An improved method for determining enantiomeric excess by 13C‐NMR in chiral liquid crystal media

By using a combination of inverse gated ¹H decoupled ¹³C‐NMR experiments 1 with short acquisition times and NMR Cryo‐probe technology, the sample requirements and experimental times necessary to accurately measure enantiomeric excess of small chiral molecules has been reduced 16‐fold. Quality ¹³C‐NMR spectra can now be obtained from a 1 to 5 mg sample in 12 minutes. The enantiomeric excess determination achieved from the average integration of all the ¹³C‐resonances in the spectrum is comparable to enantiomeric excess measured by chiral SFC. The advantage of the NMR method is that enantiomeric excess can rapidly be measured in situ on practical amounts of enantioselective reaction products without the need for chromatographic separation or chemical modification and with substantially less solvent waste. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Lower-extremity muscle atrophy and fat infiltration after chronic spinal cord injury

Background:

Atrophy and fatty-infiltration of lower-extremity muscle after spinal cord injury (SCI) predisposes individuals to metabolic disease and related mortality.

Objectives:

To determine the magnitude of atrophy and fatty-infiltration of lower-extremity muscles and related factors in a group of individuals with chronic SCI and diverse impairment.

Methods:

Muscle cross-sectional area and density were calculated from peripheral quantitative computed tomography scans of the 66% site of the calf of 70 participants with chronic SCI [50 male, mean age 49 (standard deviation 12) years, C2-T12, AIS A-D] and matched controls. Regression models for muscle area and density were formed using 16 potential correlates selected a priori.

Results:

Participants with motor-complete SCI had ≈32% lower muscle area, and ≈43% lower muscle density values relative to controls. Participants with motor-incomplete SCI had muscle area and density values that were both ≈14% lower than controls. Body mass (+), tetraplegia (+), motor function (+), spasticity (+), vigorous physical activity (+), wheelchair use (-), age (-), and waist circumference (-) were associated with muscle size and/or density in best-fit regression models.

Conclusions:

There are modifiable factors related to muscle size, body composition, and activity level that may offer therapeutic targets for preserving metabolic health after chronic SCI.     

Molecularly imprinted polymers for RGD selective recognition and separation

Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N′-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid–trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K _D) value and the higher B _max% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.     

A review of FMRFamide- and RFamide-like peptides in metazoa

Neuropeptides are a diverse class of signalling molecules that are widely employed as neurotransmitters and neuromodulators in animals, both invertebrate and vertebrate. However, despite their fundamental importance to animal physiology and behaviour, they are much less well understood than the small molecule neurotransmitters. The neuropeptides are classified into families according to similarities in their peptide sequence; and on this basis, the FMRFamide and RFamide-like peptides, first discovered in molluscs, are an example of a family that is conserved throughout the animal phyla. In this review, the literature on these neuropeptides has been consolidated with a particular emphasis on allowing a comparison between data sets in phyla as diverse as coelenterates and mammals. The intention is that this focus on the structure and functional aspects of FMRFamide and RFamide-like neuropeptides will inform understanding of conserved principles and distinct properties of signalling across the animal phyla.     

A Scoping Review of Strategies for the Prevention of Hip Fracture in Elderly Nursing Home Residents

Background

Elderly nursing home residents are at increased risk of hip fracture; however, the efficacy of fracture prevention strategies in this population is unclear.

Objective

We performed a scoping review of randomized controlled trials of interventions tested in the long-term care (LTC) setting, examining hip fracture outcomes.

Methods

We searched for citations in 6 respective electronic searches, supplemented by hand searches. Two reviewers independently reviewed all citations and full-text papers; consensus was achieved on final inclusion. Data was abstracted in duplicate.

Findings

We reviewed 22,349 abstracts or citations and 949 full-text papers. Data from 20 trials were included: 7 - vitamin D (n = 12,875 participants), 2 - sunlight exposure (n = 522), 1 - alendronate (n = 327), 1 - fluoride (n = 460), 4 – exercise or multimodal interventions (n = 8,165), and 5 - hip protectors (n = 2,594). Vitamin D, particularly vitamin D₃ ≥800 IU orally daily, reduced hip fracture risk. Hip protectors reduced hip fractures in included studies, although a recent large study not meeting inclusion criteria was negative. Fluoride and sunlight exposure did not significantly reduce hip fractures. Falls were reduced in three studies of exercise or multimodal interventions, with one study suggesting reduced hip fractures in a secondary analysis. A staff education and risk assessment strategy did not significantly reduce falls or hip fractures. In a study underpowered for fracture outcomes, alendronate did not significantly reduce hip fractures in LTC.

