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Background: Africa is in an orphan-care crisis. In Zimbabwe, where one-fourth of adults are HIV-positive and one-fifth of children are orphans, AIDS and economic decline are straining society's ability to care for orphans within their extended families. Lack of stable care is putting thousands of children at heightened risk of malnourishment, emotional underdevelopment, illiteracy, poverty, sexual exploitation, and HIV infection, endangering the future health of the society they are expected to sustain. Methods: To explore barriers and possible incentives to orphan care, a quantitative cross-sectional survey in rural eastern Zimbabwe asked 371 adults caring for children, including 212 caring for double orphans, about their well-being, needs, resources, and perceptions and experiences of orphan care. Results: Survey responses indicate that: (1) foster caregivers are disproportionately female, older, poor, and without a spouse; (2) 98% of non-foster caregivers are willing to foster orphans, many from outside their kinship network; (3) poverty is the primary barrier to fostering; (4) financial, physical, and emotional stress levels are high among current and potential fosterers; (5) financial need may be greatest in single-orphan AIDS-impoverished households; and (6) struggling families lack external support. Conclusions: Incentives for sustainable orphan care should focus on financial assistance, starting with free schooling, and development of community mechanisms to identify and support children in need, to evaluate and strengthen families' capacity to provide orphan care, and to initiate and support placement outside the family when necessary. [ABSTRACT FROM AUTHOR]

