Profilin1 regulates invadopodium maturation in human breast cancer cells.

Invadopodia are actin-driven membrane protrusions that show oscillatory assembly and disassembly causing matrix degradation to support invasion and dissemination of cancer cells in vitro and in vivo. Profilin1, an actin and phosphoinositide binding protein, is downregulated in several adenocarcinomas and it is been shown that its depletion enhances invasiveness and motility of breast cancer cells by increasing PI(3,4)P2 levels at the leading edge. In this study, we show for the first time that depletion of profilin1 leads to an increase in the number of mature invadopodia and these assemble and disassemble more rapidly than in control cells. Previous work by Sharma et al. (2013a), has shown that the binding of the protein Tks5 with PI(3,4)P2 confers stability to the invadopodium precursor causing it to mature into a degradation-competent structure. We found that loss of profilin1 expression increases the levels of PI(3,4)P2 at the invadopodium and as a result, enhances recruitment of the interacting adaptor Tks5. The increased PI(3,4)P2-Tks5 interaction accelerates the rate of invadopodium anchorage, maturation, and turnover. Our results indicate that profilin1 acts as a molecular regulator of the levels of PI(3,4)P2 and Tks5 recruitment in invadopodia to control the invasion efficiency of invadopodia.

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo.

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Detection of West Nile virus-specific antibodies and nucleic acid in horses and mosquitoes, respectively, in Nuevo Leon State, northern Mexico, 2006-2007.

In the last 5 years, there has been only one reported human case of West Nile virus (WNV) disease in northern Mexico. To determine if the virus was still circulating in this region, equine and entomological surveillance for WNV was conducted in the state of Nuevo Leon in northern Mexico in 2006 and 2007. A total of 203 horses were serologically assayed for antibodies to WNV using an epitope-blocking enzyme-linked immunosorbent assay (bELISA). Seroprevalences for WNV in horses sampled in 2006 and 2007 were 26% and 45%, respectively. Mosquito collections in 2007 produced 7365 specimens representing 15 species. Culex mosquitoes were screened for WNV RNA and other genera (Mansonia, Anopheles, Aedes, Psorophora and Uranotaenia) were screened for flaviviruses using reverse-transcription (RT)-PCR. Two pools consisting of Culex spp. mosquitoes contained WNV RNA. Molecular species identification revealed that neither pool included Culex quinquefasciatus (Say) (Diptera:Culicidae) complex mosquitoes. No evidence of flaviviruses was found in the other mosquito genera examined. These data provide evidence that WNV is currently circulating in northern Mexico and that non-Cx. quinquefasciatus spp. mosquitoes may be participating in the WNV transmission cycle in this region.

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity.

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.

Interest in health information exchange in ambulatory care: a statewide survey.

OBJECTIVE: Assess the interest in and preferences of ambulatory practitioners in HIE. BACKGROUND: Health information exchange (HIE) may improve the quality and efficiency of care. Identifying the value proposition for smaller ambulatory practices may help those practices engage in HIE. METHODS: Survey of primary care and specialist practitioners in the State of Colorado. RESULTS: Clinical data were commonly (always [2%], often [29%] or sometimes [49%]) missing during clinic visits. Of 12 data types proposed as available through HIE, ten were considered "extremely useful" by most practitioners. "Clinical notes/consultation reports," "diagnosis or problem lists," and "hospital discharge summaries" were considered the three most useful data types. Interest in EKG reports, diagnosis/problem lists, childhood immunizations, and discharge summaries differed among ambulatory practitioner groups (primary care, obstetrics-gynecology, and internal medicine subspecialties). CONCLUSION: Practitioners express strong interest in most of the data types, but opinions differed by specialties on what types were most important. All providers felt that a system that provided all data types would be useful. These results support the potential benefit of HIE in ambulatory practices.

Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the diagnosis of West Nile virus infections in humans.

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.

Entomological studies along the Colorado Front Range during a period of intense West Nile virus activity.

To better understand the ecology of West Nile virus transmission in Northern Colorado, field studies were conducted in Larimer and Weld counties from September 2003 through March 2005. During summer studies, 18,540 adult mosquitoes were collected using light traps and gravid traps. West Nile virus RNA was detected in 24 of the 2,140 mosquito pools tested throughout the study area in 2003 and 2004. Culex tarsalis had the highest minimum infection rate (MIR) in both 2003 (MIR = 34.48) and in 2004 (MIR = 8.74). During winter studies, 9,391 adult mosquitoes were collected by aspirator from various overwintering sites including bridges and storm drains. The most frequently collected species was Culex pipiens. West Nile virus was not detected in our overwintering collections. The relationship between spring adult emergence and temperature inside and outside overwintering sites is described. Species composition of collections as well as the spatial and temporal distribution of West Nile virus detections are presented.

