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Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host–parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene (March1) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86(+)DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH–type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy.

Genome-wide association studies have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP, and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). As ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWAS identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor gene (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. As AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus, and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression, and altering melanocyte growth phenotypes upon exposure.

Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.

Infection by malaria parasites triggers dynamic immune responses leading to diverse symptoms and pathologies; however, the molecular mechanisms responsible for these reactions are largely unknown. We performed Trans-species Expression Quantitative Trait Locus analysis to identify a large number of host genes that respond to malaria parasite infections. Here we functionally characterize one of the host genes called receptor transporter protein 4 (RTP4) in responses to malaria parasite and virus infections. RTP4 is induced by type I IFN (IFN-I) and binds to the TANK-binding kinase (TBK1) complex where it negatively regulates TBK1 signaling by interfering with expression and phosphorylation of both TBK1 and IFN regulatory factor 3. Rtp4(−/−) mice were generated and infected with malaria parasite Plasmodiun berghei ANKA. Significantly higher levels of IFN-I response in microglia, lower parasitemia, fewer neurologic symptoms, and better survival rates were observed in Rtp4(−/−) than in wild-type mice. Similarly, RTP4 deficiency significantly reduced West Nile virus titers in the brain, but not in the heart and the spleen, of infected mice, suggesting a specific role for RTP4 in brain infection and pathology. This study reveals functions of RTP4 in IFN-I response and a potential target for therapy in diseases with neuropathology.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare bi-allelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells, and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify bi-allelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Background and aims Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. Approach and results Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. Conclusions Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies., Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoellii nigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro. The modified iRBC surface triggered PS-CD36-mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating host-pathogen interaction and innate immune responses, which may be explored for developing disease control strategies.

Cancer treatments composed of immune checkpoint inhibitors and oncogene-targeted drugs might improve cancer management, but there has been little investigation of their combined potential as yet. To estimate the fraction of cancer cases that might benefit from such combination therapy, we conducted an exploratory study of cancer genomic datasets to determine the proportion with somatic mutation profiles amenable to either immunotherapy or targeted therapy. We surveyed 13,349 genomic profiles from public databases for cases with specific mutations targeted by current agents or a burden of exome-wide nonsynonymous mutations (NsM) that exceed a proposed threshold for response to checkpoint inhibitors. Overall, 8.9% of cases displayed profiles that could benefit from combination therapy, which corresponded to approximately 11.2% of U.S. annual incident cancer cases. Frequently targetable mutations were in PIK3CA, BRAF, NF1, NRAS, and PTEN. We also noted a high burden of NsM in cases with targetable mutations in SMO, DDR2, FGFR1, PTCH1, FGFR2, and MET. Our results indicate that a significant proportion of solid tumor patients are eligible for immuno-targeted combination therapy, and they suggest prioritizing specific cancers for trials of certain targeted and checkpoint inhibitor drugs. Cancer Res; 77(13); 3666–71. ©2017 AACR.

MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley & Sons, Inc.

Cryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus., Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.

New vaccines are needed to combat the public health threat posed by M. tuberculosis (M. tb), but no correlates have been defined to aid vaccine development. Using mouse models, we previously developed an in vitro system that measures the ability of M. tb-immune lymphocytes to control bacterial replication during co-culture with M. tb-infected macrophages. We demonstrated that the degree of in vitro growth control by lymphocytes from mice given vaccines of varying efficacy reflected the relative degree of in vivo protection against lethal challenge. Further, using targeted analyses of gene expression in lymphocytes recovered from co-cultures, we found mediators whose relative expression also correlated with in vitro and in vivo outcomes. Here we advanced those findings by employing genome-wide expression analyses. We first screened splenocytes recovered from co-cultures by microarray, revealing additional genes whose expression correlated with protection. After applying pathway analyses to down-select gene candidates, we used both splenocytes and peripheral blood lymphocytes to validate microarray findings by qRT-PCR. We then subjected data from top candidates to rigorous statistical analyses. Resulting correlate candidates, including CXCL9, IFN-γ, and CCL5, significantly predicted protection with high specificity. These findings therefore refine and extend a panel of relevant immune correlates to advance vaccine development.

