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Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of ( R )- or ( S )-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2'-bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel-Crafts reaction with SCP_Q111BpyAla achieving the best selectivity with an enantioselectivity of 72% ee ( S ). Interestingly, despite using the same protein scaffold, different attachment strategies for Bpy at the same residue (Q111) led to a switch in the enantiopreference of the ArM. X-ray crystal structures of SCP_Q111CBpy and SCP_Q111BpyAla ArMs with bound Cu(ii) ions unveiled crucial differences in the orientation of the catalytic centre. Combining structural information, alanine scanning studies, and computational analysis shed light on the distinct active sites of the ArMs, clarifying that these active sites stabilise the nucleophilic substrate on different sides of the electrophile leading to the observed switch in enantioselectivity. This work underscores the importance of integrating structural studies with catalytic screening to unravel the intricacies of ArM behaviour and facilitate their development for targeted applications in biocatalysis., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Tyrosyl DNA phosphodiesterase (TDP1) is a DNA repair enzyme that hydrolyzes the phosphotyrosyl linkage between 3'-DNA-protein crosslinks such as stalled topoisomerase 1 cleavage complexes (Top1cc). Here, we present a fluorescence-resonance-energy-transfer-(FRET) based assay to estimate modulation of TDP1 activity through arginine methylation. We describe steps for TDP1 expression and purification and estimating TDP1 activity using fluorescence-quenched probes mimicking Top1cc. We then detail data analysis of real-time TDP1 activity and screening of TDP1-selective inhibitors. For complete details on the use and execution of this protocol, please refer to Bhattacharjee et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Diffuse correlation spectroscopy (DCS) is an optical technique that allows for the non-invasive measurement of blood flow. Recent work has shown that utilizing longer wavelengths beyond the traditional NIR range provides a significant improvement to signal-to-noise ratio (SNR). However, current detectors both sensitive to longer wavelengths and suitable for clinical applications (InGaAs/InP SPADs) suffer from suboptimal afterpulsing and dark noise characteristics. To overcome these barriers, we introduce a cross correlation method to more accurately recover blood flow information using InGaAs/InP SPADs., Methods: Two InGaAs/InP SPAD detectors were used for during in vitro and in vivo DCS measurements. Cross correlation of the photon streams from each detector was performed to calculate the correlation function. Detector operating parameters were varied to determine parameters which maximized measurement SNR.State-space modeling was performed to determine the detector characteristics at each operating point., Results: Evaluation of detector characteristics was performed across the range of operating conditions. Modeling the effects of the detector noise on the correlation function provided a method to correct the distortion of the correlation curve, yielding accurate recovery of flow information as confirmed by a reference detector., Conclusion: Through a combination of cross-correlation of the signals from two detectors, model-based characterization of detector response, and optimization of detector operating parameters, the method allows for the accurate estimation of the true blood flow index., Significance: This work presents a method by which DCS can be performed at longer NIR wavelengths with existing detector technology, taking advantage of the increased SNR.

Science, Technology, Engineering and Mathematics (STEM) disciplines recognize the need for leadership development, but the lack of a professionally endorsed model has led to a patchwork of programmes across the nation, each with its unique brand of skills development. Leadership programmes in six diverse STEM fields are included., (© 2022 Wiley Periodicals, LLC.)

Introduction: Self-monitoring of blood glucose (BG) is important in diabetes management, allowing people with diabetes (PWD) to assess responses to diabetes therapy and to inform if they are attaining their glycemic targets. This study assessed the accuracy and user performance (UP) of a new blood glucose monitoring system (BGMS), CONTOUR®PLUS ELITE, according to International Organization for Standardization (ISO) 15197:2013 criteria and also more stringent criteria., Methods: In laboratory Study 1, capillary fingertip blood samples from 100 PWD were evaluated using the new BGMS. In clinical Study 2, 130 PWD had Yellow Springs Instrument (YSI) analyzer reference measurements against subject-obtained fingertip and palm blood, and trial staff-obtained venous blood. The new BGMS was tested with test strips from three different lots. A UP questionnaire assessed ease of use., Results: Study 1: 100% of combined accuracy results fulfilled ISO criteria (±15 mg/dL at BG <100 mg/dL; ±15% at BG ≥100 mg/dL); 99.8% fulfilled more stringent criteria (±10 mg/dL at BG <100 mg/dL; ±10% at BG ≥100 mg/dL). Error grid analysis showed that 100% of results were within zone A. Study 2: >98% of subject- and 100% of trial staff-obtained performance results met ISO criteria. Most subjects (>96%) found the BGMS easy to use., Conclusion: The new BGMS exceeded minimum ISO 15197:2013-specified standards for both accuracy and UP criteria, along with the more stringent accuracy criteria. These data show that this new BGMS can be a useful tool in managing glycemic control for PWD.