The first-line treatment choice of EGFRIs plus doublet chemotherapy vs. bevacizumab plus doublet chemotherapy remains a topic of interest for patients with left-sided RAS WT mCRC. We conducted a systematic literature review and meta-analysis of clinical trial data published between 2015 and 2024. We evaluated the relative efficacy and safety of first-line EGFRIs plus doublet chemotherapy (FOLFIRI or FOLFOX) vs. bevacizumab plus doublet chemotherapy for patients with RAS WT left-sided mCRC, as well as in all- and right-sided tumors. We identified eight trials with 2624 patients. Five trials reported outcomes by tumor sidedness. In the left-sided population, overall survival (OS) (Hazard Ratio (HR) = 0.80, 95% Confidence Interval (CI): 0.71-0.90) and objective response rate (ORR) (Odds ratio [OR]=1.61, 95% CI: 1.30-1.99) favored EGFRI plus chemotherapy, while no statistically significant differences were observed for progression-free survival (PFS) (HR=0.93, 95% CI: 0.84-1.04) or resection rate (RR). Similar results were found in the all-sided population. In the right-sided population, PFS favored bevacizumab plus chemotherapy (HR=1.45, 95% CI: 1.19-1.78), while no statistically significant differences were observed for OS (HR=1.17, 95% CI: 0.95-1.44), ORR (OR=0.99, 95% CI: 0.69-1.41), and RR. Early tumor shrinkage in the all-sided population favored EGFRI plus chemotherapy (OR=1.72; 95% CI: 1.36-2.17); limited data precluded evaluation by sidedness. Safety was available in 6 trials for all-sided tumors and 1 trial for left-sided tumors, each demonstrating typical class-specific adverse events. This most comprehensive meta-analysis indicates a benefit for first-line EGFRI plus chemotherapy over bevacizumab plus chemotherapy in patients with left-sided RAS WT mCRC., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N. Hooda, M. Suh, H. Reichert, and J. Fryzek are employees of EpidStrategies, A Division of ToxStrategies, LLC. and received a grant from Amgen for this research. N. Hooda has also received research funding from Sanofi, and SpringWorks Therapeutics. M. Rehn, V. Chia, D. Younan, K. Mezzi are employees of Amgen and may hold shares and/or stock options in the company. J. Soeda is an employee of Takeda and may hold shares/and or stock options in the company. F. Rivera has served on advisory boards for Amgen, Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Lilly, Merck Serono, MSD, Roche, Sanofi-Aventis, and Servier. He has received research funding from Amgen, Bayer, Celgene, Lilly, Merck Serono, MSD, Roche, Sanofi-Aventis, and Servier. He has received honoraria for talks/presentations from Amgen, Bayer, Bristol Myers Squibb, Celgene, Lilly, Merck Serono, MSD, Roche, Sanofi-Aventis, and Servier. He has received grant support from Amgen, Bristol Myers Squibb, and MSD. K. Muro has received grants from Amgen, Chugai, Sanofi, Astellas, Eisai, Novartis, Pfizer, MSD, ONO Pharmaceutical, Daiichi Sankyo, Taiho. He has received personal fees from Amgen, MSD, ONO Pharmaceutical, Daiichi Sankyo, Taiho, Takeda, Bristol-Myers Squibb, Eli Lilly, and AstraZeneca, outside the submitted work. K. Shitara has received personal fees for consulting and advisory roles from Bristol Myers Squibb, Takeda, Ono Pharmaceutical, Novartis, Daiichi Sankyo, Amgen, Boehringer Ingelheim, Merck Pharmaceutical, Astellas, Guardant Health Japan, Janssen, AstraZeneca, Zymeworks Biopharmaceuticals, ALX Oncology Inc., and Bayer. He has received honoraria from Bristol-Myers Squibb, Ono Pharmaceutical, Janssen, Eli Lilly, Astellas, and AstraZeneca. He has also received research funding (all to institution) from Astellas, Ono Pharmaceutical, Daiichi Sankyo, Taiho Pharmaceutical, Chugai, Merck Pharmaceutical, Amgen, Eisai, PRA Health Sciences and Syneos Health, outside the submitted work. M. Peeters has served on advisory boards for Amgen, Bayer, Bimini, Ipsen, Merck, MSD, Qurin, Remedus, Sanofi, Sirtex, and Terumo. He has received speaker fees from Amgen, Bayer, Bristol-Myers Squibb, Merck, MSD, Roche, Sanofi, Servier, and Sirtex. He reports scientific grants from Amgen (inst.), Bayer (inst.), Bristol-Myers Squibb (inst.), Ipsen (inst.), Novartis (inst.), and Roche (inst.). S. Stintzing is an employee of Charité – Universitaetsmedizin Berlin. He has served on advisory boards for Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, ESAI, Lilly, Merck KgaA, MSD, Pierre-Fabre, Roche, Sanofi, Servier, Taiho, and Takeda. He has received honoraria for talks/presentations and advisory board participation from Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, ESAI, Leo-Pharma, Lilly, Merck KgaA, MSD, Pierre-Fabre, Roche, Sanofi, Servier, Taiho, and Takeda. He has received research funding from Merck KgaA, Pierre-Fabre, Roche, and Servier. He has educational collaborationS with Medscape Foundation and COR2ED. He is a member of ASCO, ESMO, DKG, AIO, and DHGO. T. Yoshino has received honoraria from Chugai Pharma, Takeda Pharma, Merck, Bayer Yakuhin, Ono Pharmaceutical, and MSD K.K. He has received consulting fees from Sumitomo Corp. and research grants from Amgen, Chugai, Daiichi Sankyo, Eisai, FALCO Biosystems, Genomedia Inc., Molecular Health, MSD, Nippon Boehringer Ingelheim, Ono, Pfizer, Roche Diagnostics, Sanofi, Sysmex, and Taiho. V. Heinemann has received honoraria Amgen, Merck, Roche, Sanofi, SIRTEX, Servier, Pfizer, Pierre-Fabre, Astra-Zeneca, MSD, GSK, and Seagen. He participated in consulting and advisory boards for Merck, Amgen, Roche, Sanofi, SIRTEX, Bristol-Myers Squibb, MSD, Novartis, Boehringer Ingelheim, Servier, Pierre-Fabre, Celgene, Terumo, GSK, Oncosil, Nordic, and Seagen. He reports research funding (institution) from Merck, Amgen, Roche, Sanofi, Pfizer, Boehringer-Ingelheim, Sirtex, Bayer, and Servier. He has received travel support from Merck Roche, Amgen, SIRTEX, Bayer, Servier, MSD, and GSK. All remaining authors have declared no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Spatial memory encoding depends in part on cholinergic modulation. How acetylcholine supports spatial memory encoding is not well understood. Prior studies indicate that acetylcholine release is correlated with exploration, including epochs of rearing onto hind legs. Here, to test whether elevated cholinergic tone increases the probability of rearing, we tracked rearing frequency and duration while optogenetically modulating the activity of choline acetyltransferase containing (i.e., acetylcholine producing) neurons of the medial septum in rats performing a spatial working memory task (n = 17 rats). The cholinergic neurons were optogenetically inhibited using halorhodopsin for the duration that rats occupied two of the four open arms during the study phase of an 8-arm radial arm maze win-shift task. Comparing rats' behaviour in the two arm types showed that rearing frequency was not changed, but the average duration of rearing epochs became significantly longer. This effect on rearing was observed during optogenetic inhibition but not during sham inhibition or in rats that received infusions of a fluorescent reporter virus (i.e., without halorhodopsin; n = 6 rats). Optogenetic inhibition of cholinergic neurons during the pretrial waiting phase had no significant effect on rearing, indicating a context-specificity of the observed effects. These results are significant in that they indicate that cholinergic neuron activity in the medial septum is correlated with rearing not because it motivates an exploratory state but because it contributes to the processing of information acquired while rearing., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Rickettsial pathogens are among the emerging and re-emerging vector-borne zoonoses of public health importance. Reports indicate human exposure to Rickettsial pathogens in Namibia through serological surveys, but there is a lack of data on infection rates in tick vectors, hindering the assessment of the relative risk to humans. Our study sought to screen Ixodid ticks collected from livestock for the presence of Rickettsia species in order to determine infection rates in ticks and to determine the Rickettsia species circulating in the country. We collected and pooled Hyalomma and Rhipicephalus ticks from two adjacent regions of Namibia (Khomas and Otjozondjupa) and observed an overall minimum Rickettsia infection rate of 8.6% (26/304), with an estimated overall pooled prevalence of 9.94% (95% CI: 6.5-14.3). There were no statistically significant differences in the estimated pooled prevalence between the two regions or tick genera. Based on the nucleotide sequence similarity and phylogenetic analysis of the outer membrane protein A (n = 9) and citrate synthase (n = 12) genes, BLAST analysis revealed similarity between Rickettsia africae (n = 2) and Rickettsia aeschlimannii (n = 11), with sequence identities ranging from 98.46 to 100%. Our initial study in Namibia indicates that both zoonotic R. africae and R. aeschlimannii are in circulation in the country, with R. aeschlimannii being the predominant species.

