Simultaneous acquisition of PAR and PAIN spectra

We present a scheme that allows the simultaneous detection of PAR and PAIN correlation spectra in a single two-dimensional experiment. For both spectra, we obtain almost the same signal-to-noise ratio as if a PAR or PAIN spectrum is recorded separately, which in turn implies that one of the spectra may be considered additional information for free. The experiment is based on the observation that in a PAIN experiment, the PAR condition is always also fulfilled. The performance is demonstrated experimentally using uniformly ¹³C,¹⁵N-labeled samples of N–f–MLF–OH and ubiquitin.     

Solid-state NMR sequential assignments of α-synuclein

Parkinson’s disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the first one with a well-ordered and rigid N-terminal part.     

Identification of immunogenic proteins of Waddlia chondrophila

Evidence is growing for a role of Waddlia chondrophila as an agent of adverse pregnancy outcomes in both humans and ruminants. This emerging pathogen, member of the order Chlamydiales, is also implicated in bronchiolitis and lower respiratory tract infections. Until now, the serological diagnosis of W. chondrophila infection has mainly relied on manually intensive tests including micro-immunofluorescence and Western blotting. Thus, there is an urgent need to establish reliable high throughput serological assays. Using a combined genomic and proteomic approach, we detected 57 immunogenic proteins of W. chondrophila, of which 17 were analysed by mass spectrometry. Two novel hypothetical proteins, Wim3 and Wim4, were expressed as recombinant proteins in Escherichia coli, purified and used as antigens in an ELISA test. Both proteins were recognized by sera of rabbits immunized with W. chondrophila as well as by human W. chondrophila positive sera but not by rabbit pre-immune sera nor human W. chondrophila negative sera. These results demonstrated that the approach chosen is suitable to identify immunogenic proteins that can be used to develop a serological test. This latter will be a valuable tool to further clarify the pathogenic potential of W. chondrophila.     

Chromatin as an expansive canvas for chemical biology

Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.     

Future of an "Asymptomatic" T-cell Epitope-Based Therapeutic Herpes Simplex Vaccine

Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as "asymptomatic" protective epitopes") could boost local and systemic "natural" protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging "asymptomatic" T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease.     

A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.     

High variation in foliage and leaf litter chemistry among 45 tree species of a neotropical rainforest community

Distinct ecosystem level carbon : nitrogen : phosphorus (C : N : P) stoichiometries in forest foliage have been suggested to reflect ecosystem-scale selection for physiological strategies in plant nutrient use. Here, this hypothesis was explored in a nutrient-poor lowland rainforest in French Guiana. Variation in C, N and P concentrations was evaluated in leaf litter and foliage from neighbour trees of 45 different species, and the litter concentrations of major C fractions were also measured. Litter C ranged from 45.3 to 52.4%, litter N varied threefold (0.68-2.01%), and litter P varied seven-fold (0.009-0.062%) among species. Compared with foliage, mean litter N and P concentrations decreased by 30% and 65%, respectively. Accordingly, the range in mass-based N : P shifted from 14 to 55 in foliage to 26 to 105 in litter. Resorption proficiencies indicated maximum P withdrawal in most species, but with a substantial increase in variation in litter P compared with foliage. These data suggest that constrained ecosystem-level C : N : P ratios do not preclude the evolution of highly diversified strategies of nutrient use and conservation among tropical rainforest tree species. The resulting large variation in litter quality will influence stoichiometric constraints within the decomposer food web, with potentially far-ranging consequences on nutrient dynamics and plant-soil feedbacks.     

Age-related changes in muscle fiber type frequencies and cross-sectional areas in straightbred and crossbred rabbits

This study was designed to investigate the effects of the interaction among genetic group, sex and age on the frequencies and cross-sectional areas of myofiber types in rabbits. A total of 48 straightbred and crossbred Botucatu rabbits, males and females, were involved in a split plot design with a 2 × 2 (genetic groups × genders) factorial arrangement. Young rabbits were weaned at 35 days of age and sequentially slaughtered, four per genetic group × sex combination, at 42, 63 and 84 days of age. The flexor carpi radialis muscle was dissected, histological sections (10 μm) were obtained and the frequencies and cross-sectional areas of myofiber types: I, IIA and IIB/X were determined. An effect of the genetic group × sex × slaughter age interaction was found on the frequency distribution of myofiber types. A transition from type IIA to type IIB/X fibers was observed (P < 0.01) with advancing age, except in crossbred females, but the frequency of IIA fibers was already lower (57.3%) and of IIB/X fibers numerically higher (33.7%) in this group at 42 days. The proportions of IIA fibers in straightbred males, crossbred males and straightbred females decreased from 80.1%, 89.4% and 68.8% at 42 days to 43.9%, 52.3% and 40.1% at 63 days, respectively, whereas the proportions of type IIB/X fibers, in the same groups, increased from 10.3%, 1.6% and 22.3% at 42 days to 42.2%, 37.0% and 49.8% at 63 days, respectively. In all three age points, type IIA fibers showed the largest cross-sectional areas, followed by type I and IIB/X fibers. The cross-sectional areas of IIB/X fibers were larger in crossbreds, but no differences were found between genetic groups concerning fiber types IIA and I. All three types of fibers showed positive linear association with age, but relative to the initial area type IIB/X fibers presented a higher degree of hypertrophy (144% up to 84 days) than type IIA and I fibers (86% and 85%, respectively). The flexor carpi radialis muscle was, on average, heavier in crossbred than in straightbred females, but no difference was observed between crossbred and straightbred males. Differences in the weight of flexor carpi radialis muscle were attributed to the hypertrophy of type IIB/X fibers in the crossbreds.