Sorting of early and late flagellar subunits after docking at the membrane ATPase of the type III export pathway

The bacterial flagellum assembles in a strict order, with structural subunits delivered to the growing flagellum by a type III export pathway. Early rod-and-hook subunits are exported before completion of the hook, at which point a subunit-specificity switch allows export of late filament subunits. This implies that in bacteria with multiple flagella at different stages of assembly, each export pathway can discriminate and sort unchaperoned early and chaperoned late subunits. To establish whether subunit sorting is distinct from subunit transition from the cytosol to the membrane, in particular docking at the membrane-associated FliI ATPase, the pathway was manipulated in vivo. When ATP hydrolysis by the FliI ATPase was disabled and when the pathway was locked into an early export state, both unchaperoned early and chaperoned late subunits stalled and accumulated at the inner membrane. Furthermore, a chaperone that attenuates late subunit export by stalling when docked at the wild-type ATPase also stalled at the ATPase in an early-locked pathway and inhibited export of early subunits in both native and early-locked pathways. These data indicate that the pathways for early and late subunits converge at the FliI ATPase, independent of ATP hydrolysis, before a distinct, separable sorting step. To ascertain the likely signals for sorting, the export of recombinant subunits was assayed. Late filament subunits unable to bind their chaperones were still sorted accurately, but chaperoned late subunits were directed through an early-locked pathway when fused to early subunit N-terminal export signal regions. Furthermore, while an early subunit signal directed export of a heterologous type III export substrate through both native and early-locked pathways, a late subunit signal only directed export via native pathways. These data suggest that subunits are distinguished not by late chaperones but by N-terminal export signals of the subunits themselves.     

Retinal Vascular Fractal Dimension,Childhood IQ,and Cognitive Ability in Old Age: The Lothian Birth Cohort Study 1936

Purpose

Cerebral microvascular disease is associated with dementia. Differences in the topography of the retinal vascular network may be a marker for cerebrovascular disease. The association between cerebral microvascular state and non-pathological cognitive ageing is less clear, particularly because studies are rarely able to adjust for pre-morbid cognitive ability level. We measured retinal vascular fractal dimension (D _f) as a potential marker of cerebral microvascular disease. We examined the extent to which it contributes to differences in non-pathological cognitive ability in old age, after adjusting for childhood mental ability.

Methods

Participants from the Lothian Birth Cohort 1936 Study (LBC1936) had cognitive ability assessments and retinal photographs taken of both eyes aged around 73 years (n = 648). IQ scores were available from childhood. Retinal vascular D _f was calculated with monofractal and multifractal analysis, performed on custom-written software. Multiple regression models were applied to determine associations between retinal vascular D _f and general cognitive ability (g), processing speed, and memory.

Results

Only three out of 24 comparisons (two eyes × four D _f parameters × three cognitive measures) were found to be significant. This is little more than would be expected by chance. No single association was verified by an equivalent association in the contralateral eye.

Conclusions

The results show little evidence that fractal measures of retinal vascular differences are associated with non-pathological cognitive ageing.     

Suitability of UK Biobank Retinal Images for Automatic Analysis of Morphometric Properties of the Vasculature

PurposeTo assess the suitability of retinal images held in the UK Biobank - the largest retinal data repository in a prospective population-based cohort - for computer assisted vascular morphometry, generating measures that are commonly investigated as candidate biomarkers of systemic disease.MethodsNon-mydriatic fundus images from both eyes of 2,690 participants - people with a self-reported history of myocardial infarction (n=1,345) and a matched control group (n=1,345) - were analysed using VAMPIRE software. These images were drawn from those of 68,554 UK Biobank participants who underwent retinal imaging at recruitment. Four operators were trained in the use of the software to measure retinal vascular tortuosity and bifurcation geometry.ResultsTotal operator time was approximately 360 hours (4 minutes per image). 2,252 (84%) of participants had at least one image of sufficient quality for the software to process, i.e. there was sufficient detection of retinal vessels in the image by the software to attempt the measurement of the target parameters. 1,604 (60%) of participants had an image of at least one eye that was adequately analysed by the software, i.e. the measurement protocol was successfully completed. Increasing age was associated with a reduced proportion of images that could be processed (p=0.0004) and analysed (p<0.0001). Cases exhibited more acute arteriolar branching angles (p=0.02) as well as lower arteriolar and venular tortuosity (p<0.0001).ConclusionsA proportion of the retinal images in UK Biobank are of insufficient quality for automated analysis. However, the large size of the UK Biobank means that tens of thousands of images are available and suitable for computational analysis. Parametric information measured from the retinas of participants with suspected cardiovascular disease was significantly different to that measured from a matched control group.     

Functional role of the Werner syndrome RecQ helicase in human fibroblasts

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN‐deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40‐transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.     

Parasitic nematode fatty acid- and retinol-binding proteins compromise host immunity by interfering with host lipid signaling pathways

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.     

Untying the regulation of the Raf-1 kinase

The Raf-1 kinase is the entry point to the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK-1/2) signaling pathway, which controls fundamental cellular functions including proliferation, differentiation, and survival. As such, Raf-1 is regulated by complex mechanisms that are incompletely understood. Recent results have shown that release from repression is an important event that facilitates the interaction of Raf-1 with the Ras activator and its substrate, MAPK/ERK-1/2 kinase. A number of distinct activation steps contribute in a combinatorial fashion to regulate and adjust Raf-1 activity. The efficiency of downstream signal transmission is modulated by protein:protein interactions, and new data consolidate an important role for kinase suppressor of ras (KSR) as a scaffolding protein. KSR is a dynamic scaffold whose function and localization is regulated by phosphorylation.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.