Detection of truncated virus particles in a persistent RNA virus infection in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors.     

Refolding of proteins from inclusion bodies: rational design and recipes

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.     

Structure of AscE and induced burial regions in AscE and AscG upon formation of the chaperone needle-subunit complex of type III secretion system in Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit AscF requires both putative chaperones AscE and AscG for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 A resolution and the mapping of buried regions of AscE, AscG, and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate-binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remain exposed even in the ternary AscEFG complex. On the other hand, the 35-residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.     

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.     

iTRAQ-based proteomic profiling of breast cancer cell response to doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC-MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer.     

Regional Amyloid Deposition in Amnestic Mild Cognitive Impairment and Alzheimer's Disease Evaluated by [18F]AV-45 Positron Emission Tomography in Chinese Population

Background

To compare the neocortical amyloid loads among cognitively normal (CN), amnestic mild cognitive impairment (aMCI), and Alzheimer''s disease (AD) subjects with [¹⁸F]AV-45 positron emission tomography (PET).

Materials and Methods

[¹⁸F]AV-45 PET was performed in 11 CN, 13 aMCI, and 12 AD subjects to compare the cerebral cortex-to-whole cerebellum standard uptake value ratios (SUVRs) of global and individual volumes of interest (VOIs) cerebral cortex. The correlation between global cortical [¹⁸F]AV-45 SUVRs and Mini-Mental State Examination (MMSE) scores was analyzed.

Results

The global cortical [¹⁸F]AV-45 SUVRs were significantly different among the CN (1.08±0.08), aMCI (1.27±0.06), and AD groups (1.34±0.13) (p = 0.0003) with amyloidosis positivity rates of 9%, 62%, and 92% in the three groups respectively. Compared to CN subjects, AD subjects had higher SUVRs in the global cortical, precuneus, frontal, parietal, occipital, temporal, and posterior cingulate areas; while aMCI subjects had higher values in the global cortical, precuneus, frontal, occipital and posterior cingulate areas. There were negative correlations of MMSE scores with SUVRs in the global cortical, precuneus, frontal, parietal, occipital, temporal, posterior cingulate and anterior cingulate areas on a combined subject pool of the three groups after age and education attainment adjustment.

Conclusions

Amyloid deposition occurs relatively early in precuneus, frontal and posterior cingulate in aMCI subjects. Higher [¹⁸F]AV-45 accumulation is present in parietal, occipital and temporal gyri in AD subjects compared to the aMCI group. Significant correlation between MMSE scores and [¹⁸F]AV-45 SUVRs can be observed among CN, aMCI and AD subjects.     

Microbial colonization and succession in the large intestine of newborn infants during their stay with mothers at the maternity hospital]

Formation of microflora in the large intestine of 5-day old infants was studied in one of the Moscow maternity homes. The up-to-date procedures for isolation and identification of aerobic and anaerobic organisms were used in the study and the findings were processed on a computer. In the newborns of the maternity home of the "mother-infant" type there was observed colonization of the large intestine with aerobic and anaerobic organisms. A wave-like dynamics in the formation of the symbiotic microflora was revealed. It reflected the phenomenon of the microbial succession in the infants. The attempts to detect microbial interference between the species colonizing the large intestine showed that it was extremely rare in the 5-day old infants. This was likely the reason of the low intestine resistance to the colonization in the newborns which in its turn defined the frequent colonization of the intestine mucosa with S. aureus and the organisms of the Klebsiella, Enterobacter and Citrobacter group.     

Liver fat reduction with niacin is influenced by DGAT-2 polymorphisms in hypertriglyceridemic patients

Niacin reduces plasma triglycerides, but it may increase free fatty acids and insulin resistance during long-term treatment. We examined the effect of extended-release niacin on liver fat content in Chinese patients with dyslipidemia and whether the common diacylglycerol acyltransferase-2 (DGAT2) polymorphisms influenced this effect. The 39 patients (baseline liver fat content: 12.8 ± 7.6%, triglycerides: 3.30 ± 1.67 mmol/l) were treated with niacin, gradually increasing the dose to 2 g/day for a total of 23 weeks. The liver fat content and visceral/subcutaneous fat was measured before and after treatment. Subjects were genotyped for the DGAT2 rs3060 and rs101899116 polymorphisms. There were significant (P < 0.001) reductions in plasma triglycerides (-34.9 ± 37.6%), liver fat content (-47.2 ± 32.8%), and visceral fat (-6.3 ± 15.8%, P < 0.05) after niacin treatment. Mean body weight decreased by 1.46 ± 2.7% (1.17 ± 2.44 kg, P < 0.001) during the study, but liver fat changes remained significant after adjustment for age, gender, and body weight changes [mean absolute change (95% CI): -6.1% (-8.0, -4.3), P < 0.001]. The DGAT2 variant alleles were associated with a smaller reduction in liver fat content in response to niacin after adjustment for other covariates (P < 0.01). These findings suggest that niacin treatment may reduce liver fat content in Chinese patients with dyslipidemia and that the mechanism may involve inhibition of DGAT2. However, the findings might have been confounded by the small but significant reductions in body weight during the study. Future large randomized controlled trials are needed to verify these findings.     

Lifestyle and Ice: The Relationship between Ecological Specialization and Response to Pleistocene Climate Change

Major climatic changes in the Pleistocene had significant effects on marine organisms and the environments in which they lived. The presence of divergent patterns of demographic history even among phylogenetically closely-related species sharing climatic changes raises questions as to the respective influence of species-specific traits on population structure. In this work we tested whether the lifestyle of Antarctic notothenioid benthic and pelagic fish species from the Southern Ocean influenced the concerted population response to Pleistocene climatic fluctuations. This was done by a comparative analysis of sequence variation at the cyt b and S7 loci in nine newly sequenced and four re-analysed species. We found that all species underwent more or less intensive changes in population size but we also found consistent differences between demographic histories of pelagic and benthic species. Contemporary pelagic populations are significantly more genetically diverse and bear traces of older demographic expansions than less diverse benthic species that show evidence of more recent population expansions. Our findings suggest that the lifestyles of different species have strong influences on their responses to the same environmental events. Our data, in conjunction with previous studies showing a constant diversification tempo of these species during the Pleistocene, support the hypothesis that Pleistocene glaciations had a smaller effect on pelagic species than on benthic species whose survival may have relied upon ephemeral refugia in shallow shelf waters. These findings suggest that the interaction between lifestyle and environmental changes should be considered in genetic analyses.