Human infection withCryptococcuscauses up to a quarter million AIDS-related deaths annually and is the most common cause of non-viral meningitis in the United States. As an opportunistic fungal pathogen,C. neoformansis distinguished by its ability to adapt to diverse host environments including plants, amoeba and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified a low nutrient-induced gene, an alternative carbon nutrient transporterSTL1associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role forSTL1in mammalian pathogenesis and revealed thatSTL1expression is regulated by a novel target-of-rapamycin (TOR)-related multi-gene regulatory mechanism involving theCAC2subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the TOR-related RNA chaperone,VAD1was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein Ecm15, in turn, required for nuclear transport of the Cac2 protein. Derepression ofSTL1by theCAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of theSTL1enhancer element. Derepression ofSTL1resulted in enhanced survival and growth of the fungus in the presence of low nutrient, alternative carbon sources, facilitating virulence in mice. The study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host.Author summaryThe fungusCryptococcusis a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of meningitis in the United States. The fungus is carried in about 10% of the adult population and, after re-activation, causes disease in a wide variety of individuals including HIV-infected as well as immunosuppression either from genetic defects or after immune suppressive treatments due to transplant conditioning, cancer therapy or treatment of autoimmune diseases. The fungus is widely carried in the soil and trees and can infect plants, single cell organisms and even dolphins. However, mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to infect these diverse hosts and identified a nutrient transporter,STL1to be associated with a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in environmental adaptation may help to design better methods of control and treatment of widely dispersed fungal pathogens such asCryptococcus.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense is hindered by invasive isolation methods and insufficient techniques for long-term passage of primary keratinocytes in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

Mycobacterium tuberculosis infection (Mtb) is the leading cause of death due to a single infectious agent and is among the top ten causes of all human deaths worldwide1. CD4 T cells are essential for resistance to Mtb infection, and for decades it has been thought that IFNγ production is the primary mechanism of CD4 T-cell-mediated protection2,3. However, IFNγ responses do not correlate with host protection, and several reports demonstrate that additional anti-tuberculosis CD4 T-cell effector functions remain unaccounted for4-8. Here we show that the tumour-necrosis factor (TNF) superfamily molecule CD153 (encoded by the gene Tnfsf8) is required for control of pulmonary Mtb infection by CD4 T cells. In Mtb-infected mice, CD153 expression is highest on Mtb-specific T helper 1 (TH1) cells in the lung tissue parenchyma, but its induction does not require TH1 cell polarization. CD153-deficient mice develop high pulmonary bacterial loads and succumb early to Mtb infection. Reconstitution of T-cell-deficient hosts with either Tnfsf8-/- or Ifng-/- CD4 T cells alone fails to rescue mice from early mortality, but reconstitution with a mixture of Tnfsf8-/- and Ifng-/- CD4 T cells provides similar protection as wild-type T cells. In Mtb-infected non-human primates, CD153 expression is much higher on Ag-specific CD4 T cells in the airways compared to blood, and the frequency of Mtb-specific CD153-expressing CD4 T cells inversely correlates with bacterial loads in granulomas. In Mtb-infected humans, CD153 defines a subset of highly polyfunctional Mtb-specific CD4 T cells that are much more abundant in individuals with controlled latent Mtb infection compared to those with active tuberculosis. In all three species, Mtb-specific CD8 T cells did not upregulate CD153 following peptide stimulation. Thus, CD153 is a major immune mediator of host protection against pulmonary Mtb infection and CD4 T cells are one important source of this molecule.

The Plasmodium life cycle within the mosquito involves the gamete, zygote, motile ookinete, and the oocyst stage that supports sporogony and sporozoite formation. We mapped the P. falciparum transcriptome as the parasite progresses through the oocyst stage of development on days 2, 4, 6, and 8 post-P. falciparum infectious blood meal. Through these genomic studies, we identified 212 novel transmission stage biomarkers including genes that are developmentally expressed at a single time point and genes that are pan-developmentally expressed at all four time points in P. falciparum oocysts. Validation of a small subset of genes at the transcriptional and translational level resulted in identification of a signature of genes/proteins that can detect parasites within the mosquito as early as day 2 post-infectious blood meal and can be used to distinguish early versus late stage P. falciparum oocyst development in the mosquito. Currently, circumsporozoite protein (CSP), which is detectable only after day 7 post-infection, is the only marker used for detection of P. falciparum infection in mosquitoes. Our results open the prospect to develop a non-CSP based detection assay for assessment of P. falciparum infection in mosquitoes and evaluate the effect of intervention measures on malaria transmission in an endemic setting.

