-law mass function with a slope of -1.4 +/- 0.1, which is very similar to the slope seen at lower masses in molecular clouds and cloud cores in the Galaxy. Compared to the spiral galaxy M51, which has a similar surface density and total mass of molecular gas, the Antennae contain clouds that are an order of magnitude more massive. Many of the youngest star clusters lie in the gas-rich overlap region, where extinctions as high as Av~100 imply that the clusters must lie in front of the gas. Combining data on the young clusters, thermal and nonthermal radio sources, and the molecular gas suggests that young massive clusters could have formed at a constant rate in the Antennae over the last 160 Myr and that sufficient gas exists to sustain this cluster formation rate well into the future. However, this conclusion requires that a very high fraction of the massive clusters that form initially in the Antennae do not survive as long as 100 Myr. Finally, we compare our data with two models for massive star cluster formation and conclude that the model where young massive star clusters form from dense cores within the observed super giant molecular complexes is most consistent with our current understanding of this merging system. (abbreviated)Comment: 40 pages, four figures; accepted for publication in Ap

Analysis of De Novo HOXA 13 Polyalanine Expansions Supports Replication Slippage Without Repair in Their Generation

Polyalanine repeat expansion diseases are hypothesized to result from unequal chromosomal recombination, yet mechanistic studies are lacking. We identified two de novo cases of hand‐foot‐genital syndrome (HFGS) associated with polyalanine expansions in HOXA13 that afforded rare opportunities to investigate the mechanism. The first patient with HFGS was heterozygous for a de novo nine codon polyalanine expansion. Haplotype investigation showed that the expansion arose on the maternally inherited chromosome but not through unequal crossing over between homologs, leaving unequal sister chromatid exchange during mitosis or meiosis or slipped mispairing as possible explanations. The asymptomatic father of the second patient with HFGS was mosaic for a six codon polyalanine expansion. Multiple tissue PCR and clonal analysis of paternal fibroblasts showed only expansion/WT and WT/WT clones, and haplotype data showed that two unaffected offspring inherited the same paternal allele without the expansion, supporting a postzygotic origin. Absence of the contracted allele in the mosaic father does not support sister chromatid exchange in the origin of the expansion. Mosaicism for HOXA13 polyalanine expansions may be associated with a normal phenotype, making examination of parental DNA essential in apparently de novo HFGS cases to predict accurate recurrence risks. We could not find an example in the literature where unequal sister chromatid exchange has been proven for any polyalanine expansion, suggesting that the principal mechanism for polyalanine expansions (and contractions) is slipped mispairing without repair or that the true frequency of unequal sister chromatid exchange involving these repeats is low. © 2013 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97454/1/ajmga35843.pd

Patch - Occupancy Survey of Elephant (Loxodonta africana) Surrounding Livingstone, Zambia

Wild elephants represent the biggest human–wildlife conflict issue in Livingstone, Zambia. However, little is known about their movements. This survey investigated elephants’ habitat use outside a core protected and fenced zone that forms part of Mosi-oa-Tunya National Park, Zambia. Using ‘patch-occupancy’ methodology, indications of elephant presence (feeding behaviour, dung and tracks) were surveyed. The survey aimed to assist proposed future monitoring exercises by defining the geographical extent that should be considered to improve accuracy in species abundance estimates. Results were supplemented using collected indications of elephant presence from prior monitoring exercises, and during this survey. Elephant presence was confirmed up to 8 km from the boundary of the protected core habitat, focussed in: (1) an unfenced zone of the national park, (2) along a road leading from the national park to the Dambwa Forest to the north and (3) along two rivers located to the west (Sinde River) and east (Maramba River) of the core area. Detection probability of elephant presence was high using these methods, and we recommend regular sampling to determine changes in habitat use by elephants, as humans continue to modify land-use patterns. Conservation implications: Identification of elephant ranging behaviour up to 8 km outside of the Mosi-oa-Tunya National Park in southern Zambia will assist in managing human– elephant conflict in the area, as well as in assessing this seasonal population’s abundance

