Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.     

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.     

Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples

Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.     

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

Background

Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’).

Methodology/Principal Findings

Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0.

Conclusions/Significance

Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.     

Etoposide Induces Nuclear Re-Localisation of AID

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.     

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

Exquisite Light Sensitivity of Drosophila melanogaster Cryptochrome

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.     

Focused Ultrasound-Induced Blood–Brain Barrier Opening to Enhance Temozolomide Delivery for Glioblastoma Treatment: A Preclinical Study

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.     

Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC₅₀ in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.     

Indirect Estimates of Total Fertility Rate Using Child Woman/Ratio: A Comparison with the Bogue-Palmore Method

Indirect estimation methodologies of the total fertility rate (TFR) have a long history within demography and have provided important techniques applied demographers can use when data is sparse or lacking. However new methodologies for approximating the total fertility rate have not been proposed in nearly 30 years. This study presents a novel method for indirectly approximating the total fertility rate using an algebraic rearrangement of the general fertility rate (GFR) through the known relationship between GFR and TFR. It then compares the proposed method to the well-known Bogue-Palmore method. These methods are compared in 196 countries and include overall errors as well as characteristics of the countries that contribute to fertility behavior. Additionally, these methods were compared geographically to find any geographical patterns. We find this novel method is not only simpler than the Bogue-Palmore method, requiring fewer data inputs, but also has reduced algebraic and absolute errors when compared with the Bogue-Palmore method and specifically outperforms the Bogue-Palmore method in developing countries. We find that our novel method may be useful estimation procedure for demographers.