The development of a reverse genetics system for the study of chikungunya pathogenesis in the mouse model

The genus Alphavirus is one of two within the family Togaviridae and comprises approximately 30 species, including several pathogens of humans and other animals that are of medical, veterinary and economic importance. Most alphaviruses are maintained in nature by a biological transmission cycle between susceptible vertebrate hosts and blood-feeding arthropods such as mosquitoes. In common with many Old World alphavirus pathogens Chikungunya virus (CHIKV) is the aetiological agent of a syndrome characterized by fever, skin rash and acute or chronic poly-arthralgia or arthritis. Whereas previously CHIKV outbreaks were sporadic and self-limiting, in 2004 it re-emerged in coastal Kenya and there followed a series of outbreaks that have continued until the present day, resulting in many millions of cases. Inroads into understanding the pathogenesis of CHIKV disease were until recently hampered through the lack of a convenient small animal model capable of exhibiting symptoms similar to those observed in humans in response to viral challenge. By contrast, studies with other alphaviruses, most notably Sindbis and Semliki Forest viruses (SINV and SFV), have provided insights into many aspects of the infectious process including several key determinants of virulence. In the present study a reverse genetics system was developed to investigate the pathogenesis of CHIKV disease, the virus replication cycle and its interactions with susceptible hosts. This tool was used to investigate the hypothesis that a CHIKV encoded virulence factor is located within the cleavage domain of the non-structural polyprotein precursor of the viral replicase. Results from this study indicate that a unique amino acid within this conserved site is instrumental in contributing the inhibition of the type 1 IFN response in infected hosts. It was also shown that the type 1 IFN response was induced at an earlier stage in mice challenged with virus containing an engineered mutation at this site and that joint-swelling was less severe than with wild-type virus

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics

© 2019 Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons

Reproductive Phase Locking of Mosquito Populations in Response to Rainfall Frequency

The frequency of moderate to heavy rainfall events is projected to change in response to global warming. Here we show that these hydrologic changes may have a profound effect on mosquito population dynamics and rates of mosquito-borne disease transmission. We develop a simple model, which treats the mosquito reproductive cycle as a phase oscillator that responds to rainfall frequency forcing. This model reproduces observed mosquito population dynamics and indicates that mosquito-borne disease transmission can be sensitive to rainfall frequency. These findings indicate that changes to the hydrologic cycle, in particular the frequency of moderate to heavy rainfall events, could have a profound effect on the transmission rates of some mosquito-borne diseases

Geographic Variation of Strontium and Hydrogen Isotopes in Avian Tissue: Implications for Tracking Migration and Dispersal

Background: Isotopes can provide unique solutions to fundamental problems related to the ecology and evolution of migration and dispersal because prior movements of individuals can theoretically be tracked from tissues collected from a single capture. However, there is still remarkably little information available about how and why isotopes vary in wild animal tissues, especially over large spatial scales. Methodology/Principal Findings: Here, we describe variation in both stable-hydrogen (dDF) and strontium ( 87Sr/86SrF) isotopic compositions in the feathers of a migratory songbird, the Tree Swallow (Tachycineta bicolor), across 18 sampling sites in North America and then examine potential mechanisms driving this variation. We found that dDF was correlated with latitude of the sampling site, whereas 87Sr/86SrF was correlated with longitude. dDF was related to dD of meteoric waters where molting occurred and 87Sr/86SrF was influenced primarily by the geology in the area where feathers were grown. Using simulation models, we then assessed the utility of combining both markers to estimate the origin of individuals. Using 13 geographic regions, we found that the number of individuals correctly assigned to their site of origin increased from less than 40 % using either dD or 87Sr/86Sr alone to 74 % using both isotopes. Conclusions/Significance: Our results suggest that these isotopes have the potential to provide predictable an

Stable isotope analysis provides new information on winter habitat use of declining avian migrants that is relevant to their conservation

Winter habitat use and the magnitude of migratory connectivity are important parameters when assessing drivers of the marked declines in avian migrants. Such information is unavailable for most species. We use a stable isotope approach to assess these factors for three declining African-Eurasian migrants whose winter ecology is poorly known: wood warbler Phylloscopus sibilatrix, house martin Delichon urbicum and common swift Apus apus. Spatially segregated breeding wood warbler populations (sampled across a 800 km transect), house martins and common swifts (sampled across a 3,500 km transect) exhibited statistically identical intra-specific carbon and nitrogen isotope ratios in winter grown feathers. Such patterns are compatible with a high degree of migratory connectivity, but could arise if species use isotopically similar resources at different locations. Wood warbler carbon isotope ratios are more depleted than typical for African-Eurasian migrants and are compatible with use of moist lowland forest. The very limited variance in these ratios indicates specialisation on isotopically restricted resources, which may drive the similarity in wood warbler populations' stable isotope ratios and increase susceptibility to environmental change within its wintering grounds. House martins were previously considered to primarily use moist montane forest during the winter, but this seems unlikely given the enriched nature of their carbon isotope ratios. House martins use a narrower isotopic range of resources than the common swift, indicative of increased specialisation or a relatively limited wintering range; both factors could increase house martins' vulnerability to environmental change. The marked variance in isotope ratios within each common swift population contributes to the lack of population specific signatures and indicates that the species is less vulnerable to environmental change in sub-Saharan Africa than our other focal species. Our findings demonstrate how stable isotope research can contribute to understanding avian migrants' winter ecology and conservation status

Attitude toward contraception and abortion among Curaçao women. Ineffective contraception due to limited sexual education?

