Tenomodulin promotes human adipocyte differentiation and beneficial visceral adipose tissue expansion
Proper regulation of energy storage in adipose tissue is crucial for maintaining insulin sensitivity and molecules contributing to this process have not been fully revealed. Here we show that type II transmembrane protein tenomodulin (TNMD) is upregulated in adipose tissue of insulin-resistant versus insulin-sensitive individuals, who were matched for body mass index (BMI). TNMD expression increases in human preadipocytes during differentiation, whereas silencing TNMD blocks adipogenesis. Upon high-fat diet feeding, transgenic mice overexpressing Tnmd develop increased epididymal white adipose tissue (eWAT) mass, and preadipocytes derived from Tnmd transgenic mice display greater proliferation, consistent with elevated adipogenesis. In Tnmd transgenic mice, lipogenic genes are upregulated in eWAT, as is Ucp1 in brown fat, while liver triglyceride accumulation is attenuated. Despite expanded eWAT, transgenic animals display improved systemic insulin sensitivity, decreased collagen deposition and inflammation in eWAT, and increased insulin stimulation of Akt phosphorylation. Our data suggest that TNMD acts as a protective factor in visceral adipose tissue to alleviate insulin resistance in obesity.
The Mother Centriole Appendage Protein Cenexin Modulates Lumen Formation through Spindle Orientation
Establishing apical-basal polarity is instrumental in the functional shaping of a solitary lumen within an acinus. By exploiting micropatterned slides, wound healing assays, and three-dimensional culture systems, we identified a mother centriole subdistal appendage protein, cenexin, as a critical player in symmetric lumen expansion through the control of microtubule organization. In this regard, cenexin was required for both centrosome positioning in interphase cells and proper spindle orientation during mitosis. In contrast, the essential mother centriole distal appendage protein CEP164 did not play a role in either process, demonstrating the specificity of subdistal appendages for these events. Importantly, upon closer examination we found that cenexin depletion decreased astral microtubule length, disrupted astral microtubule minus-end organization, and increased levels of the polarity protein NuMA at the cell cortex. Interestingly, spindle misorientation and NuMA mislocalization were reversed by treatment with a low dose of the microtubule-stabilizing agent paclitaxel. Taken together, these results suggest that cenexin modulates microtubule organization and stability to mediate spindle orientation.
Structural birth defects in the kidney and urinary tract are observed in 0.5% of live births and are a major cause of end-stage renal disease, but their genetic aetiology is not well understood. Here we analyse 135 lines of mice identified in large-scale mouse mutagenesis screen and show that 29% of mutations causing congenital heart disease (CHD) also cause renal anomalies. The renal anomalies included duplex and multiplex kidneys, renal agenesis, hydronephrosis and cystic kidney disease. To assess the clinical relevance of these findings, we examined patients with CHD and observed a 30% co-occurrence of renal anomalies of a similar spectrum. Together, these findings demonstrate a common shared genetic aetiology for CHD and renal anomalies, indicating that CHD patients are at increased risk for complications from renal anomalies. This collection of mutant mouse models provides a resource for further studies to elucidate the developmental link between renal anomalies and CHD.
Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction
MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)-qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids.
Assessment of and Response to Data Needs of Clinical and Translational Science Researchers and Beyond
Objective and Setting: As universities and libraries grapple with data management and “big data,” the need for data management solutions across disciplines is particularly relevant in clinical and translational science (CTS) research, which is designed to traverse disciplinary and institutional boundaries. At the University of Florida Health Science Center Library, a team of librarians undertook an assessment of the research data management needs of CTS researchers, including an online assessment and follow-up one-on-one interviews.
Design and Methods: The 20-question online assessment was distributed to all investigators affiliated with UF’s Clinical and Translational Science Institute (CTSI) and 59 investigators responded. Follow-up in-depth interviews were conducted with nine faculty and staff members.
Results: Results indicate that UF’s CTS researchers have diverse data management needs that are often specific to their discipline or current research project and span the data lifecycle. A common theme in responses was the need for consistent data management training, particularly for graduate students; this led to localized training within the Health Science Center and CTSI, as well as campus-wide training. Another campus-wide outcome was the creation of an action-oriented Data Management/Curation Task Force, led by the libraries and with participation from Research Computing and the Office of Research.
