Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

ASSESSMENT OF EVOLUTIONARY AFFINITIES IN GOSSYPIUM BY PROTEIN ELECTROPHORESIS

Protein band patterns from 25 species of Gossypium were obtained by electrophoresis of crude seed extracts on polyacrylamide gel. Band homologies between species were verified by electrophoresis of a mixture of their extracts. The patterns were found to be largely consistent with the conventional classification of the diploids into 6 genomic groups, A–F. However, G. triphyllum and G. bickii showed unique patterns differing respectively from those of the B and C groups, and G. australe showed closer affinity with the Arabian E- than with the Australian C-genome species. Affinities among the D-genome species were different from those implied by their former grouping into taxonomic sections but remarkably similar to those indicated in the most recent taxonomic revision of the genus. They were classifiable into two subgroups, β and ɛ. The clustering pattern of the diploids based on correlation coefficients calculated from densitometer curves of the electrophoretic spectra suggested that the genomic groups were derived from an African progenitor type, and that the American β and ɛ subgroups, most closely related to the African B- and the Arabian E-genome groups respectively, evolved under comparative mutual isolation, possibly separated by the Tertiary Amazonas basin.     

Chemical exchange effects during refocusing pulses in constant-time CPMG relaxation dispersion experiments

In the analysis of the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment, chemical exchange parameters, such as rate of exchange and population of the exchanging species, are typically optimized using equations that predict experimental relaxation rates recorded as a function of effective field strength. In this process, the effect of chemical exchange during the CPMG pulses is typically assumed to be the same as during the free-precession. This approximation may introduce systematic errors into the analysis of data because the number of CPMG pulses is incremented during the constant-time relaxation period, and the total pulse duration therefore varies as a function of the effective field strength. In order to estimate the size of such errors, we simulate the time-dependence of magnetization during the entire constant time period, explicitly taking into account the effect of the CPMG pulses on the spin relaxation rate. We show that in general the difference in the relaxation dispersion profile calculated using a practical pulse width from that calculated using an extremely short pulse width is small, but under certain circumstances can exceed 1 s^?1. The difference increases significantly when CPMG pulses are miscalibrated.     

Spatial and temporal deforestation dynamics in protected and unprotected dry forests: a case study from Myanmar (Burma)

Tropical dry forests are more threatened, less protected and especially susceptible to deforestation. However, most deforestation research focuses on tropical rain forests. We analyzed spatial and temporal changes in land cover from 1972 through 2005 at Chatthin Wildlife Sanctuary (CWS), a tropical dry forest in Myanmar (Burma). CWS is one of the largest protected patches of tropical dry forest in Southeast Asia and supports over half the remaining wild population of the endangered Eld’s deer. Between 1973 and 2005, 62% of forest was lost at an annual rate of 1.86% in the area, while forest loss inside CWS was only 16% (0.45% annually). Based on trends found during our study period, dry forests outside CWS would not persist beyond 2019, while forests inside CWS would persist for at least another 100 years. Analysis of temporal deforestation patterns indicates the highest rate of loss occurred between 1992 and 2001. Conversion to agriculture, shifting agriculture, and flooding from a hydro-electric development were the main deforestation drivers. Fragmentation was also severe, halving the area of suitable Eld’s deer habitat between 1973 and 2001, and increasing its isolation. CWS protection efforts were effective in reducing deforestation rates, although deforestation effects extended up to 2 km into the sanctuary. Establishing new protected areas for dry forests and finding ways to mitigate human impacts on existing forests are both needed to protect remaining dry forests and the species they support.     

Estimating abundance with sparse data: tigers in northern Myanmar

As part of a national strategy for recovering tiger populations, the Myanmar Government recently proposed its first and the world’s largest tiger reserve in the Hukaung Valley, Kachin State. During November 2002–June 2004, camera-traps were used to record tigers, identify individuals, and, using capture–recapture approaches, estimate density in the reserve. Despite extensive (203 trap locations, 275–558 km² sample plots) and intensive (>4,500 trap nights, 9 months of sampling) survey efforts, only 12 independent detections of six individual tigers were made across three study sites. Due to the sparse data, estimates of tiger abundance generated by Program CAPTURE could not be made for all survey sites. Other approaches to estimating density, based on numbers of tigers caught, or derived from borrowed estimates of detection probability, offer an alternative to capture–recapture analysis. Tiger densities fall in the range of 0.2–2.2 tigers/100 km², with 7–71 tigers inside a 3,250 km² area of prime tiger habitat, where efforts to protect tigers are currently focused. Tiger numbers might be stabilized if strict measures are taken to protect tigers and their prey from seasonal hunting and to suppress illegal trade in wildlife. Efforts to monitor abundance trends in the tiger population will be expensive given the difficulty with which tiger data can be obtained and the lack of available surrogate indices of tiger density. Monitoring occupancy patterns, the subject of a separate ongoing study, may be more efficient.     

Allele Distributions and Frequencies of the Six Prion Protein Gene (PRNP) Polymorphisms in Asian Native Cattle, Japanese Breeds, and Mythun (Bos frontalis)

Six polymorphic sites of the bovine prion protein gene (PRNP) were genotyped in 569 animals of Asian native cattle, Japanese breeds, purebred mythun (Bos frontalis), and mythun × cattle composite animals. At the 23-bp indel site, a deletion (23?) allele was a major allele in all populations except mythun. At the 12-bp indel site, an insertion (12+) allele was a major allele in all populations. The 14-bp indel site was polymorphic in all Asian native cattle. In the octapeptide repeat region, a six-repeat allele was a major allele in all populations, and 5/5 and 4/6 genotypes were detected in Japanese Black and Mongolian cattle and in mythun, respectively. Two nonsynonymous single nucleotide polymorphisms (SNPs) (K3T and S154N) were detected in Asian native cattle and mythun. Haplotype analysis using the genotypes of the six sites estimated 33 different haplotypes. The haplotype 23? 12? K 6 S 14+ was found in all populations.     

Visual Responses of Crayfish Ocular Motoneurons: An Information Theoretical Analysis

Motoneuron responses were elicited by global visual motion and stepwise displacements of an illuminated stripe. Stimulus protocols were identical to those used in previous behavioral studies of compensatory eyestalk reflexes. The firing rates and directional selectivity of the motoneuron responses were measured with respect to four stimulus dimensions (spatial frequency, contrast, angular displacement and velocity). The directional selectivity of the motoneuron response was correlated to the previously measured gain of the reflex for each stimulus dimension. The information theoretical analysis is based upon Kullback-Leibler (K-L) distances which measure the dissimilarity of responses to different stimuli. K-L distances for single neurons are strongly influenced by the mean rate difference of the responses to any pair of stimuli. Because of redundancy, the joint K-L distances of pairs of neurons were less than the sum of the K-L distances of the individual neurons. Furthermore, the joint K-L distances were only weakly influenced by correlations among coactivated neurons. For most of the stimulus dimensions, the K-L distances of single motoneurons were not sufficient to account for the stimulus discriminations exhibited by the eyestalk reflex which typically required the summed output of 2 to 5 motoneurons. Thus the behaviorally relevant information is encoded in the motoneuron ensemble. The minimum time required to discriminate the direction of motion (the encoding window) for a single motoneuron is about 380 to 480 ms (including a 175 ms response latency) for stepwise displacements and up to 1.0 s for global motion. During this period a motoneuron fires 2 to 3 impulses.