Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Aortic input impedance during Mueller maneuver: an evaluation of "effective length"

Aortic input impedance was calculated in seven subjects in the control state (normal reflection) and during the Mueller maneuver (increased reflection) to evaluate "effective arterial length" under altered physiological conditions. Regional foot-to-foot pulse wave velocities and pressure waveforms along the aorta were used to define an "apparent anatomic length" or distance to a dominant discrete site of reflection "seen" by the ejecting ventricle. Time of wave travel was taken to be one-half the interval from the foot of the incident wave to the midsystolic inflection point. Knowing the time of travel from the returning reflection and velocity, distances calculated to the "apparent anatomic length" were 35 +/- 2 and 34 +/- 2 during control and Mueller maneuver, respectively (P = NS). The frequency of the first minimum of the modulus (fmin) and the first zero crossing of the phase angle (f phi) were determined from the input impedance spectra. During baseline conditions, fmin (3.9 +/- 0.2 Hz) approximately equaled f phi (4.2 +/- 0.2 Hz), and the resulting "effective lengths" calculated using the quarter-wavelength formula were similar to the apparent anatomic length. These data suggested that the aortic region incorporating the renal arterial branches as a site of discrete reflection and that terminal load was not significantly frequency dependent. During Mueller maneuver, however, f min (3.3 +/- 0.2 Hz) and f phi (5.1 +/- 0.2 Hz) were significantly discordant, the terminal load became strongly frequency dependent, and effective length calculated from f min was dissimilar (P less than 0.05) from the unchanged apparent anatomic length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of gene expression during preimplantation development between diploid and haploid mouse embryos

Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.