Spatial memory encoding depends in part on cholinergic modulation. How acetylcholine supports spatial memory encoding is not well understood. Prior studies indicate that acetylcholine release is correlated with exploration, including epochs of rearing onto hind legs. Here, to test whether elevated cholinergic tone increases the probability of rearing, we tracked rearing frequency and duration while optogenetically modulating the activity of choline acetyltransferase containing (i.e., acetylcholine producing) neurons of the medial septum in rats performing a spatial working memory task (n = 17 rats). The cholinergic neurons were optogenetically inhibited using halorhodopsin for the duration that rats occupied two of the four open arms during the study phase of an 8-arm radial arm maze win-shift task. Comparing rats' behavior in the two arm types showed that rearing frequency was not changed but the average duration of rearing epochs became significantly longer. This effect on rearing was observed during optogenetic inhibition but not during sham inhibition or in rats that received infusions of a fluorescent reporter virus (i.e., without halorhodopsin; n = 6 rats). Optogenetic inhibition of cholinergic neurons during the pre-trial waiting phase had no significant effect on rearing, indicating a context-specificity of the observed effects. These results are significant in that they indicate that cholinergic neuron activity in the medial septum is correlated with rearing not because it motivates an exploratory state but because it contributes to the processing of information acquired while rearing., Competing Interests: Competing Interests The authors have no competing interests to declare.

In this study, we demonstrate three-dimensional (3D) hollow nanosphere electrocatalysts for CO 2 conversion into formate with excellent H-Cell performance and industrially-relevant current density in a 25 cm 2 membrane electrode assembly electrolyzer device. Varying calcination temperature maximized formate production via optimizing the crystallinity and particle size of the constituent SnO 2 nanoparticles. The best performing SnO 2 nanosphere catalysts contained ~ 7.5 nm nanocrystals and produced 71-81% formate Faradaic efficiency (FE) between -0.9 V and -1.3 V vs. the reversible hydrogen electrode (RHE) at a maximum formate partial current density of 73 ± 2 mA cm geo -2 at -1.3 V vs. RHE. The higher performance of nanosphere catalysts over SnO 2 nanoparticles and commercially-available catalyst could be ascribed to their initial structure providing higher electrochemical surface area and preventing extensive nanocrystal growth during CO 2 reduction. Our results are among the highest performance reported for SnO 2 electrocatalysts in aqueous H-cells. We observed an average 68 ± 8% FE over 35 h of operation with multiple on/off cycles. In situ Raman and time-dependent X-ray diffraction measurements identified metallic Sn as electrocatalytic active sites during long-term operation. Further evaluation in a 25 cm 2 electrolyzer cell demonstrated impressive performance with a sustained current density of 500 mA cm geo -2 and an average 75 ± 6% formate FE over 24 h of operation. Our results provide additional design concepts for boosting the performance of formate-producing catalysts., (© 2022. The Author(s).)

Objective: To assess the effect of the adherence to medical treatment on urinary parameters in the 24-h metabolic study of patients with kidney stones., Methods: A retrospective, longitudinal, descriptive, and observational study was carried out by reviewing the hospital electronic medical record from 2014 to 2018. The adherence to drug treatment was measured 6 months after its initiation, and the numerical values of the metabolic studies were compared. Wilcoxon tests were performed to compare the difference before and after treatment., Results: Ninety patients were evaluated, with 73.3% of adherence. The 180-day overall adherence rate was 61.2% in patients treated with a single drug and 85.4% in patients treated with multiple drugs. There is a statistically significant increase in citrate levels in patients with good adherence in comparison with non-adherent patients ( p =0.031 vs. p =0.528)., Conclusions: Medical treatment and dietary measures in patients with kidney stones have an initial impact at 6 months on the values of the main urinary metabolic alterations that predispose to calculi formation; the most significant is seen in those patients with adherence to medical treatment for hypocitraturia., (© 2021 Editorial Office of Asian Journal of Urology. Production and hosting by Elsevier B.V.)

Understanding the temperature dependence of functional properties in high-temperature gas sensors is vital for applications in combustion environments. Temperature effect on the electronic structure due to electron-phonon coupling is a key property of interest as this influences other responses of sensors. In this work, we assess the impact of temperature on band gap renormalization of pristine and oxygen-vacant LaCrO 3-δ perovskite employing Allen-Heine-Cardona theory with first-principles simulations and corroborate with experimental observation. Antiferromagnetic cubic LaCrO 3 shows a direct ground-state band gap of 2.62 eV that is reduced by over 1 eV due to the presence of oxygen vacancies, which can form endothermically. We find excellent agreement in temperature-dependent band gap shift in LaCrO 3 between theory and an in-house experiment, proving that the theory can adequately predict renormalization on the band gap in a magnetic system. Band gaps in cubic LaCrO 3-δ are found to monotonically narrow by 1.13 eV in pristine and by around 0.62 eV in oxygen-vacant structures as temperature increases from 0 to 1500 K. The predicted band gap variations are rationalized using an analytical model. The experimental zero-temperature band gaps are extracted from the model fits that can provide useful insights on the simulated band gaps.

Introduction: There are limited data regarding the effect of thulium laser (Tm:YAG) and holmium laser (Ho:YAG) on upper urinary tract. The aim of this study was to compare soft tissue effects of these two lasers at various settings, with a focus on incision depth (ID) and coagulation area (CA). Materials and Methods: An ex vivo experimental study was performed in a porcine model. The kidneys were dissected to expose the upper urinary tract and the block samples containing urothelium and renal parenchyma were prepared. The laser fiber, fixed on a robotic arm, perpendicular to the target tissue was used with a 100 W Ho:YAG and a 200 W Tm:YAG. Incisions were made with the laser tip in contact with the urothelium and in continuous movement at a constant speed of 2 mm/s over a length of 1.5 cm. Total energy varied from 5 to 30 W. Incision shape was classified as follows: saccular, triangular, tubular, and irregular. ID, vaporization area (VA), CA, and total laser area (TLA = VA + CA) were evaluated. Statistical analysis was performed using the SPSS V23 package, p -values <0.05 were considered statistically significant. Results: A total of 216 experiments were performed. Incision shapes were saccular (46%), triangular (38%), and irregular (16%) with the Ho:YAG, while they were tubular (89%) and irregular (11%) with the Tm:YAG. ID was significantly deeper with the Ho:YAG ( p  = 0.024), while CA and TLA were larger with the Tm:YAG ( p  < 0.001 and p  < 0.005). Conclusion: ID was deeper with Ho:YAG, whereas CA and TLA were larger with the Tm:YAG. Considering surgical principles for endoscopic ablation of upper tract urothelial carcinoma, these results suggest that Tm:YAG may have a lower risk profile (less depth of incision) while also being more efficient at tissue destruction. Future in vivo studies are necessary to corroborate these findings.