The self-renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site-specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF-independent self-renewal. In contrast, phosphomimetic substitution of S131 enhances LIF-independent self-renewal, without influencing DNA binding. Modelling the DNA-homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self-renewal., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Animal studies are essential to biomedical research and the cornerstone is a reproducible animal model. While there are many reports on rodent peripheral nerve injury models, a large animal model is essential to confirm the effects of nerve regeneration over the longer distances of regeneration required in humans. Swine have long been used as a large animal model for other surgical and biomedical studies. This paper represents a novel neurovascular injury model in the Sus scrofa domesticus swine (American Yorkshire pig). This paper will describe our experience and recommendations with pre-operative, operative and post-operative protocols and our refinements to produce an effective model., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Objective: To compare time efficiency and nociceptive input between digital strumming (DS) and sharp transection (ST) of the suspensory ligament during ovariohysterectomy (OVH)., Study Design: Randomized controlled trial., Animals: 30 adult female dogs., Methods: Dogs were randomly assigned to ST or DS procedures. Measures of nociception were assessed through measurements of preoperative and intraoperative heart rate during manipulation of the suspensory ligament. Measures of pain were assessed through preoperative and postoperative pain scores by using the short form Glasgow Composite Pain Scale. Time efficiency was measured through total surgical time and the time to release each suspensory ligament., Results: After body weight was accounted for, the total surgical time was 1.1 minutes (P = .06) faster for ST than for DS, and each additional kilogram of body weight increased total surgical time by 0.1 minutes (P = .02). Digital strumming had 30.6-fold greater odds of taking greater than 1 minute compared with ST (P = .001). The heart rate from baseline to peak was 7.4 beats per minute lower in the ST group than in the DS group (P = .06). No complications were observed, and there was no difference in postoperative pain scores between treatments., Conclusion: Sharp transection was faster and generated less intraoperative acceleration in heart rate but did not differ in postoperative outcomes compared with DS., Clinical Significance: Sharp transection is a viable alternative to DS for breakdown of the suspensory ligament during canine OVH. Sharp transection may improve surgical efficiency, especially when performing large volumes in the spay/neuter setting and could influence veterinary student training., (© 2018 The American College of Veterinary Surgeons.)

The high rate of new HIV infections, particularly in Sub-Saharan Africa, emphasizes the need for a safe and effective vaccine to prevent acquired immunodeficiency syndrome (AIDS). To date, the only HIV vaccine trial that has exhibited protective efficacy in humans was the RV144 study completed in Thailand. The finding that protection correlated with antibodies to gp120 suggested that increasing the quality or magnitude of the antibody response that recognize gp120 might improve the modest yet significant protection (31.2%) achieved with this immunization regimen. However, the large-scale production of rgp120 suitable for clinical trials has been challenging due, in part, to low productivity and difficulties in purification. Moreover, the antigens that are currently available were produced largely by the same technology used in the early 1990s and fail to incorporate unique carbohydrates presented on HIV virions required for the binding of several major families of broadly neutralizing antibodies (bNAbs). Here we describe the development of a high-yielding CHO cell line expressing rgp120 from a clade C isolate (TZ97008), representative of the predominant circulating HIV subtype in Southern Africa and Southeast Asia. This cell line, produced using robotic selection, expresses high levels (1.2 g/L) of the TZ97008 rgp120 antigen that incorporates oligomannose glycans required for binding to multiple glycan dependent bNAbs. The resulting rgp120 displays a lower degree of net charge and glycoform heterogeneity as compared to rgp120s produced in normal CHO cells. This homogeneity in net charge facilitates purification by filtration and ion exchange chromatography methods, eliminating the need for expensive custom-made lectin, or immunoaffinity columns. The results described herein document the availability of a novel cell line for the large-scale production of clade C gp120 for clinical trials. Finally, the strategy used to produce a TZ97008 gp120 in the MGAT - CHO cell line can be applied to the production of other candidate HIV vaccines.