Conclusions: Initiating conversations with affected stakeholders and campus leadership about best practices in data management and implications for institutional policy shows the library’s proactive leadership and furthers our goal to provide concrete guidance to our users in this area.
BACKGROUND: Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.
RESULTS: We present slncky, a lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we developed a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.
CONCLUSION: Our results highlight that lncRNAs are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological mechanism and/or roles. Our novel comparative methods for lncRNAs reveals 233 constrained lncRNAs out of tens of thousands of currently annotated transcripts, which we make available through the slncky Evolution Browser.
Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.
Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo
The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.
In 2016 the National Science Foundation (NSF) rolled out its new online public access repository, NSF-PAR for investigators funded by the NSF to deposit their manuscripts to comply with its new Public Access Policy. The NSF’s policy and its new publications repository differ in several key ways from the National Institutes of Health’s (NIH) public access policy and PMC, particularly in terms of requirements for compliance and procedures for deposit. While NIH grants may make up the majority of biomedical institutions’ research funds, the NSF is also an important source of biomedical funding, especially for career awards, research training grants, and translational research. In this webinar we will walk participants through the requirements for compliance and the process for deposit and share insights provided by the NSF Policy Office.
Data Management Plan Requirements for Campus Grant Competitions: Opportunities for Research Data Services Assessment and Outreach
Objective: To examine the effects of research data services (RDS) on the quality of data management plans (DMPs) required for a campus-level faculty grant competition, as well as to explore opportunities that the local DMP requirement presented for RDS outreach.
Methods: Nine reviewers each scored a randomly assigned portion of DMPs from 82 competition proposals. Each DMP was scored by three reviewers, and the three scores were averaged together to obtain the final score. Interrater reliability was measured using intraclass correlation. Unpaired t-tests were used to compare mean DMP scores for faculty who utilized RDS services with those who did not. Unpaired t-tests were also used to compare mean DMP scores for proposals that were funded with proposals that were not funded. One-way ANOVA was used to compare mean DMP scores among proposals from six broad disciplinary categories.
Results: Analyses showed that RDS consultations had a statistically significant effect on DMP scores. Differences between DMP scores for funded versus unfunded proposals and among disciplinary categories were not significant. The DMP requirement also provided a number of both expected and unexpected outreach opportunities for RDS services.
Conclusions: Requiring DMPs for campus grant competitions can provide important assessment and outreach opportunities for research data services. While these results might not be generalizable to DMP review processes at federal funding agencies, they do suggest the importance, at any level, of developing a shared understanding of what constitutes a high quality DMP among grant applicants, grant reviewers, and RDS providers.
Based off our workshop series at Ghanaian churches in Worcester, MA, the Healthy Ghanaian Cookbook is a collection of recipes created by Peggy Akufo from the Apostolic Church at Bethsaida Christian Center. The recipes were altered to increase nutritional value. Calorie and nutrition breakdown for each recipe is included.
Introduction: Individuals with excess weight have increased morbidity and mortality compared to those of normal weight, and there are differences in disease risk between overweight and obese men and women. However, limited information on how physicians counsel these groups and on patients’ experiences with weight management counseling (WMC) is available. The goals of this study are to describe specific WMC approaches provided to patients, reported benefit of these strategies, and study participants’ WMC preferences.
Methods:103 participants, stratified by BMI (Overweight: 25.0 ≤ BMI ≤ 29.9; Obese: BMI ≥ 30.0) and gender, completed surveys. Survey questions focused on WMC approaches (e.g., discussions about diet, generation of specific weight loss goals) currently provided by physicians, reported benefit of these methods, and patients’ WMC preferences for future care. Frequency counts were used in analysis of all questions. Chi-square and Fisher’s exact test (p < .05) were performed to assess significance between stratified groups.
Results: Participants reported receiving a wide-range of WMC, from discussions about diet to surgery. Overweight participants and women reported less counseling compared to obese individuals and men, respectively. Compared to men, women reported fewer discussions in areas such as past weight loss attempts (p=0.014) and effects of weight on long-term health (p=0.008). In general, participants found scheduling follow-up appointments most beneficial (72.8%). There were no significant differences by BMI or gender. Overall, participants most preferred that physicians increase support in generating specific strategies to assist in weight loss (74.8%) and in helping them to develop specific weight loss goals (65.1%). By gender, men most preferred increased development of weight loss strategies (70.0%) by their physicians and desired more discussions about the effects of weight on long-term health (63.3%). Women most preferred increased development of specific weight loss strategies (79.2%) as well as increased generation of specific weight loss goals (67.9%) by their physicians. Both overweight and obese participants (68.6% and 80.7%, respectively) sought increased development of weight loss strategies.
