Vector competence of Coquillettidia linealis (Skuse) (Diptera: Culicidae) for Ross River and Barmah Forest viruses

Abstract Coquillettidia linealis is a severe pest on some of the Moreton Bay islands in Queensland, Australia, but little is known of its breeding habitats and biology. Because of its high abundance and its association with Ross River (RR) and Barmah Forest (BF) viruses by field isolation, its vector competence was evaluated in the laboratory by feeding dilutions of both viruses in blood. For RR, Cq. linealis was of comparable efficiency to Ochlerotatus vigilax (Skuse), recognised as being a major vector. Results were as follows for Cq. linealis and Oc. vigilax , respectively: dose to infect 50%, 10^2.2 and <10^1.7 CCID₅₀/mosquito; 88% and 90% disseminated infection at 4 days postinfection; transmission at 4 days with rates of 68−92% and 25−60%. For BF dose to infect 50%, 10^2.7 and 10^2.0; disseminated infection rates on first transmission day (day 6), 40% and 70%; transmission rates of 8−16% and 0−10%. As a capillary-tube method was used rather than suckling mice to demonstrate transmission, transmission rates may be underestimates. This, the first study of the vector competence of Cq. linealis in Australia, demonstrates that this species deserves control on the southern Moreton Bay islands.     

Modeling analysis of GST (glutathione-S-transferases) from Wuchereria bancrofti and Brugia malayi

GST (glutathione S-transferases) are a family of detoxification enzymes that catalyze the conjugation of reduced GSH (glutathione) to xenobiotic (endogenous electrophilic) compounds. GST from Wb (Wuchereria bancrofti) and Bm (Brugia malayi) are significantly different from human GST in sequence and structure. Thus, Wb-GST and Bm-GST are potential chemotherapeutic targets for anti-filarial treatment. Comparison of modeled Wb and Bm GST with human GST show structural difference between them. Analysis of the active site residues for the binding of electrophilic co-substrates provides insight towards the design of parasite specific GST inhibitors.     

Comparative immunogenicity analysis of intradermal versus intramuscular administration of SARS-CoV-2 RBD epitope peptide-based immunogen In vivo

COVID-19 pandemic has caused severe disruption of global health and devastated the socio-economic conditions all over the world. The disease is caused by SARS-CoV-2 virus that belongs to the family of Coronaviruses which are known to cause a wide spectrum of diseases both in humans and animals. One of the characteristic features of the SARS-CoV-2 virus is the high reproductive rate (R₀) that results in high transmissibility of the virus among humans. Vaccines are the best option to prevent and control this disease. Though, the traditional intramuscular (IM) route of vaccine administration is one of the effective methods for induction of antibody response, a needle-free self-administrative intradermal (ID) immunization will be easier for SARS-CoV-2 infection containment, as vaccine administration method will limit human contacts. Here, we have assessed the humoral and cellular responses of a RBD-based peptide immunogen when administered intradermally in BALB/c mice and side-by-side compared with the intramuscular immunization route. The results demonstrate that ID vaccination is well tolerated and triggered a significant magnitude of humoral antibody responses as similar to IM vaccination. Additionally, the ID immunization resulted in higher production of IFN-γ and IL-2 suggesting superior cellular response as compared to IM route. Overall, our data indicates immunization through ID route provides a promising alternative approach for the development of self-administrative SARS-CoV-2 vaccine candidates.     

Prevalence of non-strongyle gastrointestinal parasites of horses in Riyadh region of Saudi Arabia

This study aimed to provide recent data on the occurrence of non-strongyle intestinal parasite infestation in horses in the Riyadh region of Saudi Arabia as a basis for developing parasite control strategies. We conducted necropsy for 45 horses from September 2006 to November 2007 in the Riyadh region, Saudi Arabia. 39 out of 45 horses were infected with intestinal parasites with an infestation rate of 86.6%. Infestations with seven nematode species and two species of Gasterophilus larva were found. The most prevalent parasites were Strongyloides westeri (64.4%) and Parascaris equorum (28.8%) followed by Habronema muscae (22.2%). Trichostrongylus axei and Oxyuris equi were less common at (11.1%) and (8.8%), respectively. Habronema megastoma and Setaria equine were found in two horses only (4.4%). Gasterophilus intestinalis larvae were recovered from 39 horses (86.6%) and Gasterophilus nasalis larvae were found in 17 horses (37.7%). Season had a significant effect on the prevalence of P. equorum and G. nasalis, while age of horses had a significant effect only on the prevalence of P. equorum. The husbandry in Saudi Arabia appears to be conductive to parasites transmitted in stables or by insects rather than in pasture.     

Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization

The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G₀/G₁ phase of the cell cycle, the block occurs primarily at the G₂/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC₅₀ <1 μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G₀/G₁. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development.A link between development of human cancers and cellular pathways where the retinoblastoma protein (pRb) has a major role is well established.^{1, 2} One of the frequent events associated with human tumour progression is abnormality in the pathway that links pRb, p16^INK4A, cyclin D1 and Cdk4 (cyclin-dependent kinase 4).³ Cdk4 along with its activating cyclin partner D1 has a key role in cell cycle control.^{4, 5} The naturally occurring inhibitor of Cdk4-cyclin D1, p16^INK4a (p16), is a tumour supressor protein. Deletion or inactivating mutations in the p16 gene are observed in many human cancers.^{6, 7} The catalytic activity of Cdk4 depends on its activation by the protein cyclin D1, which is expressed during the G₀/G₁ phase of the cell cycle. Many cancers are characterised by abnormal overproduction of cyclin D1.^{8, 9} As Cdk4 inhibitors target a pathway that links pRb, p16INK4A, cyclin D1 and Cdk4, it makes inhibition of Cdk4-cyclin D1 enzyme a crucially important target for cancer chemotherapy.^{10, 11, 12, 13} However, Rb mutations, consistent with loss of Rb function, have been identified in a wide spectrum of tumours including osteosarcomas, small-cell lung carcinomas, breast carcinomas and others, and the Cdk4 inhibitors cannot inhibit such pathway involving Rb-mutated tumours.A number of potential anticancer agents that selectively modulate the activity of Cdk4-cyclin D1 in vitro have been reported.¹⁴ These molecules also show the genotypic consequences of Cdk4 enzyme inhibition at the cellular level, that is, growth inhibition of cancer cells in vitro, arrest of asynchronous cells at G₀/G₁ and prevention of pRb phosphorylation at Cdk4-specific serine residues.^{15, 16, 17} Usually, competing with ATP molecules for binding at the protein kinase active site is the normal mechanism by which most small molecules inhibit kinase enzyme activity. Successful attempts to identify selective Cdk4 inhibitors using structure-based chemical design and molecular modelling have been reported.^{18, 19, 20} Furthermore, the success of Cdk4 inhibitors at clinical stages^{21, 22, 23, 24, 25} has indicated it as a promising therapeutic target for anticancer drug discovery.¹⁴Fascaplysin (1), a natural product originally isolated from a marine sponge, specifically inhibits the Cdk4 enzyme.^{26, 27} It inhibits Cdk4-cyclin D1 with an IC₅₀ of ~0.35 μM and blocks growth of cancer cells at the G₀/G₁ phase of the cell cycle. Similar to cryptolepine and ellipticine,²⁸ fascaplysin is also a planar structure and thus it intercalates double-stranded (d-s) DNA and shows unusual toxicity at the cellular level. It has been suggested that fascaplysin''s planar structure is the possible explanation for its ability to intercalate d-s DNA and also its unusual toxicity at the cellular level. To overcome this unusual toxicity, recently we reported CA224 (2), a non-planar analogue of fascaplysin exhibiting selective Cdk4 inhibition with no DNA-intercalating property.²⁹ In continuation to these efforts, herein we report identification of tryptoline-based compounds CA198 (3), CA199 (4), CA211 (5) and N-(biphenyl-2-yl)-tryptoline (BPT, 6) as selective Cdk4 inhibitors with no DNA-intercalating property. Based on the molecular modelling design, a number of non-planar analogues of fascaplysin were synthesized. They show specificity towards Cdk4-cyclin D1 enzyme activity and blocks the growth of cancer cells at the G₀/G₁ phase. Although BPT was also designed using a homology model of Cdk4, based on the X-ray crystallographic structures of Cdk2, Cdk4 and Cdk6,^{30, 31, 32, 33} further investigations showed that BPT blocks growth of cells at the G₂/M phase, in a Cdk-independent manner, through inhibition of tubulin polymerization. BPT shows potent cytotoxicity in a panel of cancer cells and is efficacious against human tumours derived from HCT-116 and NCI-H460 cells in SCID mice models. Here, we present the biological activity of BPT in detail. BPT (6) was synthesized using a one-step procedure by coupling tryptoline with biphenyl 2-carboxylic acid. The chemical synthesis of BPT and chemical structures of 1-6 are shown in Figure 1.Open in a separate windowFigure 1(a) Chemical structures of fascaplysin (1), CA224 (2) and its tryptoline analogues 3–6. (b) Synthetic scheme for BPT (6). (c) Molecular modelling studies to understand Cdk4 selectivity versus Cdk2: interactions of cis/trans conformations of BPT with Cdk2 and Cdk4, respectively (orange conformation is with Cdk2 and green with Cdk4)     

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus

Background

The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3–16); hence, the mechanisms responsible for changes in copy number at this locus are unclear.

Results

In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure.

Conclusions

Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-614) contains supplementary material, which is available to authorized users.