Fabrication of metal-organic framework (MOF) thin films over macroscopic surface areas is a subject of great interest for gas sensor application platforms such as optics and microelectronics. However, a direct synthesis of MOF films at ambient conditions, in particular pillared-layer MOF films due to their anisotropic structures, remains a significant challenge. Herein, we demonstrate for the first time a facile construction of dense and continuous pillared-layer MOF thin films on a centimeter scale via an aluminum-doped zinc oxide template and hydroxy double salt (HDS) intermediates at room temperature. A series of Cu(II)-based pillared MOFs with different 1,4-benzenedicarboxylic acid (bdc) ligands were explored for optimizing MOF film formation for CO 2 sensor applications. Nonpolar ligands with lower water solubility preferentially formed crystalline pillared MOF structures from HDS intermediates. A Cu 2 (ndc) 2 (dabco) (ndc = 1,4-naphthalene-bdc; dabco = 1,4-diazabicyclo[2.2.2]octane) MOF demonstrated the most dense and uniform film growth with micrometer thickness over one square centimeter area. This synthetic approach for growing Cu 2 (ndc) 2 (dabco) MOF thin films was successfully translated toward two sensing platforms: a quartz crystal microbalance and an optical fiber sensor. These Cu 2 (ndc) 2 (dabco) MOF-coated sensors displayed sensitivity toward CO 2 and response/recovery time on the scale of seconds, even at moderate humidity levels. This work provides a road map for producing continuous and anisotropic crystalline MOF thin films over a centimeter scale area on various substrates, which will greatly facilitate their utilization in MOF-based sensor devices, among other applications.

Pelletization of basic immobilized amine sorbent (BIAS) particles is required to improve their mechanical strength and facilitate their practical CO 2 capture application under fixed or dynamic reactor conditions. Herein, we utilized two methods to prepare amine-functionalized BIAS pellets. Method (ii-a) involved combining latex polychloroprene (PC)/polyamine solutions with fly ash (FA)/BIAS powder to form sorbent pellets. Alternatively, method (ii-b) entailed shaping and drying wet pastes of binder solution plus FA/SiO 2 powder into pellet supports. These supports were then functionalized with leach-resistant polyethylenimine MW = 800 (PEI 800 )/ N - N -diglycidyl-4-glycidyloxyaniline (tri-epoxide cross-linker, E3) or ethylenamine E100/E3 mixtures. All pellets were screened for CO 2 capture by thermogravimetric analysis (dry 14% CO 2 /N 2 , 55-75 °C), H 2 O stability by accelerated water washing, and mechanical strength by crush and ball-mill attrition testing. The mechanism of superior method (ii-b) pellet formation was uncovered by N 2 physisorption measurements, diffuse reflectance infrared Fourier transform spectroscopy, and scanning electron microscopy. Extended fixed bed testing of optimum E3/PEI 800 -0.13/1 pellets under practical conditions revealed complete CO 2 capture stability of 1.5 mmol CO 2 /g after 48 h of continuous steam exposure (7.2% H 2 O/He, 105 °C) and minimal 14.6% loss in capacity after 75 hours of combined CO 2 capture cycling and steam treating (48 h). This slight oxidative degradation could be alleviated by incorporating a K 2 CO 3 antioxidant into the pellet formulation. Overall, the robust physiochemical properties of the polyamine/cross-linker method (ii-b) pellets confirm their suitability for pilot-scale testing.

Background:: many undergraduate student nurses have the opportunity to undertake an international elective-that is, a placement in another country. The benefits of undertaking an elective include developing cultural competence, understanding different healthcare systems and experiencing a different culture., Aim:: the aim of the study was to evaluate the impact of a faculty-structured international travel elective to Zambia for undergraduate UK nursing students., Methods:: a descriptive phenomenology approach was used to discover and explore the students' experiences of their elective., Findings:: 6 students participated in semi-structured interviews. Themes that emerged included the importance of preparing for the elective, different nursing cultures, realities and patient safety concerns., Conclusion:: this study found that students involved in an international elective to Zambia overall had a positive experience but did report some challenges; the findings contribute to the body of evidence relating to international electives.

Substantial evidence has accumulated linking epigenome change to alterations in stem cell function during postnatal development and aging. Yet much remains to be learned about causal relationships, and large gaps remain in our understanding of epigenome-transcriptome interactions. Here we investigate structural features of large histone H3K27me3-enriched regions in human stem cell-like monocytes and their dendritic cell derivatives, where the H3K27me3 modification is considered to demarcate Polycomb (PcG) domains. Both differentiation- and postnatal development-related change are explored, initially by confirming expected reciprocal relationships between transcript abundance and span of PcG domains overlapping transcribed regions. PcG-associated postnatal transcriptome change specific to the stem cell-like monocytes is found to be incompletely explained by conventional measures of PcG region structure. To address this, we introduce algorithms that quantify local nucleosome-scale conservation of PcG-region topology. It is shown that topology-based comparisons can reveal broad statistical linkage between postnatal gene down-regulation and epigenome remodeling; further, such comparisons provide access to a previously unexplored dimension of epigenome architecture.