Background In Curaçao is a high incidence of unintended pregnancies and induced abortions. Most of the induced abortions in Curaçao are on request of the woman and performed by general practitioners. In Curaçao, induced abortion is strictly prohibited, but since 1999 there has been a policy of connivance. We present data on the relevance of economic and socio-cultural factors for the high abortion-rates and the ineffective use of contraception. Methods Structured interviews to investigate knowledge and attitudes toward sexuality, contraception and abortion and reasons for ineffective use of contraceptives among women, visiting general practitioners. Results Of 158 women, 146 (92%) participated and 82% reported that their education on sexuality and about contraception was of good quality. However 'knowledge of reliable contraceptive methods' appeared to be - in almost 50% of the cases - false information, misjudgements or erroneous views on the chance of getting pregnant using coitus interruptus and about the reliability and health effects of oral contraceptive pills. Almost half of the interviewed women had incorrect or no knowledge about reliability of condom use and IUD. 42% of the respondents risked by their behavior an unplanned pregnancy. Most respondents considered abortion as an emergency procedure, not as contraception. Almost two third experienced emotional, physical or social problems after the abortion. Conclusions Respondents had a negative attitude toward reliable contraceptives due to socio-cultural determined ideas about health consequences and limited sexual education. Main economic factors were costs of contraceptive methods, because most health insurances in Curaçao do not cover contraceptives. To improve the effective use of reliable contraceptives, more adequate information should be given, targeting the wrong beliefs and false information. The government should encourage health insurance companies to reimburse contraceptives. Furthermore, improvement of counseling during the abortion procedure is important

Identification of Potential Therapeutic Drugs for Huntington's Disease using Caenorhabditis elegans

The prolonged time course of Huntington's disease (HD) neurodegeneration increases both the time and cost of testing potential therapeutic compounds in mammalian models. An alternative is to initially assess the efficacy of compounds in invertebrate models, reducing time of testing from months to days.We screened candidate therapeutic compounds that were identified previously in cell culture/animal studies in a C. elegans HD model and found that two FDA approved drugs, lithium chloride and mithramycin, independently and in combination suppressed HD neurotoxicity. Aging is a critical contributor to late onset neurodegenerative diseases. Using a genetic strategy and a novel assay, we demonstrate that lithium chloride and mithramycin remain neuroprotective independent of activity of the forkhead transcription factor DAF-16, which mediates the effects of the insulin-like signaling pathway on aging.These results suggest that pathways involved in polyglutamine-induced degeneration are distinct from specific aging pathways. The assays presented here will be useful for rapid and inexpensive testing of other potential HD drugs and elucidating pathways of drug action. Additionally, the neuroprotection conferred by lithium chloride and mithramycin suggests that these drugs may be useful for polyglutamine disease therapy

Antiangiogenic agents in the treatment of recurrent or newly diagnosed glioblastoma: Analysis of single-agent and combined modality approaches

Surgical resection followed by radiotherapy and temozolomide in newly diagnosed glioblastoma can prolong survival, but it is not curative. For patients with disease progression after frontline therapy, there is no standard of care, although further surgery, chemotherapy, and radiotherapy may be used. Antiangiogenic therapies may be appropriate for treating glioblastomas because angiogenesis is critical to tumor growth. In a large, noncomparative phase II trial, bevacizumab was evaluated alone and with irinotecan in patients with recurrent glioblastoma; combination treatment was associated with an estimated 6-month progression-free survival (PFS) rate of 50.3%, a median overall survival of 8.9 months, and a response rate of 37.8%. Single-agent bevacizumab also exceeded the predetermined threshold of activity for salvage chemotherapy (6-month PFS rate, 15%), achieving a 6-month PFS rate of 42.6% (p < 0.0001). On the basis of these results and those from another phase II trial, the US Food and Drug Administration granted accelerated approval of single-agent bevacizumab for the treatment of glioblastoma that has progressed following prior therapy. Potential antiangiogenic agents-such as cilengitide and XL184-also show evidence of single-agent activity in recurrent glioblastoma. Moreover, the use of antiangiogenic agents with radiation at disease progression may improve the therapeutic ratio of single-modality approaches. Overall, these agents appear to be well tolerated, with adverse event profiles similar to those reported in studies of other solid tumors. Further research is needed to determine the role of antiangiogenic therapy in frontline treatment and to identify the optimal schedule and partnering agents for use in combination therapy

A Yersinia Effector with Enhanced Inhibitory Activity on the NF-κB Pathway Activates the NLRP3/ASC/Caspase-1 Inflammasome in Macrophages

A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJKIM) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJKIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJKIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJKIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJCO92, YopJKIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJKIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJCO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways