Intensity of eccentric exercise, shift of optimum angle, and the magnitude of repeated-bout effect.

This study compared the effect of four different intensities of initial eccentric exercise (ECC1) on optimum angle shift and extent of muscle damage induced by subsequent maximal eccentric exercise. Fifty-two male students were placed into 100%, 80%, 60%, or 40% groups (n = 13 per group), performing 30 eccentric actions of the elbow flexors of 100%, 80%, 60%, or 40% of maximal isometric strength [maximal voluntary contraction (MVC)] for ECC1, followed 2-3 wk later by a similar exercise (ECC2) that used 100% MVC load. MVC at six elbow joint angles, range of motion, upper arm circumference, serum creatine kinase activity, myoglobin concentration, and muscle soreness were measured before and for 5 days following ECC1 and ECC2. A rightward shift of optimum angle following ECC1 was significantly (P < 0.05) greater for the 100% and 80% than for the 60% and 40% groups, and it decreased significantly (P < 0.05) from immediately to 5 days postexercise. By the time ECC2 was performed, only the 100% group kept a significant shift (4 degrees). Changes in most of the criterion measures following ECC1 were significantly greater for the 100% and 80% groups compared with the 60% and 40% groups. Changes in the criterion measures following ECC2 were significantly (P < 0.05) greater for the 40% group compared with other groups. Although the magnitude of repeated bout effect following ECC2 was significantly (P < 0.05) smaller for the 40% and 60% groups, all groups showed significantly (P < 0.05) reduced changes in criterion measures following ECC2 compared with the ECC1 100% bout. We conclude that the repeated-bout effect was not dependent on the shift of optimum angle.     

Studies on a novel doughnut-shaped minitablet for intraocular drug delivery

The objective of this study was to evaluate the effect of 2 independent formulation variables on the drug release from a novel doughnut-shaped minitablet (DSMT) in order to optimize formulations for intraocular drug delivery. Formulations were based on a 3(2) full-factorial design. The 2 independent variables were the concentration of Resomer (% wt/wt) and the type of Resomer grade (RG502, RG503, and RG504), respectively. The evaluated response was the drug release rate constant computed from a referenced marketed product and in vitro drug release data obtained at pH 7.4 in simulated vitreous humor. DSMT devices were prepared containing either of 2 model drugs, ganciclovir or foscarnet, using a Manesty F3 tableting press fitted with a novel central-rod, punch, and die setup. Dissolution data revealed biphasic drug release behavior with 55% to 60% drug released over 120 days. The inherent viscosity of the various Resomer grades and the concentration were significant to achieve optimum release rate constants. Using the resultant statistical relationships with the release rate constant as a response, the optimum formulation predicted for devices formulated with foscarnet was 70% wt/wt of Resomer RG504, while 92% wt/wt of Resomer RG503 was ideal for devices formulated with ganciclovir. The results of this study revealed that the full-factorial design was a suitable tool to predict an optimized formulation for prolonged intraocular drug delivery.     

Proteomic Analysis of Two Non-Bronchoscopic Methods of Sampling the Lungs of Patients with the Acute Respiratory Distress Syndrome (ARDS)

Objective

The collection of lung fluid using a suction catheter (s-Cath) and non-bronchoscopic bronchoalveolar lavage (mini-BAL) are two minimally invasive methods of sampling the distal airspaces in patients with the acute respiratory distress syndrome (ARDS). The objective of this study was to determine the similarity of the lung fluid samples recovered by these methods using proteomic analysis.

Methods

Distal lung fluid samples were collected from seven mechanically ventilated patients with ARDS using both s-Cath and mini-BAL in each patient and compared using two-dimensional difference gel electrophoresis. Protein spots of interest were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Main Results

An average of 2,164 spots was detected in the s-Cath and mini-BAL samples. Of these, 68.4% of the protein spots were similar between the s-Cath and mini-BAL samples, 13.2% were increased in s-Cath compared to mini-BAL, and 18.4% were decreased in s-Cath compared to mini-BAL. For each of the seven subjects, overabundance analysis showed that the actual number of differentially expressed spots in the mini-BAL and s-Cath sample was more than the expected number if the samples were identical. There were nine proteins that were consistently differentially expressed between the mini-BAL and s-Cath samples. Of these nine proteins, five are abundantly found in neutrophils or airway epithelial cells, suggesting that the s-Cath may sample the bronchial airways to a greater extent than mini-BAL.

Conclusion

Proteomic analysis of mini-BAL and s-Cath samples shows for the first time that, although these two methods for sampling the lungs of critically ill patients are generally similar, the s-Cath method oversamples the distal airways compared to the mini-BAL method.     

Gene expression programs of human smooth muscle cells: tissue-specific differentiation and prognostic significance in breast cancers

Smooth muscle is present in a wide variety of anatomical locations, such as blood vessels, various visceral organs, and hair follicles. Contraction of smooth muscle is central to functions as diverse as peristalsis, urination, respiration, and the maintenance of vascular tone. Despite the varied physiological roles of smooth muscle cells (SMCs), we possess only a limited knowledge of the heterogeneity underlying their functional and anatomic specializations. As a step toward understanding the intrinsic differences between SMCs from different anatomical locations, we used DNA microarrays to profile global gene expression patterns in 36 SMC samples from various tissues after propagation under defined conditions in cell culture. Significant variations were found between the cells isolated from blood vessels, bronchi, and visceral organs. Furthermore, pervasive differences were noted within the visceral organ subgroups that appear to reflect the distinct molecular pathways essential for organogenesis as well as those involved in organ-specific contractile and physiological properties. Finally, we sought to understand how this diversity may contribute to SMC-involving pathology. We found that a gene expression signature of the responses of vascular SMCs to serum exposure is associated with a significantly poorer prognosis in human cancers, potentially linking vascular injury response to tumor progression.     

Genetic quality and offspring performance in Chinook salmon: implications for supportive breeding

Each year salmon and other fishes are caught and used for supportive breeding programs that attempt to augment natural populations that are threatened with extinction. These programs typically mate individuals randomly and as such they overlook the importance of genetic quality to offspring fitness and ultimately to ensuring population health. Here, we use Chinook salmon (Oncorhynchus tshawytscha) and a fully crossed quantitative genetic breeding design to partition genetic variance in offspring performance (growth and survival) to additive and non-additive genetic effects as well as maternal effects. We show that these three effects contribute about equally to the variation in survival, but only non-additive genetic and maternal effects contribute to variation in growth. Some of the genetic effects could be assigned to variation at the class IIB locus of the major histocompatibility complex, but the maternal effects were not associated with egg size and we found no relationship between dam phenotypic measures and offspring survival or growth. We also found no relationship between sire sexually selected characters and offspring survival or growth, which is inconsistent with a “good genes” hypothesis. Finally, we show that incorporation of genetic quality into supportive breeding programs can increase offspring growth or survival by between 3% and 19% during the endogenous feeding stage alone, and projections to adulthood suggest that survivorship could be over four fold higher. Electronic Supplementary Material  Supplementary material is available in the online version of this article at and is accessible for authorised users.     

Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography-tandem mass spectrometry

A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography-tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50-5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from -5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were < or =10.1%.     

The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals.     

Structural differences within the loop E motif imply alternative mechanisms of viroid processing

Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multibranched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson-Crick GC pairs results in a significant overall stabilization of the CVd-III loop E-like motif. Unlike PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5' side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3' sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5' and 3' termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5' and 3' termini of CVd-III in position for the host ligase.