Effect of La Crosse virus infection on insemination rates in female Aedes triseriatus (Diptera: Culicidae).

Aedes triseriatus (Say) (Diptera: Culicidae) females orally infected with La Crosse virus after ingesting an infectious bloodmeal were compared for mating efficiency with females that ingested a noninfectious bloodmeal. After 14-d extrinsic incubation to allow for dissemination of the infection, all females were offered a second noninfectious bloodmeal and were placed in cages with age-matched males for 5 d. After 6 d, insemination rates were determined by detection of sperm in the spermathecae. Insemination rates of the La Crosse virus-infected females were significantly greater than in uninfected females.

Dengue 2 genotypes in the state of Oaxaca, Mexico.

To genetically characterize dengue 2 (DEN-2) viruses in Oaxaca, Mexico, the C protein, and a portion of the prM protein genes of 8 isolates from the 2001 DEN epidemic were sequenced. The sequences were compared to those of prototype DEN-2 viruses from various parts of the world. Phylogenetic analysis suggested that the 2001 isolates of DEN-2 were of the American/Asian genotype and were most similar to the Jamaica and Venezuelan isolates MARA3, LARD1996 and LARD1910. Molecular analyses confirmed the origin of the isolates. This study indicates that DEN-2 strains of American/Asian genotype probably from Southeast Asian are circulating in Oaxaca.

Control of arbovirus diseases: is the vector the weak link?

Arthropod-borne virus (arbovirus) diseases (ABVDs) remain major threats to human health and well-being and, as an epidemiologic group, inflict an unacceptable health and economic burden on humans and animals, including livestock. The developed world has been fortunate to have escaped much of the burden that arboviruses and their arthropod vectors inflict on humans in disease endemic countries, but the introduction and rapid spread of West Nile virus in the Western Hemisphere demonstrated that we can no longer be complacent in the face of these emerging and resurging vector-borne diseases. Unfortunately, as the burdens and threats of ABVDs have increased, the U.S. and international public health capacity to address them has decreased. Vaccines are not available for most of these agents. Previously successful strategies to control ABVDs emphasized vector control, but source reduction and vector control strategies using pesticides have not been sustainable. New insights into vector biology and vector pathogen interactions, and the novel targets that likely will be forthcoming in the vector post-genomics era, provide new targets and opportunities for vector control and disease reduction programs. These findings and approaches must be incorporated into existing strategies if we are to control these important pathogens.

Recurrent early childhood injuries among disadvantaged children in primary care settings.

OBJECTIVE: To examine differences in social risk factors and health care use between young children with and without recurrent injuries. DESIGN: Retrospective cohort study using administrative claims and medical records. Children with no, one, or more than one injury were compared. SETTING: Integrated public healthcare system. SUBJECTS: All children born at Denver Health Medical Center (DHMC) in 1993 who continued care there beyond 15 months of age. Children were followed to 36 months. OUTCOME MEASURES: Sociodemographic information, service use, injury episodes, cause of injury, and social risk factors. RESULTS: 371 injury episodes occurred among 817 children. In the study cohort, 7% had >1 injury episode, 26% had one injury, and 67% had none. Among children with >1 injury episode, 78% had at least one social risk factor compared with 63% of children with one injury and 52% of children with none (p<0.0001). Risk factors for >1 injury included maternal substance abuse (p = 0.0003), maternal age under 18 years (p = 0.04), a primary caregiver who was single (p<0.0001) or mentally ill (p = 0.03), and a history of family violence (p = 0.01). Multiple injury episodes were associated with increased non-injury service use, including primary care visits (p<0.0001), emergency department visits (p<0.0001), and total non-injury encounters (p<0.0001). CONCLUSIONS: Children with recurrent injury were more likely to have social risk factors, and used DHMC more frequently, than children with one or no injuries. Children with risk factors for recurrent injury can be identified and injury prevention counseling can be delivered to their families at their multiple visits to the system.

Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species.

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5'dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5'dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2-3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5'dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically.

Effect of mosquito salivary gland treatment on vesicular stomatitis New Jersey virus replication and interferon alpha/beta expression in vitro.

The sensitivity of vesicular stomatitis (VS) viruses to interferon (IFN)-mediated antiviral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN alpha/beta expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN alpha/beta response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN alpha/beta neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN alpha2 expression was reduced in L929 cells treated with SGH. Modulation of IFN alpha/beta by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.

Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti.