In this study, passive detectors for Pb were prepared by immobilizing powdered rock phosphate in agarose beads. Rock phosphate has been used to treat Pb-contaminated waters and soil by fixing the metal as an insoluble pyromorphite mineral. Under lab conditions, Pb was rapidly adsorbed from aqueous solution by the beads over time, consistent with the acidic dissolution of rock phosphate, the precipitation of pyromorphite within the pore space of the agarose gel matrix, and surface exchange reactions. Net accumulation of Pb occurred when beads were exposed to simulated periodic releases of Pb over time. Under field conditions, beads in mesh bags were effective at detecting dissolved Pb being transported as surface runoff from a site highly contaminated with Pb. Rates of Pb accumulation in beads under field conditions appeared to be correlated with the frequency of storm events and total rainfall. The rock phosphate agarose bead approach could be an inexpensive way to carry out source-tracking of Pb pollution, to verify the successful remediation of sites with Pb-contaminated soil, and to routinely monitor public water systems for potential Pb contamination., (Published by Elsevier B.V.)

Strain imaging is a sensitive marker of myocardial dysfunction and may be underused in Takotsubo cardiomyopathy (TC). We present a case of biventricular TC in which early improvement in left ventricular longitudinal strain predated subsequent improvement in ejection fraction. Early temporal patterns of strain of the left and right ventricles have not previously been described in TC. Our case illustrates how strain can be a sensitive marker for myocardial dysfunction and recovery in TC. Increased use of strain in TC may have further implications on prognosis and management., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Recovering aqueous rare earth elements (REEs) from domestic water sources is one key strategy to diminish the U.S.'s foreign reliance of these precious commodities. Herein, we synthesized an array of porous, amine-epoxy monolith and particle REE recovery sorbents from different polyamine, namely tetraethylenepentamine, and diepoxide (E2), triepoxide (E3), and tetra-epoxide (E4) monomer combinations via a polymer-induced phase separation (PIPS) method. The polyamines provided -NH 2 (primary amine) plus -NH (secondary amine) REE adsorption sites, which were partially reacted with C-O-C (epoxide) groups at different amine/epoxide ratios to precipitate porous materials that exhibited a wide range of apparent porosities and REE recoveries/affinities. Specifically, polymer particles (ground monoliths) were tested for their recovery of La 3+ , Nd 3+ , Eu 3+ , Dy 3+ , and Yb 3+ (Ln 3+ ) species from ppm-level, model REE solutions (pH ≈ 2.4, 5.5, and 6.4) and a ppb-level, simulated acid mine drainage (AMD) solution (pH ≈ 2.6). Screening the sorbents revealed that E3/TEPA-88 (88% theoretical reaction of -NH 2 plus -NH) recovered, overall, the highest percentage of Ln 3+ species of all particles from model 100 ppm- and 500 ppm-concentrated REE solutions. Water swelling (monoliths) and ex situ, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) (ground monoliths/particles) data revealed the high REE uptake by the optimized particles was facilitated by effective distribution of amine and hydroxyl groups within a porous, phase-separated polymer network. In situ DRIFTS results clarified that phase separation, in part, resulted from polymerization of the TEPA-E3 (N-N-diglycidyl-4-glycidyloxyaniline) species in the porogen via C-N bond formation, especially at higher temperatures. Most importantly, the E3/TEPA-88 material cyclically recovered >93% of ppb-level Ln 3+ species from AMD solution in a recovery-strip-recovery scheme, highlighting the efficacy of these materials for practical applications.

This report describes a school-based screening project to improve early identification of children at risk for attention-deficit/hyperactivity disorder (ADHD) and communicate these concerns to parents, recommending that they contact their child's primary care provider (PCP). Of 17,440 eligible children in first through fifth grades in five school districts, 47.0% of parents provided required written consent, and teachers completed 70.4% of the online screeners (using the Vanderbilt AD/HD Diagnostic Teacher Rating Scale). Of 5,772 screeners completed, 18.1% of children ( n = 1,044) were identified as at risk. Parents of at-risk children were contacted to explain risk status and recommended to visit their child's PCP for further evaluation. It was not possible to contact 39.1% of parents of at-risk children. Of the 636 parents of at-risk children who could be contacted, 53.1% ( n = 338) verbally accepted the recommendation to follow-up with their PCP, which was not related to ADHD symptom severity. Parents of children with IEPs or related services were more likely to accept the recommendation to visit the PCP. Our exploration of the potential for school-based screening for ADHD identified a number of barriers to successful execution, but the data also indicated that this is an important problem to address.

Diminished ovarian function occurs early and is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated the roles that genome and epigenome structure play in age-related changes in gene expression and ovarian function, using human ovarian granulosa cells as an experimental system. DNA methylomes were compared between two groups of women with distinct age-related differences in ovarian functions, using both Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) and Reduced Representation Bisulfite Sequencing (RRBS); their transcriptomes were investigated using mRNA-seq. Significant, non-random changes in transcriptome and DNA methylome features are observed in human ovarian granulosa cells as women age and their ovarian functions deteriorate. The strongest correlations between methylation and the age-related changes in gene expression are not confined to the promoter region; rather, high densities of hypomethylated CpG-rich regions spanning the gene body are preferentially associated with gene down-regulation. This association is further enhanced where CpG regions are localized near the 3'-end of the gene. Such features characterize several genes crucial in age-related decline in ovarian function, most notably the AMH (Anti-Müllerian Hormone) gene. The genome-wide correlation between the density of hypomethylated intragenic and 3'-end regions and gene expression suggests previously unexplored mechanisms linking epigenome structure to age-related physiology and pathology.

TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB-TFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed 'bootprints'. Global analysis reveals that the TFIIIB-TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.

Context: Nattokinase is a serine protease and is derived from natto, a traditional Japanese, fermented, soybean food meal. Multiple authors have described the significant fibrinolytic, antithrombotic, and antihypertensive effects of natto. Nattokinase has been growing in popularity for use as a dietary supplement for the benefit of cardiovascular health. Little is known regarding the pharmacokinetic and pharmacodynamic properties of this enzyme, and the bioavailability of nattokinase is currently unknown., Objective: This study intended to (1) detect nattokinase directly and immunologically, (2) show that nattokinase and/or its metabolites were detectable in human blood following ingestion of a commercial preparation, and (3) chart a pharmacokinetic dosing effect for nattokinase., Design: The research team designed the pilot study as an in vivo, human clinical trial. Healthy human subjects responded to an advertisement and were screened. Subjects who satisfied both inclusion and exclusion criteria were enrolled into the study. Subjects were then instructed to orally ingest a single capsule containing a known concentration of nattokinase immediately following a baseline blood draw. Subsequent blood draws occurred over a 24-h period., Setting: This study was conducted in Oakland, California, at a clinical reference laboratory and was performed with the approval of an institutional review board (IRB) to ensure that appropriate ethical standards were met., Participants: Eleven healthy participants (five male, six female, ages 21-65), who met eligibility criteria, were enrolled., Intervention(s): Administration of nattokinase occurred orally with the ingestion of a single daily dose (2000 FU) of nattokinase. Capsules, each containing approximately 100 mg of nattokinase, in softgel form (NSK-SD, Japan Bio Science Laboratory, Osaka, Japan), were used in the study., Outcome Measure(s): Baseline blood samples were collected, and participants were observed swallowing a single capsule of the nattokinase supplement before returning at 2, 4, 8, 12, 24, and 48 h post ingestion for subsequent blood draws. The presence of nattokinase in serum was measured by an enzyme-linked immunosorbent assay (ELISA), using a rabbit, polyclonal, antinattokinase-capture antibody. A pharmacokinetic pattern was observed for nattokinase between baseline and 48 h postdose., Results: Peak serum levels of nattokinase were observed at approximately 13.3 h ± 2.5 h (mean ± standard error) postdose. Statistically significant increases in binding were detectable from baseline when comparing averaged data at time points t = 2 h-t = 24 h., Conclusions: These results provided the first evidence that nattokinase can be measured directly in the blood of humans. The results further suggest that a larger, more extensive, pharmacokinetic study of nattokinase is warranted. Additionally, looking for intact enzyme and bioactive nattokinase peptides should be a consideration for future studies investigating the pharmacokinetic profile of nattokinase.

Alterations in DNA methylation have been reported to occur during development and aging; however, much remains to be learned regarding post-natal and age-associated epigenome dynamics, and few if any investigations have compared human methylome patterns on a whole genome basis in cells from newborns and adults. The aim of this study was to reveal genomic regions with distinct structure and sequence characteristics that render them subject to dynamic post-natal developmental remodeling or age-related dysregulation of epigenome structure. DNA samples derived from peripheral blood monocytes and in vitro differentiated dendritic cells were analyzed by methylated DNA Immunoprecipitation (MeDIP) or, for selected loci, bisulfite modification, followed by next generation sequencing. Regions of interest that emerged from the analysis included tandem or interspersed-tandem gene sequence repeats (PCDHG, FAM90A, HRNR, ECEL1P2), and genes with strong homology to other family members elsewhere in the genome (FZD1, FZD7 and FGF17). Our results raise the possibility that selected gene sequences with highly homologous copies may serve to facilitate, perhaps even provide a clock-like function for, developmental and age-related epigenome remodeling. If so, this would represent a fundamental feature of genome architecture in higher eukaryotic organisms.

The DNA of eukaryotic cells is packaged into chromatin by histone proteins, which play a central role in regulating access to genetic information. The nucleosome core is the basic structural unit of chromatin: it is composed of an octamer of the four major core histones (two molecules each of H2A, H2B, H3, and H4), around which are wrapped ∼1.75 negative superhelical turns of DNA, a total of 145-147bp. Nucleosome cores are regularly spaced along the DNA in vivo, separated by linker DNA. Nucleosomes are compact structures capable of blocking access to the DNA that they contain. For example, they may prevent the binding of transcription factors to their cognate sites. It is therefore very important to obtain quantitative information on the positions of nucleosomes with respect to regulatory regions in vivo. The advent of high-throughput sequencing methods has revolutionized this field. We describe the use and advantages of paired-end sequencing to map nucleosomal DNA obtained by micrococcal nuclease digestion of budding yeast nuclei. This approach provides high-quality genome-wide nucleosome occupancy and position maps., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have used paired-end sequencing of yeast nucleosomal DNA to obtain accurate genomic maps of nucleosome positions and occupancies in control cells and cells treated with 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. In control cells, 3AT-inducible genes exhibit a series of distinct nucleosome occupancy peaks. However, the underlying position data reveal that each nucleosome peak actually consists of a cluster of mutually exclusive overlapping positions, usually including a dominant position. Thus, each nucleosome occupies one of several possible positions and consequently, different cells have distinct local chromatin structures. Induction results in a major disruption of nucleosome positioning, sometimes with altered spacing and a dramatic loss of occupancy over the entire gene, often extending into a neighbouring gene. Nucleosome-depleted regions are generally unaffected. Genes repressed by 3AT show the same changes, but in reverse. We propose that yeast genes exist in one of several alternative nucleosomal arrays, which are disrupted by activation. We conclude that activation results in gene-wide chromatin remodelling and that this remodelling can even extend into the chromatin of flanking genes., (Published by Oxford University Press.)