Conclusions

The intervention with the strongest evidence for reduction of hip fractures in LTC is Vitamin D supplementation; more research on other interventions is needed.     

Experimentally induced orchitis associated with Arcanobacterium pyogenes: clinical, ultrasonographic, seminological and pathological features

The objectives of this study were to describe the features of experimentally induced orchitis associated with Arcanobacterium pyogenes and confirm the pathogenicity of the organism for the ovine testicle. One testicle of each of nine rams was inoculated with 1.3 +/- 10(4) colony-forming-units of an A. pyogenes isolate and regular clinical, ultrasonographic, bacteriological and seminological examinations were carried out up to 204 days after challenge. The rams were sequentially euthanatized 3, 6, 9, 18, 30, 50, 71, 113 and 204 days after challenge and a gross- and histopathological examination of their testicles was performed. All rams developed clinical orchitis and general signs. The initial ultrasonographic findings were changes of size and echogenicity of the genitalia, whilst in the long-standing phase they were wider appearance of the mediastinum testis, presence of hyperechogenic foci, changes of echogenicity of the genitalia and increased echogenicity of the scrotum and tunics. The following changes in semen evaluation parametres were recorded: the pH, the percentage of dead sperms, the percentage of abnormal sperms and the number of nonsperm round cells increased, whilst the mass motility, the individual motility and the sperm concentration decreased; the following sperm defects were observed: misshapen or piriform heads, sperms with coiled tails, sperms without tail and sperms with proximal cytoplasmic droplet; at the early stages neutrophils were the prevailing nonsperm round cell type, later the proportion of immature germ cells increased and in the long-standing phase there were enlogated spermatids and leucocytes; it is noteworthy that semen evaluation parametres were restored to normal at the late stages of the disease. A. pyogenes was consistently isolated from the semen samples after challenge, as well as from the dissected genitalia. The salient post-mortem findings were: initially, subcutaneous oedema, fluid into the vaginal cavity, congested and distended vessels, increased size of the genitalia and a hard dark area inside the testicles; subsequently, there were changes of size of the genitalia, thickening of scrotum and tunics and presence of fibrin on the testicular surface; in the long-standing phase of the disorder, there were induration of scrotum and tunics with adhesion between the tunics and discolouration of the surface of the genitalia. The prominent histopathological changes were observed in the inoculated testicles; milder changes were seen in the respective epididymides; interstitial oedema, diffuse neutrophilic infiltration and extravasation were observed in the early stages after challenge; lymphocytic infiltration with concurrent fibrosis, mineralization and inspissation of the tubular elements of the seminiferous tubules and presence of vacuolated Sertoli cells were seen later; finally, regeneration of the epithelium and presence of Sertoli cells and spermatogonia with various degrees of spermatogenic activity were evident. These findings, allied to the isolation of A. pyogenes from field cases of ovine orchitis, provide clear evidence that A. pyogenes is pathogenic for the ovine genitalia; however, the mechanisms of transition of the organism from commensal to pathogenic state are not clear. It is also noteworthy that some degree of fertility was restored in the late stages of the disorder. Ultrasonography appeared to be useful for the diagnosis of intra-scrotal abnormalities, especially during investigation of the long-standing stage of the disease, after clinical findings have subsided.     

Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm

FGF4, a member of the fibroblast growth factor (FGF) family, is absolutely required for periimplantation mouse development, although its precise role at this stage remains unknown. The nature of the defect leading to postimplantation lethality of embryos lacking zygotic FGF4 is unclear and little is known about downstream targets of FGF4-initiated signaling within the various cellular compartments of the blastocyst. Here we report that postimplantation lethality of Fgf4(-/-) embryos is unlikely to reflect strictly mitogenic requirements for FGF4. Rather, our results suggest that FGF4 is required to maintain trophectoderm and primitive endoderm identity at embryonic day 4.5. This result is consistent with the reported in vitro activity of FGF4 in maintaining trophoblast stem cells and with the requirement for receptor tyrosine kinase signaling in primitive endoderm formation. Thus, postimplantation lethality of Fgf4(-/-) embryos likely results from the failure of proper differentiation and function of extraembryonic cell types.     

Protein-protein cross-linking in the use of the eukaryotic eGST-fusion system

We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins. Protein complexes are involved in a large number of regulatory mechanisms. Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes. For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells. After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein. These bands appeared specifically in the GST-Alien transfected cell preparations. Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself. We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes. The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer. Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners. Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.