Extracellular vesicles (EVs) mediate the intercellular transfer of RNAs, which alter gene expression in target cells. EV heterogeneity has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing Jurkat T cell EVs captured using CD47, CD63, and MHC1 antibodies. These EV subsets have similar sizes but divergent RNA contents. Apart from differences in numbers of nonannotated transcripts, CD63+, MHC1+, and CD47+ EVs have similar overall contents of most noncoding RNA classes, but the relative enrichment of specific RNAs differs. The enrichment of micro-RNAs is highly divergent, and some including miR320a are selectively concentrated in CD47+ EVs. Small nucleolar RNAs including SNORD116@ and SNHG10 are also selectively enriched in CD47+ EVs, whereas no small nuclear RNAs are enriched in CD47+ EVs. Conversely, MHC1+ EVs are selectively enriched in a subset of tRNAs including TRE-CTC and TRR-CCG. This heterogeneity in RNA composition suggests multiple sorting mechanisms that direct specific RNAs into subsets of EVs that express specific surface markers.

A balance between excitation and inhibition is necessary to maintain stable brain network dynamics. Traditionally, seizure activity is believed to arise from the breakdown of this delicate balance in favor of excitation with loss of inhibition. Surprisingly, recent experimental evidence suggests that this conventional view may be limited, and that inhibition plays a prominent role in the development of epileptiform synchronization. Here, we explored the role of the KCC2 co-transporter in the onset of inhibitory network-induced seizures. Our experiments in acute mouse brain slices, of either sex, revealed that optogenetic stimulation of either parvalbumin- or somatostatin-expressing interneurons induced ictal discharges in rodent entorhinal cortex during 4-aminopyridine application. These data point to a proconvulsive role of GABAA receptor signaling that is independent of the inhibitory input location (i.e., dendritic vs. somatic). We developed a biophysically realistic network model implementing dynamics of ion concentrations to explore the mechanisms leading to inhibitory network-induced seizures. In agreement with experimental results, we found that stimulation of the inhibitory interneurons induced seizure-like activity in a network with reduced potassium A-current. Our model predicts that interneuron stimulation triggered an increase of interneuron firing, which was accompanied by an increase in the intracellular chloride concentration and a subsequent KCC2-dependent gradual accumulation of the extracellular potassium promoting epileptiform ictal activity. When the KCC2 activity was reduced, stimulation of the interneurons was no longer able to induce ictal events. Overall, our study provides evidence for a proconvulsive role of GABAA receptor signaling that depends on the involvement of the KCC2 co-transporter.

Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11bhighCD14+F4/80+ macrophages that express TCRβ. This population expands dramatically during a Plasmodium berghei ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRβ transcript and protein levels in macrophages in wildtype versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRβ on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vβ usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRβ and peripheral parasitemia. In addition, presence of TCRβ on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRβ expression by the macrophage. Expansion of TCRβ-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

SummaryInvading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways.

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, whereby the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional ‘futile cycle’ where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novo synthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformans results in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a key role in the facilitation of microbial pathogenesis.

Th17 cells, which express the chemokine receptor CCR6, are implicated in many immune-mediated disorders, such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4+ T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive upregulation of ZBTB16, which encodes the broad complex, tramtrack, bric-à-brac–zinc finger transcription factor promyelocytic leukemia zinc finger protein (PLZF). Using chromatin immunoprecipitation for modified histones, p300, and PLZF, we identified enhancer-like sites at −9/−10 and −13/−14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6+ cells. For Th cells from adult blood, both in the CCR6+ memory population and in naive cells activated ex vivo, knockdown of ZBTB16 downregulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the “master regulator” RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6+ cells. In naive Th cells from cord blood, ZBTB16 expression was confined to CD161+ cells, which are Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells, and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest.

