Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation

Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.     

Plants from disturbed savannah vegetation and their usage by Bakongo tribes in Uíge,Northern Angola

Background

This study represents the first in-depth ethnobotanical study in the province of Uíge in northern Angola and documents the traditional knowledge of the Bakongo people living in the area. Due to deforestation and frequent fires, degraded savannahs dominate the landscape in the study region. Here we provide a list of useful plants from these savannahs including quantitative data about cultural importance of the respective species, aiming on the one hand to conserve the local knowledge and on the other hand to create a reliable basis for research projects in the region.

Methods

Field work was conducted in April and May 2014 in 5 municipalities of Uíge province. The study is based on 32 semi-structured and free-listing interviews, group discussions of varying scope and 14 field trips, involving a total of 82 informants. Throughout the course of the study herbarium specimens of the useful species were collected for later identification. Cultural importance index was applied to analyse the data sets recorded and to determine the best-known useful species in the region. All data sets were compared to the literature available for the region.

Results

The study documents a total of 498 citations for the use of 122 plants from 48 families, 34.0 % of which were unknown according to the literature used for comparison. The high amount (71 %) of medical use-reports indicates that plants still play a crucial role in rural health care. We identified 14 plant species of special interest for pharmacological analysis. Species of highest cultural importance are Annona senegalensis Pers. and Sarcocephalus latifolius (Sm.) E.A., both of which are frequently found in disturbed savannahs.

Conclusions

The study points out the importance of savannahs even if degraded in terms of useful plants and provides a valuable addition to current knowledge of plant use in Northern Angola. This is not only essential for further studies, i.e. regarding pharmaceutical agents, but also for the design of a planned botanical garden of the University Kimpa Vita in Uíge, which aims at communicating the findings to the local people.

     

Evaluation of Nutritional Gel Supplementation in C57BL/6J Mice Infected with Mouse-Adapted Influenza A/PR/8/34 Virus

Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.

Influenza A virus (IAV) infections place a major strain on public health services world-wide. The World Health Organization estimates that influenza viral infections cause severe illness in 3 to 5 million people and death estimated at 145,000 to 650,000 people each year.^{22,27,35,40,48} Due to the significant burden that influenza places on public health, the need to learn more about the interactions of this virus with the mammalian immune system continues. This need has led to studies that use a variety of species to examine the host response to IAV. Mice are the most common species used to study influenza.^10,47 Clinical signs of influenza in mice include anorexia, dehydration, respiratory distress, hypothermia, hunched posture, unkempt hair coat and ocular discharge.^10,43,49,51 Anorexia and dehydration are common signs of influenza infection that lead to excessive weight loss in mice.Weight loss in mice infected with influenza virus is associated with anorexia induced by systemic inflammation. Proinflammatory cytokines have been shown to inhibit normal feeding behavior in mice with IAV, specifically through increased expression of tumor necrosis factor-α (TNFα), interleukin-1β (IL1β) and interleukin-6 (IL6).^{8,12,16,19,24,38,51} For example, after infection with IAV, the levels of both TNFα and IL6 begin to rise around 2 to 3 d post infection (dpi) and peak at 7 dpi.^12,24 Mice begin to lose weight at approximately 4 dpi, with peak weight loss around 9 dpi, which correlates with the levels of TNFα, IL6 and other proinflammatory mediators.^12,24,31 Weight loss is routinely used as a euthanasia endpoint criterion for mice infected with IAV. The early removal of mice from a study can have a negative impact on sample size and may compromise the accuracy of a study by introducing survivor bias.⁴³Under the guidance of Institutional Animal Care and Use Committees (IACUCs), a 20 to 30% loss in body weight is a common endpoint criterion in studies of IAV.⁴³ However, an IACUC may be reluctant to justify a weight loss of over 30%. Based on the 3 Rs, replacement, reduction, and refinement, scientists should refine techniques used in animal models of disease to minimize discomfort.²⁰ In addition, finding ways to provide physiologic support for mice in order to reduce the loss of animals in IAV studies will lead to a reduction in the number of animals required to adequately power a study. Therefore, whether supportive care reduces weight loss and consequently mortality in mice infected with IAV is an important question.The objective of the work performed here was to identify a support strategy that resulted in reduced weight loss and increased survival in mice infected with IAV, without altering the nature or degree of their immune response. The overall goal of this investigation is to improve the standard of care for influenza infected mice. If an appropriate strategy is identified, the number of mice needed for IAV studies may be reduced and providing additional support for infected mice will refine the model. To accomplish this goal, the effects of nutritional gel (NG) supplementation were evaluated in mice infected with IAV and compared with the SOC, defined as moist chow and hydrogel. Due to its physical (soft consistency) and nutritional properties, along with the minimal handling required to provide the supplement, we hypothesized that NG (DietGel Recovery) would minimize weight loss and increase the number of mice reaching the end of study as compared with the current SOC.In this paper we show that NG supplementation benefited influenza infected mice by reducing weight loss and the number of mice euthanized based on the endpoint criteria of 30% weight loss. These effects were seen at all doses in male mice, and at the low and intermediate doses in female mice. Evaluation of the immune response to influenza infection using the 0.5 EuD50 dose revealed no significant differences in the number of immune cells in mice given SOC or NG. Measurements of cytokines in BAL fluid showed lower recovery of IL6 in female mice and TNFα in male mice supplemented with NG. Thus, to summarize, both sexes should be included in study design, and mice infected with mouse-adapted IAV and supplemented with NG demonstrate less weight loss and increased survival than do SOC mice. Our data suggest that NG should be considered as a support strategy for mice infected with IAV.     

