Comparative growth,cross stress resistance,transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.     

Measurement of Shoulder Range of Motion in Patients with Adhesive Capsulitis Using a Kinect

Range of motion (ROM) measurements are essential for the evaluation for and diagnosis of adhesive capsulitis of the shoulder (AC). However, taking these measurements using a goniometer is inconvenient and sometimes unreliable. The Kinect (Microsoft, Seattle, WA, USA) is gaining attention as a new motion detecting device that is nonintrusive and easy to implement. This study aimed to apply Kinect to measure shoulder ROM in AC; we evaluated its validity by calculating the agreement of the measurements obtained using Kinect with those obtained using goniometer and assessed its utility for the diagnosis of AC. Both shoulders of 15 healthy volunteers and affected shoulders of 12 patients with AC were included in the study. The passive and active ROM of each were measured with a goniometer for flexion, abduction, and external rotation. Their active shoulder motions for each direction were again captured using Kinect and the ROM values were calculated. The agreement between the two measurements was tested with the intraclass correlation coefficient (ICC). Diagnostic performance using the Kinect ROM was evaluated with Cohen’s kappa value. The cutoff values of the limited ROM were determined in the following ways: the same as passive ROM values, reflecting the mean difference, and based on receiver operating characteristic curves. The ICC for flexion/abduction/external rotation between goniometric passive ROM and the Kinect ROM were 0.906/0.942/0.911, while those between active ROMs and the Kinect ROMs were 0.864/0.932/0.925. Cohen’s kappa values were 0.88, 0.88, and 1.0 with the cutoff values in the order above. Measurements of the shoulder ROM using Kinect show excellent agreement with those taken using a goniometer. These results indicate that the Kinect can be used to measure shoulder ROM and to diagnose AC as an alternative to goniometer.     

Development of a Efficient and Sensitive Dispersive Liquid–Liquid Microextraction Technique for Extraction and Preconcentration of 10 β2-Agonists in Animal Urine

Dispersive liquid–liquid microextraction (DLLME) coupled with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was developed for the extraction and determination of 10 β₂-agonists in animal urine. Some experimental parameters, such as the type and volume of the extraction solvent, the concentration of the dispersant, the salt concentration, the pH value of the sample solution, the extraction time and the speed of centrifugation, were investigated and optimized. Under the optimized conditions, a good enrichment factors (4.8 to 32.3) were obtained for the extraction. The enrichment factor show that the concentration rate of DLLME is significantly higher than other pretreatment methods, and the detection sensitivity has been greatly improved. The calibration curves were linear, the correlation coefficient ranged from 0.9928 to 0.9999 for the concentration range of 0.05 to 50 ngmL^-1 and 0.1 to 50 ngmL^-1, and the relative standard deviations (RSDs, n = 15, intra and inter-day precision) at a concentration of 5 ngmL^-1 were in the range of 1.8 to 14.6%. The limits of detection (LODs) for the 10 β₂-agonists, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.01 to 0.03 ngmL^-1. The proposed method was used to identify β₂-agonists in three types of animal urine (swine, cattle, sheep), and the relative recoveries from each matrix were in the range of 89.2 to 106.8%, 90.0 to 109.8% and 89.2 to 107.2%, respectively.     

Features of replication fork blockage by the Escherichia coli terminus-binding protein.

Blockage of the progress of a DNA replication fork in Escherichia coli can be ascribed to an inhibition of helicase action at the orientation-specific binding of a termination sequence (ter) by the ter-binding protein (Lee, E.H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9104-9108). These observations have been extended to include the PriA helicase, thus confirming that blockage is general for helicases. The site of arrest of synthesis by a replication fork is at the very first nucleotide of the 22-base pair E. coli-terB sequence. Strand displacement by DNA polymerases is also inhibited, but is less profound and is orientation-specific. The ter sequences of plasmids R1-terR and -terL and of plasmids R6K and R100 have been compared with those of E. coli-terA and -terB.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.