Opposing roles of RNA receptors TLR3 and RIG-I in the inflammatory response to double-stranded RNA in a Kaposi's sarcoma cell line

Kaposi’s sarcoma (KS) is strongly associated with KS herpes virus infection, and inflammation plays an important role in this disease. We have shown that human KS biopsy-derived SLK cells, which are of endothelial origin and form KS-like tumors in nude mice, express the viral RNA pattern recognition receptors Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma-differentiation-associated gene 5 (MDA5). Furthermore, SLK cells have enhanced release of IL-6, IL-8 (CXCL8), RANTES (CCL5), and IP-10 (CXCL10) proteins in response to the synthetic viral RNA analog poly(I:C). SiRNA knockdowns demonstrated that TLR3 mediates this inflammatory response to poly(I:C) in SLK cells. Furthermore, knockdown of the RNA receptor RIG-I resulted in enhanced chemokine release, in a TLR3 pathway-dependent manner. Thus, exposure of KS cells to viral RNA ligands can result in a TLR3-mediated increase in the secretion of inflammatory proteins associated with KS cell growth that may contribute to disease.     

The joint allele-frequency spectrum in closely related species

We develop the theory for computing the joint frequency spectra of alleles in two closely related species. We allow for arbitrary population growth in both species after they had a common ancestor. We focus on the case in which a single chromosome is sequenced from one of the species. We use classical diffusion theory to show that, if the ancestral species was at equilibrium under mutation and drift and a chromosome from one of the descendant species carries the derived allele, the frequency spectrum in the other species is uniform, independently of the demographic history of both species. We also predict the expected densities of segregating and fixed sites when the chromosome from the other species carries the ancestral allele. We compare the predictions of our model with the site-frequency spectra of SNPs in the four HapMap populations of humans when the nucleotide present in the Neanderthal DNA sequence is ancestral or derived, using the chimp genome as the outgroup.     

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.     

Osteoactivin promotes breast cancer metastasis to bone

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor-negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone.     

Price of disorder in the lac repressor hinge helix

The Lac system of genes has been pivotal in understanding gene regulation. When the lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The structure of this region of the protein is well understood when it is in this bound conformation, but less so when it is not. Structural studies show that this region is flexible. Our simulations show this region is extremely flexible in solution; however, a high concentration of salt can help kinetically trap the hinge helix. Thermodynamically, disorder is more favorable without the DNA present.     

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.     

Eastern equine encephalitis virus rapidly infects and disseminates in the brain and spinal cord of cynomolgus macaques following aerosol challenge

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.     

Lymphocyte lineages at mucosal effector sites: rat salivary glands

Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3(-)/RT6(+) cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1(+)) and activated (Thy-1(-)/CD45RC(-)/RT6(-)) T cells. Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCRgammadelta(+) (28%), the CD8alphaalpha homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin alpha(E)beta(7). PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in alpha(E)beta(7)(+) T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.