Conclusions: This appears to be the first cross-sectional study comparing patients’ WMC experiences and preferences, stratified by BMI and gender. Results demonstrate that regardless of BMI and gender, patients want more WMC, with preference for certain strategies. Differences were noted between stratified groups.
Letter to the editor and case report regarding primary sclerosing cholangitis (PSC), inflammatory bowel disease (IBD), the development of colorectal cancer, cancer screening, and the SCENIC consensus statement on surveillance of colorectal dysplasia.
Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates
The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.
Loss of mTOR signaling affects cone function, cone structure and expression of cone specific proteins without affecting cone survival
Cones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 and mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 and mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal.
Combined inhibition of Bcl-2/Bcl-xL and Usp9X/Bag3 overcomes apoptotic resistance in glioblastoma in vitro and in vivo
Despite great efforts taken to advance therapeutic measures for patients with glioblastoma, the clinical prognosis remains grim. The antiapoptotic Bcl-2 family protein Mcl-1 is overexpressed in glioblastoma and represents an important resistance factor to the BH-3 mimetic ABT263. In this study, we show that combined treatment with ABT263 and GX15-070 overcomes apoptotic resistance in established glioblastoma cell lines, glioma stem-like cells and primary cultures. Moreover, this treatment regimen also proves to be advantageous in vivo. On the molecular level, GX15-070 enhanced apoptosis by posttranslational down-regulation of the deubiquitinase, Usp9X, and the chaperone Bag3, leading to a sustained depletion of Mcl-1 protein levels. Moreover, knock-down of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2/Bcl-xL inhibition. In conclusion, combined treatment with ABT263 and GX15-070 results in a significantly enhanced anti-cancer activity in vitro as well as in vivo in the setting of glioblastoma. Both drugs, ABT263 and GX15-070 have been evaluated in clinical studies which facilitates the translational aspect of taking this combinatorial approach to the clinical setting. Furthermore we present a novel mechanism by which GX15-070 counteracts Mcl-1 expression which may lay a foundation for a novel target in cancer therapy.
Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgr(ko) retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgr(ko) PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgr(ko) PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.
Intraflagellar transport 27 is essential for hedgehog signaling but dispensable for ciliogenesis during hair follicle morphogenesis
Hair follicle morphogenesis requires precisely controlled reciprocal communications, including hedgehog (Hh) signaling. Activation of the Hh signaling pathway relies on the primary cilium. Disrupting ciliogenesis results in hair follicle morphogenesis defects due to attenuated Hh signaling; however, the loss of cilia makes it impossible to determine whether hair follicle phenotypes in these cilia mutants are caused by the loss of cilia, disruption of Hh signaling, or a combination of these events. In this study, we characterized the function of Ift27, which encodes a subunit of intraflagellar transport (IFT) complex B. Hair follicle morphogenesis of Ift27-null mice was severely impaired, reminiscent of phenotypes observed in cilia and Hh mutants. Furthermore, the Hh signaling pathway was attenuated in Ift27 mutants, which was in association with abnormal ciliary trafficking of SMO and GLI2, and impaired processing of Gli transcription factors; however, formation of the ciliary axoneme was unaffected. The ciliary localization of IFT25 (HSPB11), the binding partner of IFT27, was disrupted in Ift27 mutant cells, and Ift25-null mice displayed hair follicle phenotypes similar to those of Ift27 mutants. These data suggest that Ift27 and Ift25 operate in a genetically and functionally dependent manner during hair follicle morphogenesis. This study suggests that the molecular trafficking machineries underlying ciliogenesis and Hh signaling can be segregated, thereby providing important insights into new avenues of inhibiting Hh signaling, which might be adopted in the development of targeted therapies for Hh-dependent cancers, such as basal cell carcinoma.
Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-alpha and interferon-lambda production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).
TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.