Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer

Community knowledge, attitudes and practices (KAP) on malaria in Swaziland: A country earmarked for malaria elimination

<p>Abstract</p> <p>Background</p> <p>The potential contribution of knowledge, attitudes and practices (KAP) studies to malaria research and control has not received much attention in most southern African countries. This study investigated the local communities' understanding of malaria transmission, recognition of signs and symptoms, perceptions of cause, treatment-seeking patterns, preventive measures and practices in order to inform the country's proposed malaria elimination programme in Swaziland.</p> <p>Methods</p> <p>A descriptive cross-sectional survey was undertaken in four Lubombo Spatial Development Initiative (LSDI) sentinel sites in Swaziland. These sentinel sites share borders with Mozambique. A structured questionnaire was administered to 320 randomly selected households. Only one adult person was interviewed per household. The interviewees were the heads of households and in the absence of the heads of households responsible adults above 18 years were interviewed.</p> <p>Results</p> <p>A substantial number of research participants showed reasonable knowledge of malaria, including correct association between malaria and mosquito bites, its potential fatal consequences and correct treatment practices. Almost 90% (n = 320) of the respondents stated that they would seek treatment within 24 hours of onset of malaria symptoms, with health facilities as their first treatment option. Most people (78%) perceived clinics and vector control practices as central to treating and preventing malaria disease. Indoor residual spraying (IRS) coverage and bed net ownership were 87.2% and 38.8%, respectively. IRS coverage was in agreement with the World Health Organization's (WHO) recommendation of more than 80% within the targeted communities.</p> <p>Conclusion</p> <p>Despite fair knowledge of malaria in Swaziland, there is a need for improving the availability of information through the preferred community channels, such as <it>tinkhundlas </it>(districts), as well as professional health routes. This recommendation emerges along with the documented evidence suggesting that as the level transmission and disease decreases so does the perception about the importance of malaria control activities. Finally, given the relatively moderate ownership of bed net there is a need for future studies to evaluate the distribution of insecticide-treated nets (ITNs) compared with IRS.</p

Drosophila Dynein Intermediate Chain Gene, Dic61B, Is Required for Spermatogenesis

This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051c05439 and CG7051f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051c05439 and CG7051f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051c05439 and CG7051f07138 alleles to be in 5′UTR and 4th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis

Population Health Metrics Research Consortium gold standard verbal autopsy validation study: design, implementation, and development of analysis datasets

Background: Verbal autopsy methods are critically important for evaluating the leading causes of death in populations without adequate vital registration systems. With a myriad of analytical and data collection approaches, it is essential to create a high quality validation dataset from different populations to evaluate comparative method performance and make recommendations for future verbal autopsy implementation. This study was undertaken to compile a set of strictly defined gold standard deaths for which verbal autopsies were collected to validate the accuracy of different methods of verbal autopsy cause of death assignment.Methods: Data collection was implemented in six sites in four countries: Andhra Pradesh, India; Bohol, Philippines; Dar es Salaam, Tanzania; Mexico City, Mexico; Pemba Island, Tanzania; and Uttar Pradesh, India. The Population Health Metrics Research Consortium (PHMRC) developed stringent diagnostic criteria including laboratory, pathology, and medical imaging findings to identify gold standard deaths in health facilities as well as an enhanced verbal autopsy instrument based on World Health Organization (WHO) standards. A cause list was constructed based on the WHO Global Burden of Disease estimates of the leading causes of death, potential to identify unique signs and symptoms, and the likely existence of sufficient medical technology to ascertain gold standard cases. Blinded verbal autopsies were collected on all gold standard deaths.Results: Over 12,000 verbal autopsies on deaths with gold standard diagnoses were collected (7,836 adults, 2,075 children, 1,629 neonates, and 1,002 stillbirths). Difficulties in finding sufficient cases to meet gold standard criteria as well as problems with misclassification for certain causes meant that the target list of causes for analysis was reduced to 34 for adults, 21 for children, and 10 for neonates, excluding stillbirths. To ensure strict independence for the validation of methods and assessment of comparative performance, 500 test-train datasets were created from the universe of cases, covering a range of cause-specific compositions.Conclusions: This unique, robust validation dataset will allow scholars to evaluate the performance of different verbal autopsy analytic methods as well as instrument design. This dataset can be used to inform the implementation of verbal autopsies to more reliably ascertain cause of death in national health information systems

S-D logic-informed customer engagement: Integrative framework, revised fundamental propositions, and application to CRM

Advance online in 2016</p

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer

Community engagement in maternal and newborn health in eastern Indonesia

In Indonesia, high rates of maternal and child mortality can be attributed, in part at least, to a lack of access to medical facilities, especially in rural communities. In response, the Indonesian government developed a program to address the high rate of home births. A key element of the program involved drawing upon the contribution of community members to assist with facility-based births of pregnant women in their community. The success of the program was attributed to active participation from many groups, including community members, government officials and health-care workers who collaborated successfully to achieve a reduction in the maternal and infant mortality rates

High expression of interleukin-1β in the corneal epithelium of MRL/lpr mice is under the control of their genetic background

MRL/Mp mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), spontaneously develop polyarthritis, sialoadenitis and dacryoadenitis, resembling rheumatoid arthritis (RA), and also corneal involvement such as keratopathy and scleritis, which is a major complication in RA patients. In this study, we found that the expression levels of IL-1β and MMP-1 mRNAs in cornea were high in both MRL/lpr and MRL/Mp-+/+ strains of mice at an age younger than when they develop any inflammatory lesions. This was not true of other inbred strains, even those bearing the lpr gene, and also not of (NZB × NZW) F1 lupus mice. There was no significant difference in the expression of IL-1α and TGFβ in cornea in these strains. Using crosses between MRL/lpr and C3H/HeJ-lpr/lpr (C3H/lpr) mice, at least the expression of IL-1β was found to be under the control of the MRL genetic background, likely with a recessive mode of inheritance. Considering that IL-1β in cornea was detected particularly in the epithelial layer, the high expression of IL-1β in cornea is most likely involved in the genetic predisposition for corneal involvement and possibly also for arthritis in an MRL strain of mice