Objective: To examine psychosocial mediators of the effect of a culturally tailored dietary intervention on dietary change among Hispanic/Latina breast cancer survivors., Methods: Hispanic/Latina breast cancer survivors (n = 70) were randomized to receive either a 12-week theory-based and culturally tailored dietary change program (intervention group, n = 34), or standard-of-care printed recommendations (control group, n = 36) (ClinicalTrials.gov NCT01414062). Fruit/vegetable intake (F/V), % calories from fat, and hypothesized psychosocial mediators were assessed at baseline, 6 and 12 months. Analysis of covariance assessed intervention effects on psychosocial mediators at 6 and 12 months. Mediation analysis using the bootstrap method evaluated the indirect intervention effects on dietary intake at 6 and 12 months through changes in psychosocial mediators at 6 and 12 months., Results: Compared with controls, at 6 and 12 months, the intervention group reported greater improvements in stages of change (P < .001, P < .001, respectively), self-efficacy (P = .009, P = .002, respectively), snack preference for F/snack preference for F/V (P = .045, P = .002, respectively); at 12 months, the intervention group reported a decrease in chance-oriented external locus of control (P = .02). At 6 months, mediation analysis showed that the intervention effect was associated with an increase of 1.0 (95% CI, -0.1-2.4) serving/day of F/V, compared with the control group, although no indirect effect through the hypothesized psychosocial mediators was observed. At 12 months, the intervention was associated with an increase in 0.5 serving/day F/V through improved taste/snack preference for F/V at 6 and 12 months (95% CIs, 0.1-1.3, 0.0-1.4, respectively)., Conclusions: Future programs can target improving taste/snack preference for F/V to promote dietary change in Hispanic/Latina breast cancer survivors., (© 2018 John Wiley & Sons, Ltd.)

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation., (© 2017 Evrony et al.; Published by Cold Spring Harbor Laboratory Press.)

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The periwinkle, Littorina sitkana , is found throughout the intertidal zone, often in isolated subpopulations. The majority of trematode parasites use snails as intermediate hosts, and decreased survivorship is often observed in snails infected with trematodes. Sampling L. sitkana from four sites in Barkley Sound, British Columbia, Canada, we test the effects of parasitic infection on snail survival using maximum likelihood and Bayesian approaches using the software MARK and WinBUGS. We found that survival of periwinkles and trematode community composition differed among sites, but survival and trematode prevalence were uncorrelated. WinBUGS performed better than MARK in two ways: (1) by allowing the use of information on known mortality, thus preventing survival overestimation; and (2) by giving more stable estimates while testing the effect of body size on snail survival. Our results suggest that snail survival depends heavily on local environmental factors that may vary greatly within a small geographical region. These findings are important because the majority of experimental studies on survival are done on snails from a single location.

Background: Among Hispanic breast cancer survivors, we examined the long-term effects of a short-term culturally based dietary intervention on increasing fruits/vegetables (F/V), decreasing fat, and changing biomarkers associated with breast cancer recurrence risk., Methods: Spanish-speaking women (n = 70) with a history of stage 0-III breast cancer who completed treatment were randomized to ¡Cocinar Para Su Salud! (n = 34), a culturally based 9-session program (24 hours over 12 weeks, including nutrition education, cooking classes, and food-shopping field trips), or a control group (n = 36, written dietary recommendations for breast cancer survivors). Diet recalls, fasting blood, and anthropometric measures were collected at baseline, 6, and 12 months. We report changes between groups at 12 months in dietary intake and biomarkers using 2-sample Wilcoxon t tests and generalized estimating equation (GEE) models., Results: At 12 months, the intervention group compared with the control group reported higher increases in mean daily F/V servings (total: +2.0 vs. -0.4; P < 0.01), and nonsignificant decreases in the percentage of calories from fat (-2.2% vs. -1.1%; P = 0.69) and weight (-2.6 kg vs. -1.5 kg; P = 0.56). Compared with controls, participants in the intervention group had higher increases in plasma lutein (+20.4% vs. -11.5%; P < 0.01), and borderline significant increases in global DNA methylation (+0.8% vs. -0.5%; P = 0.06)., Conclusions: The short-term ¡Cocinar Para Su Salud! program was effective at increasing long-term F/V intake in Hispanic breast cancer survivors and changed biomarkers associated with breast cancer recurrence risk., Impact: It is possible for short-term behavioral interventions to have long-term effects on behaviors and biomarkers in minority cancer patient populations. Results can inform future study designs. Cancer Epidemiol Biomarkers Prev; 25(11); 1491-502. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.

