Designing a Community Engagement Framework for a New Dengue Control Method: A Case Study from Central Vietnam

Background

The Wolbachia strategy aims to manipulate mosquito populations to make them incapable of transmitting dengue viruses between people. To test its efficacy, this strategy requires field trials. Public consultation and engagement are recognized as critical to the future success of these programs, but questions remain regarding how to proceed. This paper reports on a case study where social research was used to design a community engagement framework for a new dengue control method, at a potential release site in central Vietnam.

Methodology/Principal Findings

The approach described here, draws on an anthropological methodology and uses both qualitative and quantitative methods to design an engagement framework tailored to the concerns, expectations, and socio-political setting of a potential trial release site for Wolbachia-infected Aedes aegypti mosquitoes. The process, research activities, key findings and how these were responded to are described. Safety of the method to humans and the environment was the most common and significant concern, followed by efficacy and impact on local lives. Residents expected to be fully informed and engaged about the science, the project, its safety, the release and who would be responsible should something go wrong. They desired a level of engagement that included regular updates and authorization from government and at least one member of every household at the release site.

Conclusions/Significance

Results demonstrate that social research can provide important and reliable insights into public concerns and expectations at a potential release site, as well as guidance on how these might be addressed. Findings support the argument that using research to develop more targeted, engagement frameworks can lead to more sensitive, thorough, culturally comprehensible and therefore ethical consultation processes. This approach has now been used successfully to seek public input and eventually support for releases Wolbachia-infected mosquitoes, in two different international settings - Australia and Vietnam.     

Survey of dogs from Vietnam for antibodies to visceralizing Leishmania spp

Cases of visceral leishmaniasis, one of the most neglected tropical diseases, are increasing globally. Dogs are considered an important reservoir host for visceral leishmaniasis in people. The first cases of human visceral leishmaniasis in Vietnam have recently been reported. Blood samples were collected from 41 dogs in rural Vietnam. Sera were examined for antibodies to visceralizing Leishmania spp. by canine immunochromatographic strip assays based on recombinant K39 antigen. Antibodies to Leishmania spp. were not detected in any of the dogs tested. Results from this study suggest that rural dogs are not likely to be involved in the emergence of human visceral leishmaniasis in Vietnam.     

Subcapsular sinus macrophage fragmentation and CD169+ bleb acquisition by closely associated IL-17-committed innate-like lymphocytes

Subcapsular sinus macrophages (SSMs) in lymph nodes are rapidly exposed to antigens arriving in afferent lymph and have a role in their capture and display to B cells. In tissue sections SSMs exhibit long cellular processes and express high amounts of CD169. Here, we show that many of the cells present in lymph node cell suspensions that stain for CD169 are not macrophages but lymphocytes that have acquired SSM-derived membrane blebs. The CD169 bleb(+) lymphocytes are enriched for IL-17 committed IL-7Rα(hi)CCR6(+) T cells and NK cells. In addition, the CD169 staining detected on small numbers of CD11c(hi) dendritic cells is frequently associated with membrane blebs. Counter intuitively the CD169 bleb(+) lymphocytes are mostly CD4 and CD8 negative whereas many SSMs express CD4. In situ, many IL-7Rα(hi) cells are present at the subcapsular sinus and interfollicular regions and migrate in close association with CD169(+) macrophages. These findings suggest SSMs undergo fragmentation during tissue preparation and release blebs that are acquired by closely associated cells. They also suggest an intimate crosstalk between SSMs and IL-17 committed innate-like lymphocytes that may help provide early protection of the lymph node against lymph-borne invaders.     

The positive impact of cytological specimens for EGFR mutation testing in non‐small cell lung cancer: a single South East Asian laboratory’s analysis of 670 cases

B. Pang, D. Matthias, C.W. Ong, A.N. Dhewar, S. Gupta, G.L. Lim, M.‐E. Nga, J.E. Seet, A. Qasim, T.‐M. Chin, R. Soo, R. Soong and M. Salto‐Tellez The positive impact of cytological specimens for EGFR mutation testing in non‐small cell lung cancer: a single South East Asian laboratory’s analysis of 670 cases Objectives: To compare the rejection rates of non‐small cell lung cancer (NSCLC) samples obtained by differing sampling methods for testing by Sanger sequencing for epidermal growth factor receptor (EGFR) mutations. To assess the association between unsatisfactory outcomes and the quantity of DNA extracted from cytological versus histological samples. Methods: Six hundred and seventy NSCLC samples referred to our centre from 2008 to 2010 were reviewed as a consequence of sample rejection, presence of EGFR mutations, cytological versus histological sampling methods, DNA quantity and the unsatisfactory genotyping rate. Results: Eighty samples were rejected for testing in similar proportions of histological and cytological samples (11.9% versus 10.9%) usually (n = 75) because the amount of cellular material was judged insufficient in small biopsies or cytology samples. The remaining 590 samples on which EGFR testing was attempted yielded 51 (8.6%) unsatisfactory test outcomes caused by failure of the polymerase chain reaction (PCR) (n = 47 cases), uninterpretable Sanger chromatograms (n = 3 cases) and insufficient DNA extracted for PCR (n = 1 case). The difference in rates of unsatisfactory outcomes between cytological samples (seven of 147 samples or 4.7%) versus tissue samples (44 of 443 samples or 9.9%) was clinically relevant but not statistically significant (Mann–Whitney test; P < 0.081). There was no association between the concentration of DNA extracted and the likelihood of an unsatisfactory analysis; which was similar in all types of sections (large and small) while 0% of 37 cytology slides were unsatisfactory. Conclusions: Utilizing cytology samples for EGFR testing avoids unnecessary patient re‐biopsing and yields a clinically superior satisfactory rate to the overall satisfactory rate of tissue biopsies of NSCLC. The quality rather than quantity of DNA extracted may be a more important determinant of a satisfactory result.     

