Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cmr, Tcr and Emr determinants that are expressed in E. coli. Only the Emr determinant is expressed in S. sanguis. Both the Cmr and the Tcr of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345-353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25067/1/0000498.pd

Quantifying HIV transmission flow between high-prevalence hotspots and surrounding communities: a population-based study in Rakai, Uganda

Background International and global organisations advocate targeting interventions to areas of high HIV prevalence (ie, hotspots). To better understand the potential benefits of geo-targeted control, we assessed the extent to which HIV hotspots along Lake Victoria sustain transmission in neighbouring populations in south-central Uganda. Methods We did a population-based survey in Rakai, Uganda, using data from the Rakai Community Cohort Study. The study surveyed all individuals aged 15–49 years in four high-prevalence Lake Victoria fishing communities and 36 neighbouring inland communities. Viral RNA was deep sequenced from participants infected with HIV who were antiretroviral therapy-naive during the observation period. Phylogenetic analysis was used to infer partial HIV transmission networks, including direction of transmission. Reconstructed networks were interpreted through data for current residence and migration history. HIV transmission flows within and between high-prevalence and low-prevalence areas were quantified adjusting for incomplete sampling of the population. Findings Between Aug 10, 2011, and Jan 30, 2015, data were collected for the Rakai Community Cohort Study. 25 882 individuals participated, including an estimated 75·7% of the lakeside population and 16·2% of the inland population in the Rakai region of Uganda. 5142 participants were HIV-positive (2703 [13·7%] in inland and 2439 [40·1%] in fishing communities). 3878 (75·4%) people who were HIV-positive did not report antiretroviral therapy use, of whom 2652 (68·4%) had virus deep-sequenced at sufficient quality for phylogenetic analysis. 446 transmission networks were reconstructed, including 293 linked pairs with inferred direction of transmission. Adjusting for incomplete sampling, an estimated 5·7% (95% credibility interval 4·4–7·3) of transmissions occurred within lakeside areas, 89·2% (86·0–91·8) within inland areas, 1·3% (0·6–2·6) from lakeside to inland areas, and 3·7% (2·3–5·8) from inland to lakeside areas. Interpretation Cross-community HIV transmissions between Lake Victoria hotspots and surrounding inland populations are infrequent and when they occur, virus more commonly flows into rather than out of hotspots. This result suggests that targeted interventions to these hotspots will not alone control the epidemic in inland populations, where most transmissions occur. Thus, geographical targeting of high prevalence areas might not be effective for broader epidemic control depending on underlying epidemic dynamics. Funding The Bill & Melinda Gates Foundation, the National Institute of Allergy and Infectious Diseases, the National Institute of Mental Health, the National Institute of Child Health and Development, the Division of Intramural Research of the National Institute for Allergy and Infectious Diseases, the World Bank, the Doris Duke Charitable Foundation, the Johns Hopkins University Center for AIDS Research, and the President's Emergency Plan for AIDS Relief through the Centers for Disease Control and Prevention

A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis

A plasmid that is able to replicate in both Escherichia coli and Streptococcus sanguis has been constructed by the in vitro joining of the pACYC184 (Cmr Tcr) and pVA749 (Emr) replicons. This plasmid, designated pVA838, is 9.2 kb in size and expresses Emr in both E. coli and S. sanguis. Its Cmr marker is expressed only in E. coli and may be inactivated by addition of DNA inserts at its internal EcoRI or PvuII sites. The pVA838 molecule also contains unique SalI, SphI, BamHI, NruI and XbaI cleavage sites suitable for molecular cloning. pVA838 may be amplified in E. coli but not in S. sanguis. We have used the pVA838 plasmid as a shuttle vector to clone streptococcai plasmid fragments in E. coli. Such chimeras isolated from E. coli were readily introduced into S. sanguis by transformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23855/1/0000094.pd

N-terminal B-type natriuretic peptide urinary concentrations and retinopathy of prematurity

BACKGROUND: To validate the findings of a single-center pilot study showing elevated urinary N-terminal B-type natriuretic peptide (NTproBNP) concentrations in preterm infants subsequently developing severe retinopathy of prematurity (ROP) in a multicenter setting across eight European and Middle East countries. METHODS: Prospective observational study in 967 preterm infants <30 weeks' gestational age assessing the capacity of urinary NTproBNP on days of life (DOLs) 14 and 28 to predict ROP requiring treatment. RESULTS: Urinary NTproBNP concentrations were markedly elevated in infants who developed ROP requiring treatment (n = 94) compared with survivors without ROP treatment (n = 837), at both time points (median (interquartile range) DOL14: 8,950 (1,925-23,783) vs. 3,083 (1,193-17,393) vs. 816 (290-3,078) pg/ml, P <0.001) and DOL28 (2,203 (611-4,063) vs. 1,671 (254-11,340) vs. 408 (162-1,126) pg/ml, P <0.001). C-statistic of NTproBNP for treated ROP or death was 0.731 (95% confidence interval 0.654-0.774) for DOL14 and 0.683 (0.622-0.745) for DOL28 (P <0.001). Threshold scores were calculated, potentially enabling around 20% of infants with low NTproBNP scores never to be screened with ophthalmoscopy. CONCLUSION: There is a strong association between early urinary NTproBNP and subsequent ROP development, which can be used to further refine subgroups of patients with high or low risk of severe RO

Inferring HIV-1 transmission networks and sources of epidemic spread in Africa with deep-sequence phylogenetic analysis

To prevent new infections with human immunodeficiency virus type 1 (HIV-1) in sub-Saharan Africa, UNAIDS recommends targeting interventions to populations that are at high risk of acquiring and passing on the virus. Yet it is often unclear who and where these ‘source’ populations are. Here we demonstrate how viral deep-sequencing can be used to reconstruct HIV-1 transmission networks and to infer the direction of transmission in these networks. We are able to deep-sequence virus from a large population-based sample of infected individuals in Rakai District, Uganda, reconstruct partial transmission networks, and infer the direction of transmission within them at an estimated error rate of 16.3% [8.8–28.3%]. With this error rate, deep-sequence phylogenetics cannot be used against individuals in legal contexts, but is sufficiently low for population-level inferences into the sources of epidemic spread. The technique presents new opportunities for characterizing source populations and for targeting of HIV-1 prevention interventions in Africa