Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] < [Ca(2+)], [Mg(2+)], [Mn(2+)] < [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.     

Tunable pores for measuring concentrations of synthetic and biological nanoparticle dispersions

Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210-710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ~1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus, with a diameter of ~600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Choosy moral punishers

The punishment of social misconduct is a powerful mechanism for stabilizing high levels of cooperation among unrelated individuals. It is regularly assumed that humans have a universal disposition to punish social norm violators, which is sometimes labelled "universal structure of human morality" or "pure aversion to social betrayal". Here we present evidence that, contrary to this hypothesis, the propensity to punish a moral norm violator varies among participants with different career trajectories. In anonymous real-life conditions, future teachers punished a talented but immoral young violinist: they voted against her in an important music competition when they had been informed of her previous blatant misconduct toward fellow violin students. In contrast, future police officers and high school students did not punish. This variation among socio-professional categories indicates that the punishment of norm violators is not entirely explained by an aversion to social betrayal. We suggest that context specificity plays an important role in normative behaviour; people seem inclined to enforce social norms only in situations that are familiar, relevant for their social category, and possibly strategically advantageous.     

Sexual dimorphism in healthy aging and mild cognitive impairment: a DTI study

Previous PET and MRI studies have indicated that the degree to which pathology translates into clinical symptoms is strongly dependent on sex with women more likely to express pathology as a diagnosis of AD, whereas men are more resistant to clinical symptoms in the face of the same degree of pathology. Here we use DTI to investigate the difference between male and female white matter tracts in healthy older participants (24 women, 16 men) and participants with mild cognitive impairment (21 women, 12 men). Differences between control and MCI participants were found in fractional anisotropy (FA), radial diffusion (DR), axial diffusion (DA) and mean diffusion (MD). A significant main effect of sex was also reported for FA, MD and DR indices, with male control and male MCI participants having significantly more microstructural damage than their female counterparts. There was no sex by diagnosis interaction. Male MCIs also had significantly less normalised grey matter (GM) volume than female MCIs. However, in terms of absolute brain volume, male controls had significantly more brain volume than female controls. Normalised GM and WM volumes were found to decrease significantly with age with no age by sex interaction. Overall, these data suggest that the same degree of cognitive impairment is associated with greater structural damage in men compared with women.     

Laboratory evaluation of large-scale decontamination approaches

Aims: To evaluate the effectiveness of two spray‐based decontamination methods for surface contamination reduction and to determine the potential for contamination spread by these methods. Methods and Results: Material coupons (treated plywood and concrete) were contaminated with c. 1 × 10⁷ spores of Bacillus atrophaeus by aerosol deposition. Decontaminants (pH‐adjusted bleach or Spor‐Klenz^® RTU) were applied to coupons by either backpack sprayer or gas‐powered sprayer. Contact time, reapplication frequency and rinse method were also varied. In addition to surface removal efficacy, partitioning of contamination between the rinsate and aerosol fractions was determined. Results indicated that pH‐adjusted bleach was effective (≥6 logs reduction) when two applications and a 30 min contact time were administered, regardless of the decontaminant application method or material. Spor‐Klenz^® RTU was effective on wood, but achieved ≤3 logs reduction on concrete. A shortened application procedure with pH‐adjusted bleach resulted in lower efficacy on wood, and a greater apparent potential for contamination spread. Conclusions: Consideration of material surface type is important when selecting a decontaminant. Also, achieving conditions that effectively inactivate surface biological contamination are critical to preventing the spread of contamination. Significance and Impact of the Study: Results presented here are intended to help development of remediation plans following a biological contamination incident.