Considerations of Noise and Measurement Reproducibility of Circular Dichroism Measurements Using Na[Co(III) (EDDS)

Although circular dichroism (CD) spectroscopy has become ubiquitous in biological, chemical, and pharmacological laboratories, several issues with instrument calibration and standardization, measurements quality, and reproducibility still remain. In the context of validating and developing a wide-wavelength-range stable standard for CD that is available in both enantiomeric forms, (R,R)- and (S,S)-Na[Co(III) (EDDS)] (where EDDS is ethylenediaminedisuccinato), we have become aware of sources of error in CD spectroscopy in addition to instrument calibration and user issues. This article summarizes those errors and suggests procedures for minimizing them by averaging over sufficient independent data accumulations with appropriate parameters. Chirality 24:699-705, 2012. ? 2012 Wiley Periodicals, Inc.     

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.     

Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1β in the pancreas

IL-1β is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1β in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1β transgenic [Tg(IL1β)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1β) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1β) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1β)-Tg(Psti1)] mice expressing IL-1β and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.     

Phylogenetic beta diversity of native and alien species in European urban floras

Aim Human activities have weakened biogeographical barriers to dispersal, increasing the rate of introduction of alien plants. However, their impact on beta diversity and floristic homogenization is poorly understood. Our goal is to compare the phylogenetic beta diversity of native species with that of two groups of alien species, archaeophytes and neophytes (introduced before and after ad 1500, respectively), across European urban floras to explore how biological invasions affect phylogenetic turnover at a continental scale. Location Twenty European cities located in six countries between 49 and 53° N latitude in continental Europe and the British Isles. Methods To compare the phylogenetic beta diversity of native and alien species we use the average phylogenetic dissimilarity of individual floras from their group centroid in multivariate space. Differences in phylogenetic beta diversity among different species groups are then assessed using a randomization test for homogeneity of multivariate dispersions. Results Across European urban floras, and when contrasted with natives, archaeophytes are usually associated with lower levels of phylogenetic beta diversity while neophytes tend to increase phylogenetic differentiation. Main conclusions While archaeophytes tend to promote limited homogenization in phylogenetic beta diversity, because of their diverse geographical origin together with short residence times in the invaded regions, neophytes are not promoting biotic homogenization of urban floras across Europe. Therefore, in spite of the increasing rate of alien invasion, an intense phylogenetic homogenization of urban cities is not to be expected soon.     

Incidence of and risk factors for Motor Neurone Disease in UK women: a prospective study

ABSTRACT: BACKGROUND: Motor neuron disease (MND) is a severe neurodegenerative disease with largely unknown etiology. Most epidemiological studies are hampered by small sample sizes and/or the retrospective collection of information on behavioural and lifestyle factors. METHODS: 1.3 million women from the UK Million Women Study, aged 56 years on average at recruitment, were followed up for incident and/or fatal MND using NHS hospital admission and mortality data. Adjusted relative risks were calculated using Cox regression models. FINDINGS: During follow-up for an average of 9.2 years, 752 women had a new diagnosis of MND. Age-specific rates increased with age, from 1.9 (95% CI 1.3 - 2.7) to 12.5 (95% CI 10.2 - 15.3) per 100,000 women aged 50-54 to 70-74, respectively, giving a cumulative risk of diagnosis with the disease of 1. 74 per 1000 women between the ages of 50 and 75 years. There was no significant variation in risk of MND with region of residence, socio-economic status, education, height, alcohol use, parity, use of oral contraceptives or hormone replacement therapy. Ever-smokers had about a 20% greater risk than never smokers (RR 1.19 95% CI 1.02 to 1.38, p=0.03). There was a statistically significant reduction in risk of MND with increasing body mass index (pfor trend=0.009): obese women (body mass index, 30 kg/m2 or more) had a 20% lower risk than women of normal body mass index (20 to <25 Kg/m2)(RR 0.78 95% CI 0.65-0.94; p=0.03). This effect persisted after exclusion of the first three years of follow-up. Interpretation. MND incidence in UK women rises rapidly with age, and an estimated 1 in 575 women are likely to be affected between the ages of 50 and 75 years. Smoking slightly increases the risk of MND, and adiposity in middle age is associated with a lower risk of the disease.     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

Introduction

B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.

Methods

In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.

Results

Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.

Conclusion

By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.     

Morphological Characteristics of Motor Neurons Do Not Determine Their Relative Susceptibility to Degeneration in a Mouse Model of Severe Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, resulting primarily from the degeneration and loss of lower motor neurons. Studies using mouse models of SMA have revealed widespread heterogeneity in the susceptibility of individual motor neurons to neurodegeneration, but the underlying reasons remain unclear. Data from related motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), suggest that morphological properties of motor neurons may regulate susceptibility: in ALS larger motor units innervating fast-twitch muscles degenerate first. We therefore set out to determine whether intrinsic morphological characteristics of motor neurons influenced their relative vulnerability to SMA. Motor neuron vulnerability was mapped across 10 muscle groups in SMA mice. Neither the position of the muscle in the body, nor the fibre type of the muscle innervated, influenced susceptibility. Morphological properties of vulnerable and disease-resistant motor neurons were then determined from single motor units reconstructed in Thy.1-YFP-H mice. None of the parameters we investigated in healthy young adult mice – including motor unit size, motor unit arbor length, branching patterns, motor endplate size, developmental pruning and numbers of terminal Schwann cells at neuromuscular junctions - correlated with vulnerability. We conclude that morphological characteristics of motor neurons are not a major determinant of disease-susceptibility in SMA, in stark contrast to related forms of motor neuron disease such as ALS. This suggests that subtle molecular differences between motor neurons, or extrinsic factors arising from other cell types, are more likely to determine relative susceptibility in SMA.