Snake mortality and cover board effectiveness along exclusion fencing in British Columbia, Canada

We report on snake mortalities along exclusion fencing in southern British Columbia, showing Western Yellow-bellied Racer (Coluber constrictor mormon) deaths were disproportionately higher than our encounter rates with the species within the snake community. This suggests racers were susceptible to fence mortality more so than Northern Pacific Rattlesnakes (Crotalus o. oreganus) or Great Basin Gophersnakes (Pituouphis catenifer deserticola). Datalogger recordings revealed temperatures under cover boards were well above the tolerable temperatures of the three snake species, although the boards appeared to temper ambient heat more efficiently than natural vegetation. We caution that the effects of fencing and cover boards may vary across ecosystems and snake species

Increased Expression of Cytotoxic T-Lymphocyte-Associated Protein 4 by T Cells, Induced by B7 in Sera, Reduces Adaptive Immunity in Patients With Acute Liver Failure.

BACKGROUND & AIMS: Patients with acute liver failure (ALF) have defects in innate immune responses to microbes (immune paresis) and are susceptible to sepsis. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which interacts with the membrane receptor B7 (also called CD80 and CD86), is a negative regulator of T-cell activation. We collected T cells from patients with ALF and investigated whether inhibitory signals down-regulate adaptive immune responses in patients with ALF. METHODS: We collected peripheral blood mononuclear cells from patients with ALF and controls from September 2013 through September 2015 (45 patients with ALF, 20 patients with acute-on-chronic liver failure, 15 patients with cirrhosis with no evidence of acute decompensation, 20 patients with septic shock but no cirrhosis or liver disease, and 20 healthy individuals). Circulating CD4+ T cells were isolated and analyzed by flow cytometry. CD4+ T cells were incubated with antigen, or agonist to CD3 and dendritic cells, with or without antibody against CTLA4; T-cell proliferation and protein expression were quantified. We measured levels of soluble B7 molecules in supernatants of isolated primary hepatocytes, hepatic sinusoidal endothelial cells, and biliary epithelial cells from healthy or diseased liver tissues. We also measured levels of soluble B7 serum samples from patients and controls, and mice with acetaminophen-induced liver injury using enzyme-linked immunosorbent assays. RESULTS: Peripheral blood samples from patients with ALF had a higher proportion of CD4+ CTLA4+ T cells than controls; patients with infections had the highest proportions. CD4+ T cells from patients with ALF had a reduced proliferative response to antigen or CD3 stimulation compared to cells from controls; incubation of CD4+ T cells from patients with ALF with an antibody against CTLA4 increased their proliferative response to antigen and to CD3 stimulation, to the same levels as cells from controls. CD4+ T cells from controls up-regulated expression of CTLA4 after 24-48 hours culture with sera from patients with ALF; these sera were found to have increased concentrations of soluble B7 compared to sera from controls. Necrotic human primary hepatocytes exposed to acetaminophen, but not hepatic sinusoidal endothelial cells and biliary epithelial cells from patients with ALF, secreted high levels of soluble B7. Sera from mice with acetaminophen-induced liver injury contained high levels of soluble B7 compared to sera from mice without liver injury. Plasma exchange reduced circulating levels of soluble B7 in patients with ALF and expression of CTLA4 on T cells. CONCLUSIONS: Peripheral CD4+ T cells from patients with ALF have increased expression of CTLA4 compared to individuals without ALF; these cells have a reduced response to antigen and CD3 stimulation. We found sera of patients with ALF and from mice with liver injury to have high concentrations of soluble B7, which up-regulates CTLA4 expression by T cells and reduces their response to antigen. Plasma exchange reduces levels of B7 in sera from patients with ALF and might be used to restore antimicrobial responses to patients