Gao et al. show that E-box proteins dampen the generation and function of Foxp3+ regulatory T cells in part by inhibiting IL-2Rα expression and IL-2 responsiveness., E-proteins are TCR-sensitive transcription factors essential for intrathymic T cell transitions. Here, we show that deletion of E-proteins leads to both enhanced peripheral TGF-β–induced regulatory T (iT reg) cell and thymic naturally arising T reg cell (nT reg cell) differentiation. In contrast, deletion of Id proteins results in reduced nT reg cell differentiation. Mechanistic analysis indicated that decreased E-protein activity leads to de-repression of signaling pathways that are essential to Foxp3 expression. Decreased E-protein binding to an IL-2Rα enhancer locus facilitated TCR-induced IL-2Rα expression. Similarly, decreased E-protein activity facilitated TCR-induced NF-κB activation and generation of c-Rel. Consistent with this, microarray analysis indicated that cells with E-protein depletion that are not yet expressing Foxp3 exhibit activation of the IL-2 and NF-κB signaling pathways as well as enhanced expression of many of the genes associated with Foxp3 induction. Finally, studies using Nur77-GFP mice to monitor TCR signaling showed that TCR signaling strength sufficient to induce Foxp3 differentiation is accompanied by down-regulation of E-protein levels. Collectively, these data suggest that TCR stimulation acts in part through down-regulation of E-protein activity to induce T reg cell lineage development.

Background Systemic Capillary Leak Syndrome (SCLS) is an extremely rare and life-threatening vascular disorder of unknown etiology. SCLS is characterized by abrupt and transient episodes of hypotensive shock and edema due to plasma leakage into peripheral tissues. The disorder has garnered attention recently because its initial presentation resembles more common vascular disorders including systemic anaphylaxis, sepsis, and acute infections with the Ebola/Marburg family of filoviruses. Although approximately 70–85% of patients with SCLS have a concurrent monoclonal gammopathy of unknown significance (MGUS), any contribution of the paraprotein to acute flares is unknown. Procedure To identify circulating factors that might trigger acute SCLS crises, we profiled transcriptomes of paired peripheral blood mononuclear cell fractions obtained from patients during acute attacks and convalescent intervals by microarray. Results This study uncovered 61 genes that were significantly up- or downregulated more than 2.5-fold in acute samples relative to respective baselines. One of the most upregulated genes was ADM, which encodes the vasoactive peptide adrenomedullin. A stable ADM protein surrogate (pro-ADM) was markedly elevated in SCLS acute sera compared to remission samples or sera from healthy controls. Monocytes and endothelial cells (ECs) from SCLS subjects expressed significantly more ADM in response to proinflammatory stimuli compared to healthy control cells. Application of ADM to ECs elicited protective effects on vascular barrier function, suggesting a feedback protective mechanism in SCLS. Conclusions Since ADM has established hypotensive effects, differentiating between these dual actions of ADM is crucial for therapeutic applications aimed at more common diseases associated with increased ADM levels.

Epigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo. Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses., IMPORTANCE A significant proportion of the world’s population is infected with herpes simplex virus. Primary infection and subsequent recurrent reactivation can result in diseases ranging from mild lesions to severe ocular or neurological damage. Herpesviruses are subject to epigenetic regulation that modulates viral gene expression, lytic replication, and latency-reactivation cycles. Thus, epigenetic pharmaceuticals have the potential to alter the course of infection and disease. Here, while the histone methyltransferases EZH2/1 are implicated in the suppression of herpesviruses, inhibitors of these repressors unexpectedly suppress viral infection in vitro and in vivo by induction of key components of cellular innate defense pathways. These inhibitors suppress infection by multiple viral pathogens, indicating their potential as broad-spectrum antivirals.

We investigated the set of common single nucleotide polymorphisms (SNPs) that are highly correlated with 20 renal cancer (RCC) susceptibility regions identified by Genome-wide Association Study (GWAS). Together extensive fine-mapping and in silico functional analyses, based on publicly available resources, we used an integrated approach to pursue regulatory elements in which one or more variant allele could confer differential functional activity. To this end, we conducted an initial screening Massively Parallel Reporter Assay (MPRA), followed by three additional approaches: 1. Assay for Transposase-Accessible Chromatin (ATAC-seq); 2. RCC eQTL analysis;3. Capture-HiC. We confirmed the effects reported for three regions (8q24, 11q13 and 12p12). We selected 784 SNPs with an r2>0.4 or D’>0.5 and MAF Citation Format: Leandro M. Colli, Lea Jessop J. Chanock, Timothy Myers, Mitchell Machiela, Jiyeon Choi, Mark Purdue, Kevin Brown, Stephen J. Chanock. Identification of enhancer elements at kidney cancer susceptibility loci using genome-wide approaches in which post-GWAS functional studies implicate the SWI/SNF DPF3 gene for the 14q24 risk locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY25-01.

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, where by the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional ‘futile cycle’ where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novosynthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformansresults in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a keyrole in the facilitation of microbial pathogenesis.

Mycobacterium abscessus ( M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii , and Mycobacterium massiliense . We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus , M. massiliense , and M. bolletii . Based on the M. abscessus ATCC 19977 T genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus , 15 M. massiliense , and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm (41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm (41) PCR for straightforward identification of M. abscessus , M. massiliense , and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.

Expression microarray analysis of Candida glabrata following phagocytosis by human neutrophils was performed, and results were compared with those from C. glabrata incubated under conditions of carbohydrate or nitrogen deprivation. Twenty genes were selected to represent the major cell processes altered by phagocytosis or nutrient deprivation. Quantitative real-time PCR (qRT-PCR) with TaqMan chemistry was used to assess expression of the same genes in spleens of mice infected intravenously with Candida glabrata . The results in spleen closely paralleled gene expression in neutrophils or following carbohydrate deprivation. Fungal cells responded by upregulating alternative energy sources through gluconeogenesis, glyoxylate cycle, and long-chain fatty acid metabolism. Autophagy was likely employed to conserve intracellular resources. Aspartyl protease upregulation occurred and may represent defense against attacks on cell wall integrity. Downregulated genes were in the pathways of protein and ergosterol synthesis. Upregulation of the sterol transport gene AUS1 suggested that murine cholesterol may have been used to replace ergosterol, as has been reported in vitro. C. glabrata isolates in spleens of gp91 phox−/− knockout mice with reduced oxidative phagocyte defenses were grossly similar although with a reduced level of response. These results are consistent with reported results of other fungi responding to phagocytosis, indicating that a rapid shift in metabolism is required for growth in a carbohydrate-limited intracellular environment.

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Cellular lysates from PPD+ donors have been reported to transfer tuberculin reactivity to naïve recipients, but not diphtheria reactivity, and vice versa. A historically controversial topic, the terms “transfer factor” and “DLE” were used to characterize the reactivity-transferring properties of lysates. Intrigued by these reported phenomena, we found that the cellular extract derived from antigen-specific memory CD8+ T cells induces IL-6 from antigen-matched APCs. This ultimately elicits IL-17 from bystander memory CD8+ T cells. We have identified that dialyzable peptide sequences, S100a9, and the TCR β chain from CD8+ T cells contribute to the molecular nature of this activity. We further show that extracts from antigen-targeted T cells enhance immunity to Staphylococcus aureus and Candida albicans. These effects are sensitive to immunization protocols and extraction methodology in ways that may explain past discrepancies in the reproducibility of passive cellular immunity.

Immune checkpoint inhibitor treatment represents a promising approach toward treating cancer and has been shown to be effective in a subset of melanoma, non–small cell lung cancer (NSCLC), and kidney cancers. Recent studies have suggested that the number of nonsynonymous mutations (NsM) can be used to select melanoma and NSCLC patients most likely to benefit from checkpoint inhibitor treatment. It is hypothesized that a higher burden of NsM generates novel epitopes and gene products, detected by the immune system as foreign. We conducted an assessment of NsM across 7,757 tumor samples drawn from 26 cancers sequenced in the Cancer Genome Atlas (TCGA) Project to estimate the subset of cancers (both types and fractions thereof) that fit the profile suggested for melanoma and NSCLC. An additional independent set of 613 tumors drawn from 5 cancers were analyzed for replication. An analysis of the receiver operating characteristic curves of published data on checkpoint inhibitor response in melanoma and NSCLC data estimates a cutoff of 192 NsM with 74% sensitivity and 59.3% specificity to discriminate potential clinical benefit. Across the 7,757 samples of TCGA, 16.2% displayed an NsM count that exceeded the threshold of 192. It is notable that more than 30% of bladder, colon, gastric, and endometrial cancers have NsM counts above 192, which was also confirmed in melanoma and NSCLC. Our data could inform the prioritization of tumor types (and subtypes) for possible clinical trials to investigate further indications for effective use of immune checkpoint inhibitors, particularly in adult cancers. Cancer Res; 76(13); 3767–72. ©2016 AACR.

