Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine

Rethinking drug design in the artificial intelligence era

Artificial intelligence (AI) tools are increasingly being applied in drug discovery. While some protagonists point to vast opportunities potentially offered by such tools, others remain sceptical, waiting for a clear impact to be shown in drug discovery projects. The reality is probably somewhere in-between these extremes, yet it is clear that AI is providing new challenges not only for the scientists involved but also for the biopharma industry and its established processes for discovering and developing new medicines. This article presents the views of a diverse group of international experts on the 'grand challenges' in small-molecule drug discovery with AI and the approaches to address them

Soil Moisture and Fungi Affect Seed Survival in California Grassland Annual Plants

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival

Cancer survivors’ experiences of a community-based cancer-specific exercise programme: results of an exploratory survey

Purpose Exercise levels often decline following cancer diagnosis despite growing evidence of its benefits. Treatment side-effects, older age, lack of confidence and opportunity to exercise with others in similar circumstances influence this. Our study explored the experiences of people attending a cancer-specific community-based exercise programme (CU Fitter™). Methods A survey distributed to those attending the programme gathered demographic/clinical information, self-reported exercise levels, information provision and barriers to/benefits of exercise. Results Sixty surveys were evaluable from 65/100 returned (62% female, 68% >60yrs, 66% breast/prostate cancer). Most (68%) were receiving treatment. 68% attended classes once or twice weekly. 55% received exercise advice after diagnosis, usually from their hospital doctor/nurse. More (73%) had read about exercising, but less used the internet to source information (32%). Self-reported exercise levels were higher currently than before diagnosis (p=0.05). 48% said their primary barrier to exercising was the physical impact of cancer/treatment. Improving fitness/health (40%) and social support (16%) were the most important gains from the programme. Many (67%) had made other lifestyle changes and intented to keep (50%), or increase (30%) exercising. Conclusions This community-based cancer-specific exercise approach engaged people with cancer and showed physical, psychological, and social benefits. Implications for cancer survivors Community grown exercise initiatives bring cancer survivors together creating their own supportive environment. Combining this with instructors familiar with the population and providing an open-ended service may prove particularly motivating and beneficial. Further work is required to provide evidence for this

Multiple Dendritic Cell Populations Activate CD4+ T Cells after Viral Stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8α DC play a prominent, and sometimes exclusive, role in driving amplification of CD8+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4+ and CD8+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8α DC populations in the amplification of CD8+ and CD4+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8+ T cells are dominated by presentation of antigen by CD8α DC but can involve non-CD8α DC. In contrast, CD4+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity

Medulloblastoma outcome is adversely associated with overexpression of EEF1D, RPL30, and RPS20 on the long arm of chromosome 8

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. Improvements in clinical outcome require a better understanding of the genetic alterations to identify clinically significant biological factors and to stratify patients accordingly. In the present study, we applied cytogenetic characterization to guide the identification of biologically significant genes from gene expression microarray profiles of medulloblastoma. METHODS: We analyzed 71 primary medulloblastomas for chromosomal copy number aberrations (CNAs) using comparative genomic hybridization (CGH). Among 64 tumors that we previously analyzed by gene expression microarrays, 27 were included in our CGH series. We analyzed clinical outcome with respect to CNAs and microarray results. We filtered microarray data using specific CNAs to detect differentially expressed candidate genes associated with survival. RESULTS: The most frequent lesions detected in our series involved chromosome 17; loss of 16q, 10q, or 8p; and gain of 7q or 2p. Recurrent amplifications at 2p23-p24, 2q14, 7q34, and 12p13 were also observed. Gain of 8q is associated with worse overall survival (p = 0.0141), which is not entirely attributable to MYC amplification or overexpression. By applying CGH results to gene expression analysis of medulloblastoma, we identified three 8q-mapped genes that are associated with overall survival in the larger group of 64 patients (p < 0.05): eukaryotic translation elongation factor 1D (EEF1D), ribosomal protein L30 (RPL30), and ribosomal protein S20 (RPS20). CONCLUSION: The complementary use of CGH and expression profiles can facilitate the identification of clinically significant candidate genes involved in medulloblastoma growth. We demonstrate that gain of 8q and expression levels of three 8q-mapped candidate genes (EEF1D, RPL30, RPS20) are associated with adverse outcome in medulloblastoma

Molecular Investigations of a Locally Acquired Case of Melioidosis in Southern AZ, USA

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification

<i>Trypanosoma brucei</i> DHRF-TS revisited:characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase

<div><p>Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA^- <i>Escherichia coli</i>, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (<i>K</i>_i 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (<i>K</i>_i 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.</p></div

Bortezomib/docetaxel combination therapy in patients with anthracycline-pretreated advanced/metastatic breast cancer: a phase I/II dose-escalation study

The aim of this study was to determine the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of bortezomib plus docetaxel in patients with anthracycline-pretreated advanced/metastatic breast cancer. Forty-eight patients received up to eight 21-day cycles of docetaxel (60–100 mg m−2 on day 1) plus bortezomib (1.0–1.5 mg m−2 on days 1, 4, 8, and 11). Pharmacodynamic and pharmacokinetic analyses were performed in a subset of patients. Five patients experienced DLTs: grade 3 bone pain (n=1) and febrile neutropenia (n=4). The MTD was bortezomib 1.5 mg m−2 plus docetaxel 75 mg m−2. All 48 patients were assessable for safety and efficacy. The most common adverse events were diarrhoea, nausea, alopecia, asthenia, and vomiting. The most common grade 3/4 toxicities were neutropenia (44%), and febrile neutropenia and diarrhoea (each 19%). Overall patient response rate was 29%. Median time to progression was 5.4 months. In patients with confirmed response, median time to response was 1.3 months and median duration of response was 3.2 months. At the MTD, response rate was 38%. Pharmacokinetic characteristics of bortezomib/docetaxel were comparable with single-agent data. Addition of docetaxel appeared not to affect bortezomib inhibition of 20S proteasome activity. Mean alpha-1 acid glycoprotein concentrations increased from baseline at nearly all time points across different bortezomib dose levels. Bortezomib plus docetaxel is an active combination for anthracycline-pretreated advanced/metastatic breast cancer. The safety profile is manageable and consistent with the side effects of the individual agents