Sex-determining systems are remarkably diverse and may evolve rapidly. Polygenic sex-determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male- and female-biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus., (© 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.)

We have investigated the various morphological changes of helical nanofilament (HNF; B4) phases in multiscale nanochannels made of porous anodic aluminum oxide (AAO) film. Single or multihelical structures could be manipulated depending on the AAO pore size and the higher-temperature phase of each molecule. Furthermore, the nanostructures of HNFs affected by the chemical affinity between the molecule and surface were drastically controlled in surface-modified nanochannels. These well-controlled hierarchical helical structures that have multidimensions can be a promising tool for the manipulation of chiral pores or the nonlinear optical applications.

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3' base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3' end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Helical nanofilaments (HNFs) have attracted much interest because of their unique optical properties, but there have been many hurdles to overcome in using them for the practical applications due to their structural complexity. Here we demonstrate that the molecular configuration and layer conformation of a modulated HNF (HNFs(mod)) can be studied using a physicochemical confinement system. The layer directions affected by the chemical affinity between the mesogen and surface were drastically controlled in surface-modified nanochannels. Furthermore, an in situ experiment using grazing-incidence X-ray diffraction (GIXD) was carried out to investigate in detail the structural evolution through thermal transitions. The results demonstrate that the HNF(mod) structure can be perfectly controlled for functional HNF device applications, and a combined system with chemical and physical confinement effects will be helpful to better understand the fundamentals of soft matter.

Background: There is a need for culturally relevant nutrition programs targeted to underserved cancer survivors., Objective: Our aim was to examine the effect of a culturally based approach to dietary change on increasing fruit/vegetable (F/V) intake and decreasing fat intake among Hispanic breast cancer survivors., Design: Participants were randomized to Intervention and Control groups. Diet recalls, detailed interviews, fasting blood, and anthropometric measures were collected at baseline, 3, 6, and 12 months., Participants/setting: Hispanic women (n=70) with stage 0 to III breast cancer who completed adjuvant treatment and lived in New York City were randomized between April 2011 and March 2012., Intervention: The Intervention group (n=34) participated in ¡Cocinar Para Su Salud!, a culturally based nine-session (24 hours over 12 weeks) intervention including nutrition education, cooking classes, and food-shopping field trips. The Control group (n=36) received written dietary recommendations for breast cancer survivors., Main Outcome Measures: Change at 6 months in daily F/V servings and percent calories from total fat were the main outcome measures., Statistical Analyses: Linear regression models adjusted for stratification factors and estimated marginal means were used to compare changes in diet from baseline to 3 and 6 months., Results: Baseline characteristics were the following: mean age 56.6 years (standard deviation 9.7 years), mean time since diagnosis 3.4 years (standard deviation 2.7 years), mean body mass index (calculated as kg/m²) 30.9 (standard deviation 6.0), 62.9% with annual household income ≤$15,000, mean daily servings of all F/V was 5.3 (targeted F/V 3.7 servings excluding legumes/juices/starchy vegetables/fried foods), and 27.7% of daily calories from fat. More than 60% in the Intervention group attended seven or more of nine classes, with overall study retention of 87% retention at 6 months. At month 6, the Intervention group compared with Control group reported an increase in mean servings of F/V from baseline (all F/V: +2.0 vs -0.1; P=0.005; targeted F/V: +2.7 vs +0.5; P=0.002) and a nonsignificant decrease in percent calories from fat (-7.5% vs -4.4%; P=0.23) and weight (-2.5 kg vs +3.8 kg; P=0.22)., Conclusions: ¡Cocinar Para Su Salud! was effective at increasing short-term F/V intake in a diverse population of Hispanic breast cancer survivors., (Copyright © 2015 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals the route of an intact DNA strand through the transposase active site before second strand cleavage. The crossed architecture of this pre-second strand cleavage paired-end complex supports our proposal that second strand cleavage occurs in trans. The conserved mariner transposase WVPHEL and YSPDL motifs position the strand for accurate DNA cleavage. Base-specific recognition of the flanking DNA by conserved amino acids is revealed, defining a new role for the WVPHEL motif in mariner transposition and providing a molecular explanation for in vitro mutagenesis data. Comparison of the pre-TS cleavage and post-cleavage Mos1 transpososomes with structures of Prototype Foamy Virus intasomes suggests a binding mode for target DNA prior to Mos1 transposon integration., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polygenic sex determination (PSD) is relatively rare and theoretically evolutionary unstable, yet has been reported across a range of taxa. Evidence for multilocus PSD is provided by (i) large between-family variance in sex ratio, (ii) paternal and maternal effects on family sex ratio and (iii) response to selection for family sex ratio. This study tests the polygenic hypothesis of sex determination in the harpacticoid copepod Tigriopus californicus using the criterion of response to selection. We report the first multigenerational quantitative evidence that clutch sex ratio responds to artificial selection in both directions (selection for male- and female-biased families) and in multiple populations of T. californicus. In the five of six lines that showed a response to selection, realized heritability estimated by multigenerational analysis ranged from 0.24 to 0.58. Divergence of clutch sex ratio between selection lines is rapid, with response to selection detectable within the first four generations of selection., (© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg(2+) or Mn(2+), without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg(2+) or Mn(2+) ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.

Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg(2+) ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.

Objective: We use a case-crossover analysis to explore the association between incident cardiovascular events and residential relocation to a new home address., Methods: We conducted an ambidirectional case-crossover analysis to explore the association between incident cardiovascular events and residential relocation to a new address using data from the Cardiovascular Health Study (CHS), a community-based prospective cohort study of 5,888 older adults from four U.S. sites beginning in 1989. Relocation was assessed twice a year during follow-up. Event occurrences were classified as present or absent for the period preceding the first reported move, as compared with an equal length of time immediately prior to and following this period., Results: Older adults (65+) that experience incident cardiovascular disease had an increased probability of reporting a change of residence during the following year (OR 1.6, 95% confidence interval (CI) = 1.2-2.1). Clinical conditions associated with relocation included stroke (OR: 2.0, 95% CI: 1.2-3.3), angina (OR: 1.6, 95% CI: 1.0-2.6), and congestive heart failure (OR: 1.5, 95% CI: 1.0-2.1)., Conclusions: Major incident cardiovascular disease may increase the probability of residential relocation in older adults. Case-crossover analyses represent an opportunity to investigate triggering events, but finer temporal resolution would be crucial for future research on residential relocations.

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

The parasitic protozoan organism Trypanosoma cruzi is the causative agent of Chagas disease. The insect vector-dwelling epimastigote form of the organism expresses a low abundance glycoprotein associated with the flagellum adhesion zone, called gp72. The gp72 glycoprotein was first identified with an anti-carbohydrate IgG3 monoclonal antibody called WIC29.26 and has been shown to have an unusual sugar composition. Here, we describe a new way to isolate the WIC29.26 carbohydrate epitope of gp72. Using (1)H NMR and mass spectrometry before and after derivatization, we provide an almost complete primary chemical structure for the epitope, which is that of a complex phosphosaccharide: Galfβ1-4Rhapα1-2Fucpα1-4(Galpβ1-3)(Galpα1-2)Xylpβ1-4Xylpβ1-3(Xylpβ1-2Galpα1-4(Galpβ1-3)(Rhapα1-2)Fucpα1-4)GlcNAcp, with phosphate attached to one or other of the two Galp terminal residues and in which all residues are of the d-absolute configuration, except for fucose and rhamnose which are l. Combined with previous data (Haynes, P. A., Ferguson, M. A., and Cross, G. A. (1996) Glycobiology 6, 869-878), we postulate that this complex structure and its variants lacking one or more residues are linked to Thr and Ser residues in gp72 via a phosphodiester linkage (GlcNAcpα1-P-Thr/Ser) and that these units may form phosphosaccharide repeats through GlcNAcpα1-P-Galp linkages. The gp72 glycoprotein is associated with the flagellum adhesion zone on the parasite surface, and its ligation has been implicated in inhibiting parasite differentiation from the epimastigote to the metacyclic trypomastigote stage. The detailed structure of the unique phosphosaccharide component of gp72 reported here provides a template for future biosynthetic and functional studies.

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system., (© 2013 International Society for Neurochemistry.)

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.

We used the Integrated Microbial Genomes Annotation Collaboration Toolkit as a framework to incorporate microbial genomics research into a microbiology and biochemistry course in a way that promoted student learning of bioinformatics and research skills and emphasized teamwork and collaboration as evidenced through multiple assessment mechanisms. Student teams in microbiology used bioinformatics tools to identify and characterize gene products from Mucilaginibacter paludis necessary for the synthesis of specific amino acids and then designed and carried out growth experiments to determine if the organism could indeed synthesize the amino acids. Students in biochemistry worked to characterize one of the amino acid biosynthetic pathways reconstructed by a previous microbiology class through amplification and cloning of the M. paludis genes and complementation analysis of Escherichia coli mutants., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

The emergence of amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd) has led to the decline and extinction of numerous amphibian species. Multiple studies have observed links between climatic factors and amphibian declines apparently caused by Bd. Using outdoor experimental mesocosms, we tested the response of red-legged frog (Rana aurora) tadpoles to increased variation in temperature, a component of climate linked to amphibian declines, and Bd exposure. We included tadpoles of a sympatric competitor species, Pacific chorus frog (Pseudacris regilla), in a fully factorial design to test the effects of Bd and temperature on interspecific interactions. We found that higher variation in temperature had numerous effects in mesocosms, including interacting with Bd presence to decrease the condition of R. aurora, shifting the relative performance of competing P. regilla and R. aurora, and accelerating the development of P. regilla relative to R. aurora. Our results demonstrate that increased variation in temperature can affect amphibians in multiple ways that will be contingent on ecological context, including the presence of Bd and competing species.

Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer's disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PTB fragment reveals that the linker region forms a short α-helix that folds back onto the PTB domain and sterically hinders APP binding. This intramolecular interaction is disrupted by mutation of Tyr633 within the Mint1 autoinhibitory helix leading to enhanced APP binding and β-amyloid production. Our findings suggest that an autoinhibitory mechanism in Mint1 is important for regulating APP processing and may provide novel therapies for Alzheimer's disease.

Background: Because of local political circumstances, in 1996, the local public health department in Amarillo, Texas, divested itself of almost all personal health services and chose to retain only essential population-based public health services., Methods: We analyzed function, funding, and staffing for various health department activities in FY 1997 and again in FY 2007. The figures were adjusted for inflation and population growth. We interviewed key personnel about the motivation and effects of the changes that occurred with this 10-year period., Results: The local health department both transferred and reassumed some personal health services during this period. This was primarily in the area of immunization services and care for special population such as refugees. Public health preparedness also became a significant new area of activity. Most personal health services provided by the health department before 1996 remained the function of other health care entities in the community. When adjusted for inflation and population growth, most of the growth in the health department's personnel and budget was the result of state and federally mandated program changes., Conclusions: Growth in this local health department, which was committed to provide only essential health services, was driven primarily by state and federally mandated programs. Real growth for essential public health services did not occur over a 10-year period.

