Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

From gametogenesis to spawning: How climate-driven warming affects teleost reproductive biology

Ambient temperature modulates reproductive processes, especially in poikilotherms such as teleosts. Consequently, global warming is expected to impact the reproductive function of fish, which has implications for wild population dynamics, fisheries and aquaculture. In this extensive review spanning tropical and cold-water environments, we examine the impact of higher-than-optimal temperatures on teleost reproductive development and physiology across reproductive stages, species, generations and sexes. In doing so, we demonstrate that warmer-than-optimal temperatures can affect every stage of reproductive development from puberty through to the act of spawning, and these responses are mediated by age at spawning and are associated with changes in physiology at multiple levels of the brain–pituitary–gonad axis. Response to temperature is often species-specific and changes with environmental history/transgenerational conditioning, and the amplitude, timing and duration of thermal exposure within a generation. Thermally driven changes to physiology, gamete development and maturation typically culminate in poor sperm and oocyte quality, and/or advancement/delay/inhibition of ovulation/spermiation and spawning. Although the field of teleost reproduction and temperature is advanced in many respects, we identify areas where research is lacking, especially for males and egg quality from “omics” perspectives. Climate-driven warming will continue to disturb teleost reproductive performance and therefore guide future research, especially in the emerging areas of transgenerational acclimation and epigenetic studies, which will help to understand and project climate change impacts on wild populations and could also have implications for aquaculture.     

ADAM9 silencing inhibits breast tumor cell invasion in vitro

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.     

The tyrosine‐sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner

The tyrosine‐sulfated peptides PSKα and PSY1 bind to specific leucine‐rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss‐of‐function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen‐associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild‐type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate‐dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, ‐2 and PSY1‐receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst‐1 mutants partially restore the defense‐related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth‐promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.     

Pyrido[2,3-d]pyrimidines: Discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.     

Asparaginase Potentiates Glucocorticoid-Induced Osteonecrosis in a Mouse Model

Osteonecrosis is a common dose-limiting toxicity of glucocorticoids. Data from clinical trials suggest that other medications can increase the risk of glucocorticoid-induced osteonecrosis. Here we utilized a mouse model to study the effect of asparaginase treatment on dexamethasone-induced osteonecrosis. Mice receiving asparaginase along with dexamethasone had a higher rate of osteonecrosis than those receiving only dexamethasone after 6 weeks of treatment (44% vs. 10%, P = 0.006). Similarly, epiphyseal arteriopathy, which we have shown to be an initiating event for osteonecrosis, was observed in 58% of mice receiving asparaginase and dexamethasone compared to 17% of mice receiving dexamethasone only (P = 0.007). As in the clinic, greater exposure to asparaginase was associated with greater plasma exposure to dexamethasone (P = 0.0001). This model also recapitulated other clinical risk factors for osteonecrosis, including age at start of treatment, and association with the systemic exposure to dexamethasone (P = 0.027) and asparaginase (P = 0.036). We conclude that asparaginase can potentiate the osteonecrotic effect of glucocorticoids.     

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI^-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI^-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI^-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.     

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines

Objective

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods

Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results

Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions

We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.