Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

<p>Abstract</p> <p>Background</p> <p>The <it>Anopheles gambiae </it>heat shock cognate gene (<it>hsc70B</it>) encodes a constitutively expressed protein in the <it>hsp70 </it>family and it functions as a molecular chaperone for protein folding. However, the expression of <it>hsc70B </it>can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the <it>An. gambiae hsc70B </it>is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of <it>hsc70B </it>by ONNV infection in <it>An. gambiae</it>, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of <it>hsc70B </it>into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses.</p> <p>Results</p> <p>Serial deletion analysis of the <it>hsc70B </it>upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the <it>hsc70B </it>starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor κB (NF-κB), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the <it>hsc70B </it>promoter activity in transfected Vero cells.</p> <p>Conclusion</p> <p>Our results further validate the transcriptional activation of <it>hsc70B </it>during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection.</p

Anopheles gambiae heat shock protein cognate 70B impedes o'nyong-nyong virus replication

Background Phylogenetic and functional analysis was conducted on an Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1α (EF-1α) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1α and the agglutinin functions in ONNV replication in An. gambiae. Results Among these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of β-galactosidase (dsβ-gal). ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with dsβ-gal treated mosquitoes. Conclusion These results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector, An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.We wish to thank Dr. K. E. Olson and B. D. Foy for his kind gift of infectious clone pONNic-Foy. This research would not have been possible without the assistance of Dr. Mabel Berois with helpful guide. This project was supported by grants R01-AI44273 from NIH/NIAID to F.H.C. D.L.V. was supported by NIH T32 A10753

Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination

Yersinia pestis is a Gram-negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen-presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium-dependent (C-type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin-expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin-expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin-expressing transfectants is inhibited by purified Langerin, a DC-SIGN (DC-specific intercellular adhesion molecule 3 grabbing nonintegrin)-like molecule, an anti-CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin-mediated binding of Y. pestis to APCs may promote its dissemination and infection.Peer reviewe

Genomic Resources Notes Accepted 1 August 2014–30 September 2014

This article documents the public availability of (i) transcriptome sequence data, assembly and annotation, and single nucleotide polymorphisms ( SNP s) for the cone snail Conus miliaris ; (ii) a set of SNP markers for two biotypes from the Culex pipiens mosquito complex; (iii) transcriptome sequence data, assembly and annotation for the mountain fly Drosophila nigrosparsa ; (iv) transcriptome sequence data, assembly and annotation and SNP s for the Neotropical toads Rhinella marina and R. schneideri ; and (v) partial genomic sequence assembly and annotation for 35 spiny lizard species (Genus Sceloporus ).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/1/men12340-sup-0004-AppendixS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/2/men12340-sup-0003-AppendixS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/3/men12340-sup-0002-AppendixS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/4/men12340-sup-0005-AppendixS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/5/men12340.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110107/6/men12340-sup-0001-AppendixS1.pd

DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, AST/Pfizer Basic Science Faculty Development Grant, Ministerio de Educacióny Ciencia SAF2010-15062, SAF2013-48834-R, and Fundación Mutua Madrileñ a grants to J.O. A portion of this work appears as part of the doctoral thesis of P.C.S

Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1+ precursors and postnatally from bone marrow–derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche

Data from: "Identification and assessment of single nucleotide polymorphisms (SNPs) between Culex complex mosquitoes." in Genomic Resources Notes Accepted 1 August 2014-30 September 2014

Culex pipiens complex mosquitoes are important vectors for many human pathogens including West Nile encephalitis, Rift Valley fever and Lymphatic filariasis. In this study we characterized a set of SNP markers between two biotypes of the Culex pipiens complex, Culex pipiens form molestus and Culex pipiens form pipiens, for use in a high-resolution genetic mapping and population genetics. DNA pooled from 10 specimens of each biotype were sequenced and analyzed for variation in 28 genes. The total of 4714 bp across orthologs of C. pipiens form pipiens and C. pipiens form molestus revealed 44 SNPs in the 3279 bp coding regions, 48 SNPs in the 1435 bp non-coding regions, and 10 indels. The ratio of transitions to transversions approached 2:1, with transitions constituting the majority of synonymous coding substitutions. The informative SNP markers were successfully identified and assessed from both C. pipiens biotypes. We expect that novel SNPs characterized in this study would be useful for genetic studies to elucidate the genetic basis of diverged eco-physiological traits between the two biotypes of the C. pipiens complex

08.25.14 - Dryad submission

08.25.14 - Dryad submissio

Comparative Transcriptomics Reveals Key Gene Expression Differences between Diapausing and Non-Diapausing Adults of Culex pipiens.

Diapause is a critical eco-physiological adaptation for winter survival in the West Nile Virus vector, Culex pipiens, but little is known about the molecular mechanisms that distinguish diapause from non-diapause in this important mosquito species. We used Illumina RNA-seq to simultaneously identify and quantify relative transcript levels in diapausing and non-diapausing adult females. Among 65,623,095 read pairs, we identified 41 genes with significantly different transcript abundances between these two groups. Transcriptome divergences between these two phenotypes include genes related to juvenile hormone synthesis, anaerobic metabolism, innate immunity and cold tolerance