The Application of New Molecular Methods in the Investigation of a Waterborne Outbreak of Norovirus in Denmark, 2012

In December 2012, an outbreak of acute gastrointestinal illness occurred in a geographical distinct area in Denmark covering 368 households. A combined microbiological, epidemiological and environmental investigation was initiated to understand the outbreak magnitude, pathogen(s) and vehicle in order to control the outbreak. Norovirus GII.4 New Orleans 2009 variant was detected in 15 of 17 individual stool samples from 14 households. Norovirus genomic material from water samples was detected and quantified and sequencing of longer parts of the viral capsid region (>1000 nt) were applied to patient and water samples. All five purposely selected water samples tested positive for norovirus GII in levels up to 1.8×10⁴ genomic units per 200 ml. Identical norovirus sequences were found in all 5 sequenced stool samples and 1 sequenced water sample, a second sequenced water sample showed 1 nt (<0.1%) difference. In a cohort study, including 256 participants, cases were defined as residents of the area experiencing diarrhoea or vomiting onset on 12–14 December 2012. We found an attack rate of 51%. Being a case was associated with drinking tap-water on 12–13 December (relative risk = 6.0, 95%CI: 1.6–22) and a dose-response relation for the mean glasses of tap-water consumed was observed. Environmental investigations suggested contamination from a sewage pipe to the drinking water due to fall in pressure during water supply system renovations. The combined microbiological, epidemiological and environmental investigations strongly indicates the outbreak was caused by norovirus contamination of the water supply system.     

Genetic characterization of specific pathogen‐free rhesus macaque (Macaca mulatta) populations at the California National Primate Research Center (CNPRC)

A study based on 14 STRs was conducted to understand intergenerational genetic changes that have occurred within the California National Primate Research Center's (CNPRC) regular specific pathogen‐free (SPF) and super‐SPF captive rhesus macaque populations relative to their conventional founders. Intergenerational genetic drift has caused age cohorts of each study population, especially within the conventional population, to become increasingly differentiated from each other and from their founders. Although there is still only minimal stratification between the conventional population and either of the two SPF populations, separate derivation of the regular and super‐SPF animals from their conventional founders has caused the two SPF populations to remain marginally different from each other. The regular SPF and, especially, the super‐SPF populations have been influenced by the effects of differential ancestry, sampling, and lost rare alleles, causing a substantial degree of genetic divergence between these subpopulations. The country of origin of founders is the principal determinant of the MHC haplotype composition of the SPF stocks at the CNPRC. Selection of SPF colony breeders bearing desired genotypes of Mamu‐A^*01 or ‐B^*01 has not affected the overall genetic heterogeneity of the conventional and the SPF research stocks. Because misclassifying the ancestry of research stocks can undermine experimental outcomes by excluding animals with regional‐specific genotypes or phenotypes of importance, understanding founder/descendent genetic relationships is crucial for investigating candidate genes with distinct geographic origins. Together with demographic management, population genetic assessments of SPF colonies can curtail excessive phenotypic variation among the study stocks and facilitate successful production goals. Am. J. Primatol. 72:587–599, 2010. © 2010 Wiley‐Liss, Inc.     

Sequencing and Utilization of the Gossypium Genomes

Revealing the genetic underpinnings of cotton productivity will require understanding both the prehistoric evolution of spinnable fibers, and the results of independent domestication processes in both the Old and New Worlds. Progress toward a reference sequence for the smallest Gossypium genome is a logical stepping-stone toward revealing diversity in the remaining seven genomes (A, B, C, E, F, G, K) that permitted Gossypium species to adapt to a wide range of ecosystems in warmer arid regions of the world, and toward identifying the emergent properties that account for the superior productivity and quality of tetraploid cottons. The greatest challenge facing the cotton community is not genome sequencing per se but the conversion of sequence to knowledge.