Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 Å, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study

Maternal characteristics associated with the dietary intake of nitrates, nitrites, and nitrosamines in women of child-bearing age: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Multiple <it>N</it>-nitroso compounds have been observed in animal studies to be both mutagenic and teratogenic. Human exposure to <it>N</it>-nitroso compounds and their precursors, nitrates and nitrites, can occur through exogenous sources, such as diet, drinking water, occupation, or environmental exposures, and through endogenous exposures resulting from the formation of <it>N</it>-nitroso compounds in the body. Very little information is available on intake of nitrates, nitrites, and nitrosamines and factors related to increased consumption of these compounds.</p> <p>Methods</p> <p>Using survey and dietary intake information from control women (with deliveries of live births without major congenital malformations during 1997-2004) who participated in the National Birth Defects Prevention Study (NBDPS), we examined the relation between various maternal characteristics and intake of nitrates, nitrites, and nitrosamines from dietary sources. Estimated intake of these compounds was obtained from the Willet Food Frequency Questionnaire as adapted for the NBDPS. Multinomial logistic regression models were used to estimate odds ratios and 95% confidence intervals for the consumption of these compounds by self-reported race/ethnicity and other maternal characteristics.</p> <p>Results</p> <p>Median intake per day for nitrates, nitrites, total nitrites (nitrites + 5% nitrates), and nitrosamines was estimated at 40.48 mg, 1.53 mg, 3.69 mg, and 0.472 μg respectively. With the lowest quartile of intake as the referent category and controlling for daily caloric intake, factors predicting intake of these compounds included maternal race/ethnicity, education, body mass index, household income, area of residence, folate intake, and percent of daily calories from dietary fat. Non-Hispanic White participants were less likely to consume nitrates, nitrites, and total nitrites per day, but more likely to consume dietary nitrosamines than other participants that participated in the NBDPS. Primary food sources of these compounds also varied by maternal race/ethnicity.</p> <p>Conclusions</p> <p>Results of this study indicate that intake of nitrates, nitrites, and nitrosamines vary considerably by race/ethnicity, education, body mass index, and other characteristics. Further research is needed regarding how consumption of foods high in nitrosamines and <it>N</it>-nitroso precursors might relate to risk of adverse pregnancy outcomes and chronic diseases.</p

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress

Inflammatory Transcriptome Profiling of Human Monocytes Exposed Acutely to Cigarette Smoke

<div><h3>Background</h3><p>Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 10¹⁵ free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases.</p> <h3>Methodology/Principle findings</h3><p>To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway.</p> <h3>Conclusions</h3><p>Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes.</p> </div

What Is New for an Old Molecule? Systematic Review and Recommendations on the Use of Resveratrol