We have constructed an orally infectious Sindbis virus, ME2/5'2J/GFP, that expresses green fluorescent protein (GFP) in the midgut of Aedes aegypti and in other tissues as the virus disseminates. This virus has two unique features that are improvements over the SIN-based expression systems currently used in mosquitoes. First, a subgenomic RNA promoter and GFP coding sequence is located 5'- to the second subgenomic promoter and structural genes of the virus. Second, the E2 glycoprotein gene of TE/5'2J/GFP is replaced with the E2 gene of MRE16 SIN virus. The first feature enhances virus genome stability during virus dissemination from the midgut to other tissues and the second allows efficient virus entry into the midgut epithelial cells and then spread of the virus throughout the mosquito.

Climatic factors and the occurrence of vesicular stomatitis in New Mexico, United States of America.

Vesicular stomatitis (VS) outbreaks occurred in the southwestern United States of America in 1995, 1997 and 1998. The epidemiology of VS is not understood completely and some of the epidemiologic aspects of this disease are currently under investigation. In this study, daily maximum temperature, daily minimum temperature, daily mean temperature, daily mean relative humidity and daily total precipitation were collected at the Sevilleta Long Term Ecological Research site in central New Mexico. Discriminant analysis was used to identify the climatic variables best able to classify in which months VS would occur. The study found that the amounts of precipitation occurring two, ten, eleven and twelve months prior to the month in which cases were diagnosed, were the climatic variables that best described the occurrence of VS cases. The association of VS cases and precipitation suggests that, like numerous other arthropod-borne diseases, transmission of the disease-causing pathogen is linked to variations in climate.

Molecular characterization of California serogroup viruses isolated in Russia.

Nucleotide sequencing was used to characterize unidentified California (CAL) serogroup virus isolates from Russia. These viruses were isolated from mosquitoes and humans during epidemiologic investigations on the role of CAL serogroup viruses in the increased incidence of arboviral encephalitis in Russia. Most of the isolates were identified serologically as snowshoe hare (SSH), Inkoo (INK), and Tahyna (TAH) viruses, but some of the isolates were difficult to classify serologically, suggesting that they could be reassortant viruses. There is evidence that at least 2 of these viruses are not reassortant viruses. Sequence analysis revealed that the Russian viruses differ from other Eurasian and North American CAL serogroup viruses in all of the segments analyzed. They are most closely related to SSH virus. Whether they differ sufficiently to be considered a new group of SSH-like viruses remains to be determined.

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes.

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.

DeLee suction. Does it have clinical significance?

OBJECTIVE: To evaluate the null hypothesis that there would be no difference in neonatal outcome in infants who underwent DeLee suction vs. those who underwent bulb suction when thick meconium was present. This difference was defined as a one-day difference in length of stay, two-point difference in the five-minute Apgar score or significant difference in blood gases. STUDY DESIGN: A power calculation based on a one-day length of stay found that 115 patients would be needed in each arm. A cohort of 115 consecutive deliveries with the presence of thick meconium was reviewed during each time period when DeLee suction was utilized and then matched with deliveries that used bulb suction. RESULTS: Two hundred thirty charts were reviewed. Length of stay, one-minute Apgar score, five-minute Apgar score, blood gases, gestational age and incidence of amnioinfusion were compared. There was no significant difference in any variable. Meconium aspiration syndrome in the period studied remained stable at < 1%. CONCLUSION: There was no significant clinical change in neonatal outcome with the abandonment of nasopharyngeal DeLee suction on the perineum when meconium was present. We recommend the use of bulb suction as routine obstetric practice even in the presence of thick meconium.

Molecular cloning and characterization of a metal responsive Chironomus tentans alpha-tubulin cDNA.

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Sensitive molecular biomarkers of metal exposure are sorely needed. We have isolated a cDNA from the midge Chironomus tentans that is transcribed in all organs and developmental stages. The cDNA encodes a protein, designated Chironomus tentans alpha-tubulin 1 (CTTUB1), which has significant similarities with invertebrate and vertebrate alpha-tubulins. CTTUB1 is abundantly transcribed in embryos and to a lesser extent in adults and larvae. CTTUB1 RNA and protein abundances are increased in larvae exposed to copper or cadmium. The pattern of cellular distribution of CTTUB1 protein in the midgut epithelial cells was radically affected by cadmium. In the midgut cells of unexposed larvae, CTTUB1 was found evenly distributed throughout the cytoplasm, while in cadmium-exposed larvae, CTTUB1 was mostly concentrated along the basolateral plasma membrane. A mechanism for the regulation of alpha-tubulin synthesis by cadmium is proposed. This is the first report on the isolation of a metal responsive gene from a neartic aquatic insect.

Sindbis virus-induced silencing of dengue viruses in mosquitoes.

Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN-2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN-2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN-derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN-2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod-borne viruses in mosquito vectors, studying vector-virus interactions, and silencing endogenous mosquito genes.