The centromeres of budding yeast are ~120 bp in size and contain three functional elements: an AT-rich region flanked by binding sites for Cbf1 and CBF3. A specialized nucleosome containing the H3 variant Cse4 (CenH3) is formed at the centromere. Our genome-wide paired-end sequencing of nucleosomal DNA reveals that the centromeric nucleosome contains a micrococcal nuclease-resistant kernel of 123-135 bp, depending on the centromere, and is therefore significantly shorter than the canonical nucleosome. Unlike canonical nucleosomes, the centromeric nucleosome is essentially perfectly positioned. The entire centromere is included, together with at least 1 bp of DNA upstream of the Cbf1 site and at least 4 bp downstream of the CBF3 site. The fact that the binding sites for Cbf1 and CBF3 are included within the centromeric nucleosome has important implications for models of the centromeric nucleosome and for kinetochore function.

Background: Individuals with severe disability exiting school are likely to be enrolled in day activity and sheltered workshops at least as often as in supported employment. Such facilities are often staffed by paraprofessionals who may not have the skills to engage clients in meaningful activities. This article describes a 3-phase staff training project designed to increase client engagement in an adult habilitation centre., Method: The first phase of the project was to develop curriculum guidelines. The second phase was conducting a series of training for paraprofessional staff. And the third was the implementation of the clinical experience. The first author conducted a graduate level methods class on site at the adult habilitation centre. Staff participants (paraprofessional staff, supervisors, and graduate students) learned the components of systematic instruction and developed and implemented activities for clients. Engagement was measured before and after the 5-week clinical experience., Results and Conclusions: The engagement of clients increased with the implementation of systematic instruction following staff training. The implications are discussed.

The receptor encoded by the human TLR3 gene recognizes double-strand RNAs (dsRNAs) associated with viral infection. TLR3 expression is strongly activated upon differentiation of monocytes to dendritic cells, and can be further stimulated by the dsRNA analog polyinosine:polycytosine (PI:C). We report evidence for developmental regulation of the TLR3 gene. In dendritic cells derived from cord blood, both differentiation- and PI:C-associated TLR3 transcriptional activation are impaired as compared to cells from adults. Consistent with relative expression patterns, chromatin states and remodeling differ between newborn and adult samples. TLR3 expression in newborn dendritic cells exhibits heterocellularity and allelic imbalance (skewing), features characteristic of cis-acting epigenetic control. These findings reveal a new source for variability in innate immune system function and provide a model for further study of perinatal epigenetic transitions during development.

In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His-->Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.

Background: Africa is in an orphan-care crisis. In Zimbabwe, where one-fourth of adults are HIV-positive and one-fifth of children are orphans, AIDS and economic decline are straining society's ability to care for orphans within their extended families. Lack of stable care is putting thousands of children at heightened risk of malnourishment, emotional underdevelopment, illiteracy, poverty, sexual exploitation, and HIV infection, endangering the future health of the society they are expected to sustain., Methods: To explore barriers and possible incentives to orphan care, a quantitative cross-sectional survey in rural eastern Zimbabwe asked 371 adults caring for children, including 212 caring for double orphans, about their well-being, needs, resources, and perceptions and experiences of orphan care., Results: Survey responses indicate that: 1) foster caregivers are disproportionately female, older, poor, and without a spouse; 2) 98% of non-foster caregivers are willing to foster orphans, many from outside their kinship network; 3) poverty is the primary barrier to fostering; 4) financial, physical, and emotional stress levels are high among current and potential fosterers; 5) financial need may be greatest in single-orphan AIDS-impoverished households; and 6) struggling families lack external support., Conclusion: Incentives for sustainable orphan care should focus on financial assistance, starting with free schooling, and development of community mechanisms to identify and support children in need, to evaluate and strengthen families' capacity to provide orphan care, and to initiate and support placement outside the family when necessary.

Metal membranes play a vital role in hydrogen purification. Defect-free membranes can exhibit effectively infinite selectivity but must also provide high fluxes, resistance to poisoning, long operational lifetimes, and low cost. Alloying offers one route to improve on membranes based on pure metals such as palladium. We show how ab initio calculations and coarse-grained modeling can accurately predict hydrogen fluxes through binary alloy membranes as functions of alloy composition, temperature, and pressure. Our approach, which requires no experimental input apart from knowledge of bulk crystal structures, is demonstrated for palladium-copper alloys, which show nontrivial behavior due to the existence of face-centered cubic and body-centered cubic crystal structures and have the potential to resist sulfur poisoning. The accuracy of our approach is examined by a comparison with extensive experiments using thick foils at elevated temperatures. Our experiments also demonstrate the ability of these membranes to resist poisoning by hydrogen sulfide.

Common to numerous differentiation pathways in vertebrate organisms is the regulation of key genes through epigenetic mechanisms. Less well studied is to what extent cells of a given differentiation state, but examined at different points within the life history of an organism, are distinct at the level of the epigenome. A few instances of such variation have been reported, and it would be of considerable value to have at hand a means to characterize additional examples more efficiently. We describe an integrated approach to this task, and further present evidence for regions of age-related histone H4 acetylation change extending over tens to hundreds of kilobases. Broad similarity between two distinct regions of such change suggests a previously unsuspected link between developmental programs and aging.