Stilbenes are naturally occurring phytoalexins that generally exist as their more stable E isomers. The most well known natural stilbene is resveratrol (Res), firstly isolated in 1939 from roots of Veratrum grandiflorum (white hellebore) (1) and since then found in various edible plants, notably in Vitis vinifera L. (Vitaceae) (2). The therapeutic potential of Res covers a wide range of diseases, and multiple beneficial effects on human health such as antioxidant, anti-inflammatory and anti-cancer activities have been suggested based on several in vitro and animal studies (3). In particular, Res has been reported to be an inhibitor of carcinogenesis at multiple stages via its ability to inhibit cyclooxygenase, and is an anticancer agent with a role in antiangiogenesis (4). Moreover, both in vitro and in vivo studies showed that Res induces cell cycle arrest and apoptosis in tumor cells (4). However, clinical studies in humans evidenced that Res is rapidly absorbed after oral intake, and that the low level observed in the blood stream is caused by a fast conversion into metabolites that are readily excreted from the body (5). Thus, considerable efforts have gone in the design and synthesis of Res analogues with enhanced metabolic stability. Considering that reduced Res (dihydro- resveratrol, D-Res) conjugates may account for as much as 50% of an oral Res dose (5), and that D-Res has a strong proliferative effect on hormone-sensitive cancer cell lines such as breast cancer cell line MCF7 (6), we recently devoted our synthetic efforts to the preparation of trans-restricted analogues of Res in which the E carbon-carbon double bond is embedded into an imidazole nucleus. To keep the trans geometry, the two aryl rings were linked to the heteroaromatic core in a 1,3 fashion. Based on this design, we successfully prepared a variety of 1,4-, 2,4- and 2,5-diaryl substituted imidazoles including Res analogues 1, 2 and 3, respectively, by procedures that involve transition metal-catalyzed Suzuki-Miyaura cross-coupling reactions and highly selective N-H or C-H direct arylation reactions as key synthetic steps. The anticancer activity of compounds 1–3 was evaluated against the 60 human cancer cell lines panel of the National Cancer Institute (NCI, USA). The obtained results, that will be showed and discussed along with the protocols developed for the preparation of imidazoles 1–3, confirmed that a structural optimization of Res may provide analogues with improved potency in inhibiting the growth of human cancer cell lines in vitro when compared to their natural lead. (1) Takaoka,M.J.Chem.Soc.Jpn.1939,60,1090-1100. (2) Langcake, P.; Pryce, R. J. Physiological. Plant Patology 1976, 9, 77-86. (3) Vang, O.; et al. PLoS ONE 2011, 6, e19881. doi:10.1371/journal.pone.0019881 (4) Kraft, T. E.; et al. Critical Reviews in Food Science and Nutrition 2009, 49, 782-799. (5) Walle, T. Ann. N.Y. Acad. Sci. 2011, 1215, 9-15. doi: 10.1111/j.1749-6632.2010.05842.x (6) Gakh,A.A.;etal.Bioorg.Med.Chem.Lett.2010,20,6149-6151

Mathematics and biology: a Kantian view on the history of pattern formation theory

Driesch’s statement, made around 1900, that the physics and chemistry of his day were unable to explain self-regulation during embryogenesis was correct and could be extended until the year 1972. The emergence of theories of self-organisation required progress in several areas including chemistry, physics, computing and cybernetics. Two parallel lines of development can be distinguished which both culminated in the early 1970s. Firstly, physicochemical theories of self-organisation arose from theoretical (Lotka 1910–1920) and experimental work (Bray 1920; Belousov 1951) on chemical oscillations. However, this research area gained broader acceptance only after thermodynamics was extended to systems far from equilibrium (1922–1967) and the mechanism of the prime example for a chemical oscillator, the Belousov–Zhabotinski reaction, was deciphered in the early 1970s. Secondly, biological theories of self-organisation were rooted in the intellectual environment of artificial intelligence and cybernetics. Turing wrote his The chemical basis of morphogenesis (1952) after working on the construction of one of the first electronic computers. Likewise, Gierer and Meinhardt’s theory of local activation and lateral inhibition (1972) was influenced by ideas from cybernetics. The Gierer–Meinhardt theory provided an explanation for the first time of both spontaneous formation of spatial order and of self-regulation that proved to be extremely successful in elucidating a wide range of patterning processes. With the advent of developmental genetics in the 1980s, detailed molecular and functional data became available for complex developmental processes, allowing a new generation of data-driven theoretical approaches. Three examples of such approaches will be discussed. The successes and limitations of mathematical pattern formation theory throughout its history suggest a picture of the organism, which has structural similarity to views of the organic world held by the philosopher Immanuel Kant at the end of the eighteenth century