Belatacept and long-term outcomes in kidney transplantation

Background: in previous analyses of BENEFIT, a phase 3 study, belatacept-based immunosuppression, as compared with cyclosporine-based immunosuppression, was associated with similar patient and graft survival and significantly improved renal function in kidney-transplant recipients. Here we present the final results from this study. Methods: we randomly assigned kidney-transplant recipients to a more-intensive belatacept regimen, a less-intensive belatacept regimen, or a cyclosporine regimen. Efficacy and safety outcomes for all patients who underwent randomization and transplantation were analyzed at year 7 (month 84). Results: a total of 666 participants were randomly assigned to a study group and underwent transplantation. Of the 660 patients who were treated, 153 of the 219 patients treated with the more-intensive belatacept regimen, 163 of the 226 treated with the less-intensive belatacept regimen, and 131 of the 215 treated with the cyclosporine regimen were followed for the full 84-month period; all available data were used in the analysis. A 43% reduction in the risk of death or graft loss was observed for both the more-intensive and the less-intensive belatacept regimens as compared with the cyclosporine regimen (hazard ratio with the more-intensive regimen, 0.57; 95% confidence interval [CI], 0.35 to 0.95; P=0.02; hazard ratio with the less-intensive regimen, 0.57; 95% CI, 0.35 to 0.94; P=0.02), with equal contributions from the lower rates of death and graft loss. The mean estimated glomerular filtration rate (eGFR) increased over the 7-year period with both belatacept regimens but declined with the cyclosporine regimen. The cumulative frequencies of serious adverse events at month 84 were similar across treatment groups. Conclusions: seven years after transplantation, patient and graft survival and the mean eGFR were significantly higher with belatacept (both the more-intensive regimen and the less-intensive regimen) than with cyclosporine. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT00256750)

Comparison of early-, late-, and non-participants in a school-based asthma management program for urban high school students

<p>Abstract</p> <p>Background</p> <p>To assess bias and generalizability of results in randomized controlled trials (RCT), investigators compare participants to non-participants or early- to late-participants. Comparisons can also inform the recruitment approach, especially when working with challenging populations, such as urban adolescents. In this paper, we describe characteristics by participant status of urban teens eligible to participate in a RCT of a school-based, web-based asthma management program.</p> <p>Methods</p> <p>The denominator for this analysis was all students found to be eligible to participate in the RCT. Data were analyzed for participants and non-participants of the RCT, as well as for students that enrolled during the initially scheduled recruitment period (early-participants) and persons that delayed enrollment until the following fall when recruitment was re-opened to increase sample size (late-participants). Full Time Equivalents (FTEs) of staff associated with recruitment were estimated.</p> <p>Results</p> <p>Of 1668 teens eligible for the RCT, 386 enrolled early, and 36 enrolled late, leaving 1246 non-participants. Participants were younger (p < 0.01), more likely to be diagnosed, use asthma medication, and have moderate-to-severe disease than non-participants, odds ratios (95% Confidence Intervals) = 2.1(1.7-2.8), 1.7(1.3-2.1), 1.4(1.0-1.8), respectively. ORs were elevated for the association of late-participation with Medicaid enrollment, 1.9(0.7-5.1) and extrinsic motivation to enroll, 1.7(0.6-5.0). Late-participation was inversely related to study compliance for teens and caregivers, ORs ranging from 0.1 to 0.3 (all p-values < 0.01). Early- and late-participants required 0.45 FTEs/100 and 3.3 FTEs/100, respectively.</p> <p>Conclusions</p> <p>Recruitment messages attracted youth with moderate-to-severe asthma, but extending enrollment was costly, resulting in potentially less motivated, and certainly less compliant, participants. Investigators must balance internal versus external validity in the decision to extend recruitment. Gains in sample size and external validity may be offset by the cost of additional staff time and the threat to internal validity caused by lower participant follow-up.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00201058">NCT00201058</a></p