Background Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background. Results ERCC pools were analyzed on four different microarray platforms: Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms: the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate. Conclusions The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics: linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0281-x) contains supplementary material, which is available to authorized users.

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

Genome-wide association studies have identified multiple renal cell carcinoma (RCC) susceptibility loci. Here, we use regional imputation and bioinformatics analysis of the 12p12.1 locus to identify the single-nucleotide polymorphism (SNP) rs7132434 as a potential functional variant. Luciferase assays demonstrate allele-specific regulatory activity and, together with data from electromobility shift assays, suggest allele-specific differences at rs7132434 for AP-1 transcription factor binding. In an analysis of The Cancer Genome Atlas data, SNPs highly correlated with rs7132434 show allele-specific differences in BHLHE41 expression (trend P value=6.3 × 10−7). Cells overexpressing BHLHE41 produce larger mouse xenograft tumours, while RNA-seq analysis reveals that constitutively increased BHLHE41 induces expression of IL-11. We conclude that the RCC risk allele at 12p12.1 maps to rs7132434, a functional variant in an enhancer that upregulates BHLHE41 expression which, in turn, induces IL-11, a member of the IL-6 cytokine family., A common susceptibility haplotype for renal cell carcinoma is located on chromosome 12p12.1. Here, the authors show that the variant rs7132434 alters binding of the AP-1 transcription factor, which increases the expression of BHLHE41 in renal cells.

Exposure to ionizing radiation has a profound effect on the viability and further replication of living cells. Radiation is used for the treatment of cancer [1], generation of attenuated vaccines [2], and in food safety [3]. Radiation attenuation has also been one of the earliest methods used to generate whole parasite–based malaria vaccines [4–6]. Given the importance of ionizing radiation in the treatment of different forms of cancer, a large body of scientific data has accumulated on the effects of ionizing radiation on the cell cycle [7]. Such studies have shed light on the molecular and biochemical aspects of radiation-mediated cell death and survival mechanisms [1, 8]. These studies have also provided insight into the cell cycle checkpoints and the complex signaling pathways and associated molecules that ensure that each phase of the cell cycle is completed without errors [7, 9, 10]. Ionizing radiation is detrimental to the growth and survival of Plasmodium parasites. In murine models, asexual blood-stage parasites exposed to high doses of radiation failed to cause blood-stage parasite infection [11], and immunization with radiation-attenuated asexual-stage parasites protected against parasitemia [11–13] and experimental cerebral malaria [11]. In preerythrocytic-stage malaria, exposure of Plasmodium sporozoites to a threshold level of γ-radiation allows their invasion into liver cells and early development. However, these parasites undergo arrested development and fail to produce infectious liver-stage merozoites and, consequently, blood-stage parasite infection [14]. Irradiation-attenuated malaria sporozoites protect mice [4, 15] and humans [5, 16] against malaria in experimental challenge studies. Accordingly, despite the limited success of subunit-based malaria vaccines in clinical testing [17], there has been considerable interest in using radiation exposure to create an attenuated sporozoite-stage vaccine [14, 16]. In this study, we measured the effects of different doses of γ-irradiation on P. falciparum in short-term and long-term cultures. These studies were performed to evaluate both the immediate effect on survival and the ability of rare parasite populations to recover and repopulate. We also studied the effect of γ-irradiation on parasite structure, by fluorescence microscopy and transmission electron microscopy. In addition, we performed extensive genome-wide transcriptional profiling in P. falciparum exposed to increasing doses of γ-radiation, to identify parasite proteins that could be correlated with survival and cell death, and, on the basis of the data set, created a functionally diverse molecular signature of growth attenuation. Last, bioinformatic analyses were performed to decipher the biological systems altered by γ-irradiation that are associated with growth regulation and survival.