With completion of the human genome project, patterns of higher order chromatin structure can be easily related to other features of genome organization. A well-studied aspect of chromatin, histone H4 acetylation, is examined here on the basis of its role in setting competence for gene activation. Three applications of a new hybrid genome sampling-chromatin immunoprecipitation strategy are described. The first explores aspects of epigenome architecture in human fibroblasts. A second focuses on chromatin from HL-60 promyelocytic leukemia cells before and after differentiation into macrophage-like cells. A third application explores age-related epigenome change. In the latter, acetylation patterns are compared in human skin fibroblast chromatin from donors of various ages. Two sites are reported at which observed histone H4 acetylation differences suggest decreasing acetylation over time. The sites, located in chromosome 4p16.1 and 4q35.2 regions, appear to remodel during late fetal-early child development and from preadolescence through adult life, respectively.

Histone deacetylases are required for transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox transcription factor, HOXA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.

Background: Previous studies have documented a high prevalence of tobacco use among Native American adolescents. However, little is known about the smoking behavior of Native American adolescents who live in urban areas. This study used statewide data from California to examine the smoking behavior and related psychosocial risk factors among Native American adolescents living in urban and rural counties., Methods: The Independent Evaluation of the California Tobacco Control Program conducted three population-based statewide surveys of 10th-grade California public school students in 1996, 1998, and 2000. Past-month smoking and psychosocial correlates were examined among 22,440 respondents, including 1060 Native Americans., Results: Native Americans had a 32% excess risk of past-month smoking compared with other ethnic groups. Smoking prevalence did not differ between urban (27.7%) and rural (29.3%) Native Americans. Native Americans reported higher access to cigarettes and exposure to smoking peers than other groups. Those psychosocial variables explained some, but not all, of the excess risk of smoking among Native Americans., Conclusions: Effective smoking prevention and cessation interventions are needed for Native American adolescents in urban and rural areas of California.

Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.

Fabry disease is a lipid storage disorder resulting from mutations in the gene encoding the enzyme alpha-galactosidase A (alpha-gal A; EC ). We previously have demonstrated long-term alpha-gal A enzyme correction and lipid reduction mediated by therapeutic ex vivo transduction and transplantation of hematopoietic cells in a mouse model of Fabry disease. We now report marked improvement in the efficiency of this gene-therapy approach. For this study we used a novel bicistronic retroviral vector that engineers expression of both the therapeutic alpha-gal A gene and the human IL-2Ralpha chain (huCD25) gene as a selectable marker. Coexpression of huCD25 allowed selective immunoenrichment (preselection) of a variety of transduced human and murine cells, resulting in enhanced intracellular and secreted alpha-gal A enzyme activities. Of particular significance for clinical applicability, mobilized CD34(+) peripheral blood hematopoietic stem/progenitor cells from Fabry patients have low-background huCD25 expression and could be enriched effectively after ex vivo transduction, resulting in increased alpha-gal A activity. We evaluated effects of preselection in the mouse model of Fabry disease. Preselection of transduced Fabry mouse bone marrow cells elevated the level of multilineage gene-corrected hematopoietic cells in the circulation of transplanted animals and improved in vivo enzymatic activity levels in plasma and organs for more than 6 months after both primary and secondary transplantation. These studies demonstrate the potential of using a huCD25-based preselection strategy to enhance the clinical utility of ex vivo hematopoietic stem/progenitor cell gene therapy of Fabry disease and other disorders.

Human histone deacetylases I (HDAC1) and II (HDAC2) are homologous proteins (84% identity) that catalyze release of acetyl groups from modified N-terminal lysines of core histones. Histone deacetylation is correlated with both transient and persistent states of transcriptional inactivity (i.e. silencing) in many eukaryotes. In this study, we analyzed complexes containing HDAC1 and HDAC2 to identify the proteins most stably associated with these deacetylases. Complex cI (9.5 S) contained transcriptional corepressor CoREST/kiaa0071 and a protein homologous to FAD-dependent oxidoreductases, kiaa0601. Complex cII (15 S) contained >/=15 proteins, including CHD3/4 (Mi-2), Mta-L1, RbAp48/46, and MBD3, characteristic of vertebrate nucleosome-remodeling complexes. Under native conditions, cI and cII may contain HDAC1, HDAC2 or both; these can be dissociated to cI and cII core complexes containing only HDAC1 or HDAC2. The (m)CpG-binding protein MBD2 was associated only with the HDAC1 cII core complex. A model is proposed in which HDAC1 core complexes can be targeted to methylated DNA via MBD2 with recruitment of HDAC2 occurring through formation of HDAC1/2 cII dimers. We note that the cI component CoREST/kiaa0071 and the cII component Mta-L1 share a region of homology that includes a SANT domain; this domain may play a role in complex assembly.

We have investigated ligand-dependent negative regulation of the thyroid-stimulating hormone beta (TSHbeta) gene. Thyroid hormone (T3) markedly repressed activity of the TSHbeta promoter that had been stably integrated into GH(3 )pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH(3 )cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRbeta) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3-dependent promoter repression. We suggest that ligand-dependent histone deacetylase recruitment is a mechanism of the negative-feedback regulation, a critical function of the pituitary-thyroid axis.

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O2) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H2O2 represents a critical signal mediating replicative senescence.

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).