The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl ؊/؊ retinas, the GFP؉ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP؉ photoreceptors from wild-type and Nrl ؊/؊ retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.gene profiling ͉ gene regulation ͉ neuronal differentiation ͉ retina ͉ transcription factor E volution of higher-order sensory and behavioral functions in mammals is accompanied by increasingly complex regulation of gene expression (1). As much as 10% of the human genome is presumably dedicated to the control of transcription. Exquisitely timed expression of cell-type-specific genes, together with spatial and quantitative precision, depends on the interaction between transcriptional control machinery and extracellular signals (2, 3). Neuronal heterogeneity and functional diversity result from combinatorial and cooperative actions of regulatory proteins that form complicated yet precise transcriptional networks to generate unique gene expression profiles. A key transcription factor, combined with its cognate regulatory cis-sequence codes, specifies a particular node in the gene regulatory networks that guide differentiation and development (4).The retina offers an ideal paradigm for investigating regulatory networks underlying neuronal differentiation. The genesis of six types of neurons and Müller glia in the vertebrate retina proceeds in a predictable sequence during development (5). Subsets of multipotent retinal neuroepithelial progenitors exit the cell cycle at specific time points and acquire a particular cell fate under the influence of intrinsic genetic program and extrinsic factors (5-7). Pioneering studies using thymidine labeling and retroviral vectors established the order and birthdates of neurons in developing retina (5,(8)(9)(10)). The current model of retinal differentiation proposes that a heterogeneous pool of progenitors passes through states of competence, where it can generate a distinct subset of neurons (5). One can predict that, at the molecular level, this competence is acquired by combinatorial action of specific transcriptional regulatory proteins. Genetic ablation studies of transcription factors involved in early murine eye specification are consistent with combinatorial regulation (11-13).Rod and cone photorecep...

Background Cancer is increasingly common among HIV patients given improved survival. Objective To examine calendar trends in cumulative cancer incidence and hazard rate by HIV status. Design Cohort study Setting North American AIDS Cohort Collaboration on Research and Design during 1996–2009 Patients 86,620 HIV-infected and 196,987 uninfected adults Measurements We estimated cancer-type-specific cumulative incidence by age 75 years by HIV status and calendar era, and examined calendar trends in cumulative incidence and hazard rates. Results Cumulative incidences (%) of cancer by age 75 (HIV+/HIV−) were: Kaposi sarcoma (KS), 4.4/0.01; non-Hodgkin’s lymphoma (NHL), 4.5/0.7; lung, 3.4/2.8; anal, 1.5/0.1; colorectal, 1.0/1.5; liver, 1.1/0.4; Hodgkin lymphoma (HL), 0.9/0.1; melanoma, 0.5/0.6; and oral cavity/pharyngeal, 0.8/0.8. Among HIV-infected subjects, we observed decreasing calendar trends in cumulative incidence and hazard rate for KS and NHL. For anal, colorectal and liver cancers, increasing cumulative incidence, but not hazard rate trends, were due to the decreasing mortality rate trend (−9% per year), allowing greater opportunity to be diagnosed with these cancer types. Despite decreasing hazard rate trends for lung, HL, and melanoma, we did not observe cumulative incidence trends due to the compensating effect of the declining mortality rate on cumulative incidence. Limitations Secular trends in screening, smoking, and viral co-infections were not evaluated. Conclusions Our analytic approach helped disentangle the effects of improved survival and changing cancer-specific hazard rates on cumulative incidence trends among HIV patients. Cumulative cancer incidence by age 75, approximating lifetime risk in HIV patients, may have clinical utility in this population. The high cumulative incidences by age 75 for KS, NHL, and lung cancer supports early and sustained ART and smoking cessation. Primary Funding Source National Institutes of Health

The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed. A functional all-cone mouse retina represents a unique opportunity to investigate, at the molecular level, differences between the two photoreceptor subtypes. Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation. Comparative data analysis revealed 161 differentially expressed genes; of which, 78 exhibited significantly lower and 83 higher expression in the Nrl-/- retina. Hierarchical clustering was utilized to predict the function of these genes in a temporal context. The differentially expressed genes primarily encode proteins associated with signal transduction, transcriptional regulation, intracellular transport and other processes, which likely correspond to differences between rods and cones and/or retinal remodeling in the absence of rods. A significant number of these genes may serve as candidates for diseases involving rod or cone dysfunction. Chromatin immunoprecipitation assay showed that in addition to the rod phototransduction genes, Nrl might modulate the promoters of many functionally diverse genes in vivo. Our studies provide molecular insights into differences between rod and cone function, yield interesting candidates for retinal diseases and assist in identifying transcriptional regulatory targets of Nrl.