The state of Texas has one of the greatest records of pterosaurs in the world, surpassing all other US states and most countries in the number of occurrences. Uniquely, this record extends over the entire 150+ million history of the Pterosauria. A review of this pterosaur record confirms at least 30 pterosaurs known from 13 occurrences, including five valid species. The holotypes of two of these species have been described before and are diagnosed and erected here as the new speciesRadiodactylus langstoni, gen. et sp. nov., named in honour of Dr. Wann Langston Jr, the father of Texas pterosaurology, andAlamodactylus byrdi, gen. et sp. nov.. Phylogenetic analysis of all Texas pterosaurs that can be coded for more than one character confirms that these species are distinct from others and occupy phylogenetic positions close to their original classifications.Radiodactylus langstoniis recovered as a non-azhdarchid azhdarchoid,Quetzalcoatlus northropias an azhdarchid,Alamodactylus byrdias a non-pteranodontoid pteranodontian,Aetodactylusas a pteranodontoid, andColoborhynchus wadleighias an ornithocheirid. The presence of eudimorphodontid, dsungaripterid, as well as other azhdarchid and pteranodontoid pterosaurs, is also confirmed in Texas.

Despite its clinical significance, the mechanisms of joint morphogenesis are still elusive. Here, we show by combining laser-capture microdissection for RNA sampling with microarray analysis, that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by down-regulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in-vivo, ex-vivo and in-vitro approaches, we showed that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2Prx1KO), which lack joint development and fail chondrocyte hypertrophy, showed up-regulation of interzone-MCP-5. In-vivo and ex-vivo blockade of the sole MCP-5 receptor, CCR2, in Tgfbr2Prx1KO led to rescue of joint formation and growth plate maturation; while in control mice determined an acceleration of endochondral growth plate mineralization. Taken together, we characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.

Genetic variation on the Y chromosome has not been convincingly implicated in prostate cancer risk. To comprehensively analyze the role of inherited Y chromosome variation in prostate cancer risk in individuals of European ancestry, we genotyped 34 binary Y chromosome markers in 3,995 prostate cancer cases and 3,815 control subjects drawn from four studies. In this set, we identified nominally significant association between a rare haplogroup, E1b1b1c, and prostate cancer in stage I (P = 0.012, OR = 0.51; 95% confidence interval 0.30–0.87). Population substructure of E1b1b1c carriers suggested Ashkenazi Jewish ancestry, prompting a replication phase in individuals of both European and Ashkenazi Jewish ancestry. The association was not significant for prostate cancer overall in studies of either Ashkenazi Jewish (1,686 cases and 1,597 control subjects) or European (686 cases and 734 control subjects) ancestry (Pmeta = 0.078), but a meta-analysis of stage I and II studies revealed a nominally significant association with prostate cancer risk (Pmeta = 0.010, OR = 0.77; 95% confidence interval 0.62–0.94). Comparing haplogroup frequencies between studies, we noted strong similarities between those conducted in the US and France, in which the majority of men carried R1 haplogroups, resembling Northwestern European populations. On the other hand, Finns had a remarkably different haplogroup distribution with a preponderance of N1c and I1 haplogroups. In summary, our results suggest that inherited Y chromosome variation plays a limited role in prostate cancer etiology in European populations but warrant follow-up in additional large and well characterized studies of multiple ethnic backgrounds. Electronic supplementary material The online version of this article (doi:10.1007/s00439-012-1139-5) contains supplementary material, which is available to authorized users.

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1–dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.

The presence of pancreatic cancer (PC) in melanoma-prone families has been consistently associated with an increased frequency of CDKN2A mutations, the major high-risk susceptibility gene identified for melanoma. However, the precise relationship between CDKN2A, melanoma and PC remains unknown. We evaluated a recently identified PC susceptibility gene PALB2 using both sequencing and tagging to determine whether PALB2 might explain part of the relationship between CDKN2A, melanoma, and PC. No disease-related mutations were identified from sequencing PALB2 in multiple pancreatic cancer patients or other mutation carrier relatives of PC patients from the eight melanoma-prone families with CDKN2A mutations and PC. In addition, no significant associations were observed between 11 PALB2 tagging SNPs and melanoma risk in 23 melanoma-prone families with CDKN2A mutations or the subset of 11 families with PC or PC-related CDKN2A mutations. The results suggested that PALB2 does not explain the relationship between CDKN2A, melanoma, and pancreatic cancer in these melanoma-prone families.