Time course of the regenerative response in bupivacaine injured orbicularis oculi muscle

Local anesthetics, particularly bupivacaine, are known to be myotoxic to skeletal muscle. Injury is followed by satellite cell mediated regeneration. The eyelid is a common site for the injection of local anesthetics. Due to the complex anatomy of this region and the unique properties of facial musculature compared to limb skeletal muscle, the response of the orbicularis oculi to local injection of bupivacaine was examined to determine the time course of maximum satellite cell activation and division. The lower eyelids of rabbits were injected with two doses of a combination of bupivacaine and hyaluronidase, spaced 18 h apart. To assess the time course of satellite cell division, bromodeoxyuridine (BrdU) was injected immediately or, 1, 2, 3, 6 or 13 days after the second bupivacaine injury. The rabbits were sacrificed 24 h later. The eyelids were prepared for immunohistological examination and morphometric analysis of the presence of CD11-positive monocytes, neutrophils and macrophages, MyoD expression in satellite cells and/or myoblasts, and co-expression of BrdU and the developmental myosin heavy chain isoform. One day after bupivacaine injury of the orbicularis oculi, there was a large influx of CD11-positive cells which gradually decreased over time. Maximum activation of satellite cells, as defined by MyoD expression, occurred 2 and 3 days after the injury. Using double labeling techniques, the peak of BrdU incorporation occurred on day 3 and was identified in developmental myosin co-labeled cells 4 days after injury. The peak of satellite cell activation and division occurred 3 days after bupivacaine induced injury, as demonstrated by both MyoD expression and after pulse labeling with BrdU as identified in double labeled cells positive for BrdU and the developmental myosin heavy chain isoform. The process of regeneration in this muscle extended beyond the duration of this study. Muscle fibers remained small in cross-sectional area and positive for developmental myosin 2 weeks after injury, at a time when the fiber number had reached control, uninjured levels.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: Synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [d -Pen², d -Pen⁵]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [d -DFAB², Met⁵-NH₂]enkephalin showed μ and δ agonist potencies comparable to those of natural [Leu⁵]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While l -amino acid substitution in position 3 of DPDPE usually resulted in higher δ agonist potency than d -amino acid substitution, [d -DFAB³]DPDPE turned out to be a more potent δ agonist than [l -DFAB³]DPDPE. Furthermore, [d -DFAB³]DPDPE showed over 100-fold higher δ agonist potency than [d -Abu³]DPDPE (Abu=2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a δ opioid receptor subsite. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants

OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.     

Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.     

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.     

Role of the fungal Ras-protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis

Tpk1p, Tpk2p and Efg1p are members of the Ras-protein kinase A pathway that governs the yeast-to-hyphal transition in Candida albicans. We used tpk1Delta/tpk1Delta, tpk2Delta/tpk2Delta and efg1Delta/efg1Delta mutants to investigate the role of these proteins in regulating the interactions of C. albicans with oral epithelial cell lines in vitro and virulence in murine models of oropharyngeal candidiasis (OPC) and haematogenously disseminated candidiasis (HDC). The tpk1Delta/tpk1Delta strain adhered to, invaded and damaged oral epithelial cells in vitro similarly to the wild-type strain. In contrast, both the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains had reduced capacity to invade and damage oral epithelial cells, and the efg1Delta/efg1Delta strain also exhibited decreased adherence to these cells. Consistent with these in vitro findings, the tpk2Delta/tpk2Delta and efg1Delta/efg1Delta strains also had significantly attenuated virulence during OPC. Therefore, Tpk2p and Efg1p both govern factors that enable C. albicans to invade and damage oral epithelial cells in vitro and cause OPC. These results also suggest that hyphal formation mediated by the Ras-protein kinase A pathway is a key virulence mechanism during OPC. Interestingly, the efg1Delta/efg1Delta strain, but not the tpk2Delta/tpk2Delta had reduced virulence during HDC. Thus, Tpk2p may be more important for governing virulence during OPC than HDC.