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Objective-To assess dose-response relations between severity of iron deficiency (ID) and infant social-emotional behavior.Study design-Cohort of 9-to 10-month-old African-American infants (n = 77 with final iron status classification). Infants were provided oral iron for 3 months. Social-emotional outcomes included mother and examiner ratings at 9 and 12 months and quantitative behavioral coding from videotape at 12 months. General linear model analyses tested for linear effects of iron status group (ordered from worst to best -iron-deficient anemic (IDA), non-anemic iron-deficient (NA ID), ironsufficient (IS)) and determined thresholds for effects.Results-There were significant (p < .05) linear effects of poorer iron status for shyness (increasing, maternal rating), orientation-engagement and soothability (decreasing, examiner ratings), and the following quantitatively-coded behaviors: positive affect (decreasing) and latencies to engage with the examiner (increasing) and move away from the examiner (decreasing). The threshold for all but one effect was ID with or without anemia vs. IS.Conclusions-Infant social-emotional behavior appears to be adversely affected by ID with or without anemia. ID without anemia is not detected by common screening procedures and is more widespread than IDA. Infant social-emotional behavior can profoundly influence the caregiving environment, with repercussions for overall development.The prevalence of iron deficiency anemia (IDA) has markedly declined in US infants in the last 30 years. However, poor, minority, and immigrant infants and toddlers remain at increased Corresponding author: Betsy Lozoff, M.D., Center for Human Growth and Development, 300 N Ingalls, University of Michigan, Ann Arbor, MI 48109-5406. Tel: 734-764-2443; Fax: 734-936-9288; E-mail: blozoff@umich.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Though findings of rapid improvement in mental test scores have not been replicated, 4 socialemotional alterations are among the most consistent findings. Virtually every study that compared social-emotional behavior of IDA to non-anemic (NA) infants found them to be more wary, hesitant, solemn, unhappy, or closer to their mothers. 5 Four of 6 randomized trials of supplemental iron that assessed this domain showed affective benefits of iron (e.g., more positive affect, social interaction, etc.). 5 Notwithstanding the consistency of results, socialemotional effects have captured less attention than cognitive ones, but they could equally result from direct effects of ID on associated brain systems. Furthermore, there h...

The risk of ESRD remains high among HIV-infected individuals in care but is declining with improvements in virologic suppression. HIV-infected black persons continue to comprise the majority of cases, as a result of higher viral loads, comorbidities, and genetic susceptibility.

(See the editorial commentary by Furrer on pages 830-1.)Background. There are few recent data on the rates of AIDS-defining opportunistic infections (OIs) among human immunodeficiency virus (HIV)-infected patients in care in the United States and Canada.Methods. We studied HIV-infected participants in 16 cohorts in the North American AIDS Cohort Collaboration on Research and Design (NA-ACCORD) during 2000-2010. After excluding 16 737 (21%) with any AIDS-defining clinical events documented before NA-ACCORD enrollment, we analyzed incident OIs among the remaining 63 541 persons, most of whom received antiretroviral therapy during the observation. We calculated incidence rates per 100 person-years of observation (hereafter, "person-years") with 95% confidence intervals (CIs) for the first occurrence of any OI and select individual OIs during 2000-2003, 2004-2007, and 2008-2010.Results. A total of 63 541 persons contributed 261 573 person-years, of whom 5836 (9%) developed at least 1 OI. The incidence rate of any first OI decreased over the 3 observation periods, with 3.0 cases, 2.4 cases, and 1.5 cases per 100 person-years of observation during 2000-2003, 2004-2007, and 2008-2010, respectively (P trend <.001); the rates of most individual OIs decreased as well. During 2008-2010, the leading OIs included Pneumocystis jiroveci pneumonia, esophageal candidiasis, and disseminated Mycobacterium avium complex or Mycobacterium kansasii infection.Conclusions. For HIV-infected persons in care during 2000-2010, rates of first OI were relatively low and generally declined over this time.

Gestational and early postnatal iron deficiency occurs commonly in humans and results in altered behaviors suggestive of striatal dysfunction. We hypothesized that early iron deficiency alters the metabolome of the developing striatum and accounts for abnormalities in striatum-dependent behavior in rats. Sixteen metabolite concentrations from a 9-11 microL volume within the striatum were serially assessed in 10 iron-deficient and 10 iron-sufficient rats on postnatal days 8, 22 (peak anemia), and 37 (following recovery from anemia) using (1)H NMR spectroscopy at 9.4 tesla. Chin-elicited bilateral forelimb placing and vibrissae-elicited unilateral forelimb placing were also assessed on these days. Iron deficiency altered metabolites indexing energy metabolism, neurotransmission, glial integrity, and myelination over time (P < 0.05). Successful development of behaviors was delayed in the iron-deficient group (P < or = 0.01). Alterations in creatine, glucose, glutamine, glutamate, N-acetylaspartate, myo-inositol, and glycerophosphorylcholine + phosphorylcholine concentrations accounted for 77-83% of the behavioral variability during peak anemia on postnatal day 22 in the iron-deficient group. Correction of anemia normalized the striatal metabolome but not the behaviors on postnatal day 37. These novel data imply that alterations in the metabolite profile of the striatum likely influence later neural functioning in early iron deficiency.