SUMMARY Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ~1500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα and C/EBPβ. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes, and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs, available online, and establishes lnc-BATE1 as a novel regulator of BAT development and physiology.

BackgroundThe two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.MethodsIn this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ.FindingsThis method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.ConclusionThese methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

As a ubiquitous secondary messenger in plant signaling systems, calcium ions (Ca2+) play essential roles in plant growth and development. Within the cellular signaling network, the accurate decoding of diverse Ca2+ signal is a fundamental molecular event. Calcium-dependent protein kinases (CDPKs), identified commonly in plants, are a kind of vital regulatory protein deciphering calcium signals triggered by various developmental and environmental stimuli. This review chiefly introduces Ca2+ distribution in plant cells, the classification of Arabidopsis thaliana CDPKs (AtCDPKs), the identification of the Ca2+-AtCDPK signal transduction mechanism and AtCDPKs’ functions involved in plant growth regulation and abiotic stress responses. The review presents a comprehensive overview of AtCDPKs and may contribute to the research of CDPKs in other plants.

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), are usually involved in cell development in plants. To investigate the expression profiling as well as the role of FLA genes in fiber development, 19 GhFLA genes (cDNAs) were isolated from cotton (Gossypium hirsutum). Among them, 15 are predicted to be glycosylphosphatidylinositol anchored to the plasma membranes. The isolated cotton FLAs could be divided into four groups. Real-time quantitative reverse transcriptase polymerase chain reaction results indicated that the GhFLA genes are differentially expressed in cotton tissues. Three genes (GhFLA1/2/4) were specifically or predominantly expressed in 10 days post-anthesis fibers, and the transcripts of the other four genes (GhFLA6/14/15/18) were accumulated at relatively high levels in cotton fibers. Furthermore, expressions of the GhFLA genes are regulated in fiber development and in response to phytohormones and NaCl. The identification of cotton FLAs will facilitate the study of their roles in cotton fiber development and cell wall biogenesis.

Obesity has emerged as an alarming health crisis due to its association with metabolic risk factors such as diabetes, dyslipidemia, and hypertension. Recent work has demonstrated the multifaceted roles of lncRNAs in regulating mouse adipose development, but their implication in human adipocytes remains largely unknown. Here we present a catalog of 3149 adipose active lncRNAs, of which 909 are specifically detected in brown adipose tissue (BAT) by performing deep RNA-seq on adult subcutaneous, omental white adipose tissue and fetal BATs. A total of 169 conserved human lncRNAs show positive correlation with their nearby mRNAs, and knockdown assay supports a role of lncRNAs in regulating their nearby mRNAs. The knockdown of one of those, lnc-dPrdm16, impairs brown adipocyte differentiation in vitro and a significant reduction of BAT-selective markers in in vivo. Together, our work provides a comprehensive human adipose catalog built from diverse fat depots and establishes a roadmap to facilitate the discovery of functional lncRNAs in adipocyte development.

BackgroundTrypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.ResultsWhile the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.ConclusionUsing the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi.

Immune control of the protozoan parasite Trypanosoma cruzi requires the activation of both CD4؉ and CD8 ؉ T cells. We recently identified two T. cruzi trans-sialidase peptides that are targets of approximately 30% of all CD8 ؉ T cells during acute T. cruzi infection in mice. To determine whether CD4 ؉ T cells are required for generation of these dominant CD8 ؉ T-cell responses, major histocompatibility complex class II (MHC II)-deficient mice were infected with the Brazil strain of T. cruzi and examined for the generation of antigenspecific CD8 ؉ T cells. Strong trans-sialidase TSKB18-and TSKB20-specific CD8 ؉ T-cell responses were generated in both the presence and the absence of CD4 ؉ help. However, the magnitudes of the immunodominant TSKB20-specific CD8 ؉ T-cell responses detectable using class I MHC-peptide tetramers were consistently lower in the blood and spleens of MHC II-deficient mice. Spleen cells from infected MHC II-deficient mice produced gamma interferon after in vitro stimulation with T. cruzi peptides at levels similar to those in wild-type mice, and MHC II-deficient mice displayed strong T. cruzi peptide-specific cytotoxic T-lymphocyte activity in vivo. Thus, primary CD8؉ T-cell responses in experimental T. cruzi infection are generated in the absence of CD4 ؉ T cells, providing further evidence that T. cruzi directly activates and licenses antigenpresenting cells. Nevertheless, unhelped CD8 ؉ T cells in T. cruzi-infected mice fail to reach the frequencies achieved in the presence of CD4 T-cell help and are unable to prevent acute-phase death of these mice.Trypanosoma cruzi is a kinetoplastid protozoan parasite that causes Chagas' disease in humans, one of the most important public health threats in Latin American countries (33). Both CD8 ϩ and CD4 ϩ T cells have been shown to be essential for the control of T. cruzi infection in experimental models; mice lacking either T-cell compartment, due to gene deletion of major histocompatibility complex (MHC) molecules and components (28, 29) or T-cell coreceptors (21) or to depletion of T cells using anti-CD4 or anti-CD8 antibodies (29), succumb to acute infection and display high systemic and tissue parasite loads (27). However, the role that CD4 ϩ T cells play in the development and regulation of CD8 ϩ T-cell immunity to T. cruzi remains to be determined.The requirement for CD4 ϩ T-cell help in the induction of CD8 ϩ T-cell responses has been studied in a number of murine models of infection, using MHC II knockout (KO) mice or mice depleted of CD4 ϩ T cells by injection of anti-CD4 antibody. CD4 ϩ T-cell activity is required for priming of the CD8 ϩ T-cell response to peptide-pulsed dendritic cells (DCs) (31) and for the optimal activation of pathogen-specific CD8 ϩ Tcell responses to Listeria monocytogenes (15) and influenza virus (19) infections. Nevertheless, strong primary CD8 ϩ T-cell responses against many infectious agents, such as lymphocytic choriomeningitis virus (12) or vesicular stomatitis virus (15), can be measured readily in animals l...

The effect of the hybrid proline-rich protein (HyPRP) gene EARLI1 on the rate of germination (germinability) of Arabidopsis seeds and seedling growth under low temperature and salt stress conditions was investigated. EARLI1 was induced during germination in embryonic tissues, and was strongly expressed in certain parts of young seedlings. Comparisons of control, overexpressing (OX), and knockout (KO) lines indicated that higher than wild type levels of EARLI1 improved germinability, root elongation, and reduction of sodium accumulation in leaves under salt stress, as well as germinability under low-temperature stress. Abscisic acid (ABA) contents were relatively low after prolonged salt stress, suggesting that EARLI1 has an ABA-independent effect on germinability under these conditions. Overexpression of EARLI1 during germination enhanced the sensitivity of seeds to exogenously applied ABA, suggesting that EARLI1 has an ABA-dependent negative effect on seed germinability under high ABA stress conditions. Well-known stress response marker genes such as COR15a, KIN1, P5SC1, and RD29 were unaffected whereas P5SC2, RD22, or RAB18 were only slightly affected in OX and KO plants. The pleiotropic effects of EARLI1 during stress and an absence of strong regulatory effects on stress marker genes suggest that this HyPRP gene has an auxiliary role for various stress protection responses in Arabidopsis.