Despite the long-appreciated in vivo role of the redox-active virulence factor pyocyanin in Pseudomonas airway infections and the importance of airway epithelial cells in combating bacterial pathogens, little is known about pyocyanin's effect on airway epithelial cells. We find that exposure of bronchiolar epithelial cells to pyocyanin results in MUC2/MUC5AC induction and mucin secretion through release of inflammatory cytokines and growth factors (interleukin (IL)-1β, IL-6, heparin-bound epidermal growth factor, tissue growth factor-α, tumor necrosis factor-α) that activate the epidermal growth factor receptor pathway. These changes are mediated by reactive oxygen species produced by pyocyanin. Microarray analysis identified 286 pyocyanin-induced genes in airway epithelial cells, including many inflammatory mediators elevated in cystic fibrosis (granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF, chemokine (C-X-C motif) ligand 1 (CXCL1), serum amyloid, IL-23) and several novel pyocyanin-responsive genes of potential importance in the infection process (IL-24, CXCL2, CXCL3, CCL20, CXCR4). This comprehensive study uncovers numerous details of pyocyanin's proinflammatory action and establishes airway epithelial cells as key responders to this microbial toxin.

Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood captures the molecular and immunological events associated with the pathogenesis of disease. We find that during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore, our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory markers. Complement component C1q, β-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell antigen, DnaJC, member 15, glutathione S -transferase omega-1, and thymidine kinase 1 were overexpressed in blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM in blood. A subset of these mediators of ECM warrant validation in P. falciparum -infected young African children as diagnostic markers of CM.

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.

The chimpanzee is the only animal model for investigating the pathogenesis of viral hepatitis types A through E in humans. Studies of the host response, including microarray analyses, have relied on the close relationship between these two primate species: chimpanzee samples are commonly tested with human-based reagents. In this study, the host responses to two dissimilar viruses, hepatitis E virus (HEV) and hepatitis C virus (HCV), were compared in multiple experimentally infected chimpanzees. Affymetrix U133 + 2.0 human microarray chips were used to assess the entire transcriptome in serial liver biopsies obtained over the course of the infections. Respecting the limitations of microarray probes designed for human target transcripts to effectively assay chimpanzee transcripts, we conducted probe-level analysis of the microarray data in conjunction with a custom mapping of the probe sequences to the most recent human and chimpanzee genome sequences. Time points for statistical comparison were chosen based on independently measured viremia levels. Regardless of the viral infection, the alignment of differentially expressed genes to the human genome sequence resulted in a larger number of genes being identified when compared with alignment to the chimpanzee genome sequence. This probably reflects the lesser refinement of gene annotation for chimpanzees. In general, the two viruses demonstrated very distinct temporal changes in host response genes, although both RNA viruses induced genes that were involved in many of the same biological systems, including interferon-induced genes. The host response to HCV infection was more robust in the magnitude and number of differentially expressed genes compared to HEV infection.

Remains of a pteranodontid pterosaur are recorded in the basal Austin Group of North Texas. The specimen described here comprises a partial left wing and strongly resemblesPteranodonalthough diagnostic features of that genus are lacking. With an estimated early Coniacian age, this specimen represents the earliest occurrence of the Pteranodontidae in North America and the second earliest occurrence worldwide, predated only byOrnithostomafrom the Cambridge Greensand of England. Pterosaur material recovered from the Eagle Ford and Austin groups of Texas records an early Late Cretaceous change in the composition of North American pterosaur communities between the late Cenomanian and the early Coniacian. This faunal transition appears to be primarily a decrease in morphological disparity rather than a significant reduction in taxonomic diversity. However, the lack of Early CretaceousLagerstättenin North America may produce underestimates of true pterosaur richness during this interval, thereby obscuring a subsequent drop in diversity.