Organisms are exposed to strong selective pressures from several sources, including predators and pathogens. Response to such interacting selective pressures may vary among species that differ in life history and ecology in predictable ways. We consider the impact of multiple enemies (fish predators and trematode parasites) on the behavior of larvae of three anuran species (Lithobates (=Rana) sylvaticus, L. clamitans and L. catesbeianus). We show that the three ranid species differ in response to the trade-off imposed by the simultaneous presence of fish predators and trematode parasites in the environment. Two more permanent pond breeders (L. clamitans and L. catesbeianus), which commonly encounter parasites and fish, increased activity when in the combined presence of parasites and a fish predator, resulting in a relatively lower parasite encystment rate. In contrast, the temporary pond breeder (L. sylvaticus), which does not commonly encounter fish in the wild, decreased activity in the combined presence of a fish predator and parasites similar to when only the predator was present. For L. sylvaticus, this suggests that the presence of an unknown predator poses a greater threat than parasites. Further, the presence of fish along with parasites increased the susceptibility of both L. sylvaticus and L. clamitans to trematode infection, whereas parasite infection in L. catesbeianus was unaffected by the presence of fish. Unpalatability to fish may allow some species to respond more freely to attacking parasites in the presence of fish. The results from this study highlight the importance of considering multiple selective pressures faced by organisms and how this shapes their behavior.

The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1-3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1-4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.

Rationale: Behavior occurring during cocaine self-administration can be classified as either consummatory or appetitive. These two concepts are usually addressed independently using separate reinforcement schedules. For example, appetitive behavior can be assessed with a progressive ratio schedule, whereas consummatory behavior is typically measured using a fixed ratio schedule., Objectives: Depending on the schedule used, it is often difficult to determine whether a particular drug pretreatment is affecting self-administration through an effect on appetitive responding, consummatory responding, or perhaps both. In the present study, we tested the effect of pretreating rats with four different drugs on appetitive and consummatory behaviors., Materials and Methods: We recently developed a technique that provides an independent assessment of both behavioral concepts within the same experimental session. In this threshold procedure, rats are offered a descending series of 11 unit doses (422-1.3 μg/injection) during consecutive timed intervals under a fixed-ratio schedule. Consummatory behavior can be analyzed by assessing intake at high unit doses; an estimate of appetitive responding can be determined from responding occurring at the threshold dose. Applying behavioral economics to these data provides dependent measures of consumption when minimally constrained by price and the maximal price paid (P (max)) for cocaine., Results: Haloperidol increased cocaine consumption when minimally constrained by price but decreased P (max). In contrast, D: -amphetamine increased P (max). Fluoxetine decreased P (max) and consumption when minimally constrained by price. Baclofen selectively decreased P (max)., Conclusions: These data suggest that drug pretreatments can alter consummatory and appetitive behavior differently because each concept involves distinct neural mechanisms.

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

Ectothermic vertebrates, such as reptiles, thermoregulate behaviorally by choosing from available temperatures in their environment. As neonates, bearded dragons (Pogona vitticeps) are often observed to aggregate in vertical strata. A proximate mechanism for this behavior is the thermal advantage of heat storage (i.e., grouped lizards benefit through a decreased surface area to volume ratio), although competition for limited thermal resources, or aggregation for social reasons are alternative explanations. This study was designed to gain an understanding of how aggregation and thermoregulation interact. We observed that both isolated and grouped individuals achieved a similar level of thermoregulation (mean T(b) over trial) within a thermal gradient, but that individuals within a group had lower thermoregulatory precision. An experimental design in which light and ambient temperature (T(a)) (20 versus 30 degrees C) were altered established that a light bulb (source of heat) was a limited and valuable resource to both isolated and grouped neonatal lizards. Lizards aggregated more when the light was on at both temperatures, suggesting that individuals were equally attracted to or repelled from the heat source, depending on the ambient temperature. These data suggest aggregation occurs in neonatal bearded dragons through mutual attraction to a common resource. Further, increased variability in thermal preference occurs in groups, demonstrating the potential for agonistic behaviors to compromise optimal thermoregulation in competitive situations, potentially leading to segregation, rather than aggregation., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

A novel influenza A partly of virus of swine origin (2009 H1N1) emerged this spring, resulting in an influenza pandemic. This pandemic is anticipated to continue into the next influenza season. Given that the 2009 H1N1 and seasonal influenza A appear to be somewhat different in the human populations affected and that two influenza vaccines will be recommended this fall, those who manage long-term care facilities and treat patients in them will be faced with many uncertainties as they approach the 2009/10 influenza season. Ten specific suggestions are offered to those responsible for the care of patients in long-term care facilities regarding the upcoming influenza season. These practical suggestions are the clinical opinions of the authors and do not represent official recommendations of the American Geriatrics Society or any agency.

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.