Visuospatial Integration: Paleoanthropological and Archaeological Perspectives

The visuospatial system integrates inner and outer functional processes, organizing spatial, temporal, and social interactions between the brain, body, and environment. These processes involve sensorimotor networks like the eye–hand circuit, which is especially important to primates, given their reliance on vision and touch as primary sensory modalities and the use of the hands in social and environmental interactions. At the same time, visuospatial cognition is intimately connected with memory, self-awareness, and simulation capacity. In the present article, we review issues associated with investigating visuospatial integration in extinct human groups through the use of anatomical and behavioral data gleaned from the paleontological and archaeological records. In modern humans, paleoneurological analyses have demonstrated noticeable and unique morphological changes in the parietal cortex, a region crucial to visuospatial management. Archaeological data provides information on hand–tool interaction, the spatial behavior of past populations, and their interaction with the environment. Visuospatial integration may represent a critical bridge between extended cognition, self-awareness, and social perception. As such, visuospatial functions are relevant to the hypothesis that human evolution is characterized by changes in brain–body–environment interactions and relations, which enhance integration between internal and external cognitive components through neural plasticity and the development of a specialized embodiment capacity. We therefore advocate the investigation of visuospatial functions in past populations through the paleoneurological study of anatomical elements and archaeological analysis of visuospatial behaviors

Electrical Brain Stimulation During a Retrieval-Based Learning Task Can Impair Long-Term Memory

Anodal transcranial direct current stimulation (tDCS) to the left dorsolateral prefrontal cortex (DLPFC) has been shown to improve performance on a multitude of cognitive tasks. These are, however, often simple tasks, testing only one cognitive domain at a time. Therefore, the efficacy of brain stimulation for complex tasks has yet to be understood. Using a task designed to increase learning efficiency, this study investigates whether anodal tDCS over the left DLPFC can modulate both learning ability and subsequent long-term memory retention. Using a within-subject design, participants (N = 25) took part in 6 training sessions over consecutive days in which active or sham stimulation was administered randomly (3 of each). A computer-based task was used, containing flags from countries unknown to the participants. Each training session consisted of the repetition of 8 pairs of flag/country names. Subsequently, in three testing sessions, free, cued, and timed cued recall, participants were assessed on all 48 flags they had learnt. No difference in learning speed between active and sham tDCS was found. Furthermore, in the timed cued recall phase, flags learnt in the sham tDCS sessions were recalled significantly better than flags learnt in the active tDCS sessions. This effect was stronger in the second testing session. It was also found that for the flags answered incorrectly; thus, meaning they were presented more frequently, subsequent long-term retention was improved. These results suggest that for a complex task, anodal tDCS is ineffective at improving learning speed and potentially detrimental to long-term retention when employed during encoding. This serves to highlight the complex nature of brain stimulation, providing a greater understanding of its limitations and drawbacks

Treatment failure in pneumonia: Impact of antibiotic treatment and cost analysis

The aim of this study was to investigate treatment failure (TF) in hospitalised community-acquired pneumonia (CAP) patients with regard to initial antibiotic treatment and economic impact. CAP patients were included in two open, prospective multicentre studies assessing the direct costs for in-patient treatment. Patients received treatment either with moxifloxacin (MFX) or a nonstandardised antibiotic therapy. Any change in antibiotic therapy after >72 h of treatment to a broadened antibiotic spectrum was considered as TF. Overall, 1,236 patients (mean ± SD age 69.6 ± 16.8 yrs, 691 (55.9%) male) were included. TF occurred in 197 (15.9%) subjects and led to longer hospital stay (15.4 ± 7.3 days versus 9.8 ± 4.2 days; p < 0.001) and increased median treatment costs (€2,206 versus €1,284; p<0.001). 596 (48.2%) patients received MFX and witnessed less TF (10.9% versus 20.6%; p < 0.001). After controlling for confounders in multivariate analysis, adjusted risk of TF was clearly reduced in MFX as compared with β-lactam monotherapy (adjusted OR for MFX 0.43, 95% CI 0.27-0.68) and was more comparable with a β-lactam plus macrolide combination (BLM) (OR 0.68, 95% CI 0.38-1.21). In hospitalised CAP, TF is frequent and leads to prolonged hospital stay and increased treatment costs. Initial treatment with MFX or BLM is a possible strategy to prevent TF, and may thus reduce treatment costs