Cellobiose metabolism in Erwinia: genetic study

Summary The study of mutants of Erwinia specifically unable to ferment cellobiose indicates that the mutations are clustered between arg and ile on the chromosome of this organism. In vivo cloning of the genes responsible for cellobiose utilization lead to a plasmid, pBEC2, which complements all Erwinia Clb^- specific mutants. When introduced into wild-type E. coli it allows this organism to use cellobiose, arbutin and salicin; it also complements bglB and bglC mutants of Escherichia coli indicating that arbutin and salicin utilization is due to the products of the pBEC2 cloned genes. From the characterization of mutants pleiotropically affected in the utilization of various carbon sources, including cellobiose, arbutin and salicin, it is proposed that the three--glucosides are substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

Hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme (Short Communication)

The hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme has been investigated by using an automated ninhydrin technique. The results show an inverse relationship of hydrolysis rate with size and charge of the peptide, which parallels the inactivation in the pulmonary circulation and offers an explanation for the selectivity of metabolism of these kinins by the lungs.     

Inputs from upper airway affect firing of respiratory-associated midbrain neurons

Chen, Zibin, and Frederic L. Eldridge. Inputs fromupper airway affect firing of respiratory-associated midbrain neurons. J. Appl. Physiol. 83(1): 196-203, 1997.In 16 decerebrated unanesthetized cats, we studied effects ofneural inputs from upper airway on firing of 62 mesencephalic neuronsthat also developed respiratory-associated (RA) rhythmic firing whenrespiratory drive was high [Z. Chen, F. L. Eldridge, and P.G.Wagner. J. Physiol. (Lond.) 437:305-325, 1991] and on firing of 16 neurons that did notdevelop the rhythmic firing (non-RA neurons). Activity in RA neuronsincreased after mechanical expansion of pharynx (45% of those tested)or larynx (68%) and after stimulation of glossopharyngeal (50%) orsuperior laryngeal nerves (77%). The increased neuronal firingoccurred despite decreases or abolition of respiratory activity(expressed in phrenic nerve). Neuronal firing also increased aftermechanical stimulation of nasal mucosa (66%) or by jetsof air directed into the nares (48%) and after lightbrushing of nasal skin (~40%). Most stimuli led to decreased firingin a smaller number of neurons, and some neurons showed no response.None of the non-RA neurons developed an increase of firing after any ofthe stimuli, although one had decreased firing after stimulation of thesuperior laryngeal nerve. We conclude that inputs from the upper airwayand nasal skin have independent modulatory effects on the samemesencephalic neurons that are stimulated by ascending rhythmic RAinput from the medulla. These findings may have relevance to generationof the sensation of dyspnea.

     

Epicardial adipose tissue extent: relationship with age, body fat distribution, and coronaropathy

Epicardial fat is a relatively neglected component of the heart and could be an important risk factor of cardiac disease. The objective of our study was to assess the relationship between epicardial adipose tissue (EAT) extent, fat distribution, and coronaropathy in a group of adult victims of accidental or suspicious sudden death. In 56 cadavers, we performed 34 measurements of EAT from five computerized photographs of the heart (anterior and posterior faces, and three ventricle transversal slices) and analyzed their relationship with anthropometric markers of adiposity (BMI, waist and leg circumference, thickness of abdominal and thigh subcutaneous adipose tissue (SAT)), with the presence and staging of coronary artery disease (CAD), and with markers of myocardial hypertrophy. Simple linear regressions showed that EAT measurements are highly intercorrelated (r from 0.4 to 0.6, P < 0.001), and correlate with age, waist circumference, and heart weight, and to a lesser extent, with BMI, abdominal SAT thickness, and leg SAT thickness. Multiple regression showed that age, waist circumference, and heart weight significantly and independently correlate with EAT (P < 0.0001). No other anthropometric measurement was found independently correlated with EAT. The EAT/myocardium ratios correlated positively with age and waist circumference. Anterior and posterior areas of EAT were found significantly increased in patients with CAD and correlated positively with CAD staging (P = 0.0034, r = 0.38). Anterior EAT surface was found positively associated with CAD (P = 0.01), independently of age and other adiposity measurements. Prospective studies are needed to assess the risk of occurrence/progression of CAD that relate to EAT excess.     

Reference gene selection for quantitative real-time PCR normalization: Application in the caprine mammary gland

In dairy animals, gene expression analysis has become increasing key to understand the biological processes occurring in mammary gland development that shape future milk potential. Selecting high-stability reference genes is crucial to interpret real-time qPCR data. This study investigated the expression stability of five top-ranked candidate reference genes in the goat mammary gland through three assays comparing different experimental conditions (physiological states, sample types and experimental treatments). The expression stability of genes including β-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA, cyclophilin A and ribosomal protein large P0 was analyzed. Normalization for each experimental condition expression data revealed a different reference gene. Nevertheless, in our various assays, genes encoding for ribosomal proteins, 18S rRNA and RPLP0 presented the best expression stability. This result has been confirmed using a combined analysis of stability on the three assays. All genes showed the same distribution within and among the three assays and a different distribution between Ct variability and GeNorm normalization. In addition, the application on Catenin B1 expression using an inappropriate reference gene confirmed erroneous variations in interpretation. To conclude, there is no single ideal reference gene for caprine mammary gland studies and we recommend using a panel of top-ranked reference genes, including RPLP0, at the beginning of each experiment to validate the most stable(s) gene(s).     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

An accurate definition of the status of inactive hepatitis B virus carrier by a combination of biomarkers (FibroTest-ActiTest) and viral load

Background

The combination of transaminases (ALT), biopsy, HBeAg and viral load have classically defined the inactive status of carriers of chronic hepatitis B. The use of FibroTest (FT) and ActiTest (AT), biomarkers of fibrosis and necroinflammatory activity, has been previously validated as alternatives to biopsy. We compared the 4-year prognostic value of combining FT-AT and viral load for a better definition of the inactive carrier status.

Methods and Findings

1,300 consecutive CHB patients who had been prospectively followed since 2001 were pre-included. The main endpoint was the absence of liver-related complications, transplantation or death. We used the manufacturers'' definitions of normal FT (< = 0.27), normal AT (< = 0.29) and 3 standard classes for viral load. The adjustment factors were age, sex, HBeAg, ethnic origin, alcohol consumption, HIV-Delta-HCV co-infections and treatment.

Results

1,074 patients with baseline FT-AT and viral load were included: 41 years old, 47% African, 27% Asian, 26% Caucasian. At 4 years follow-up, 50 complications occurred (survival without complications 93.4%), 36 deaths occurred (survival 95.0%), including 27 related to HBV (survival 96.1%). The prognostic value of FT was higher than those of viral load or ALT when compared using area under the ROC curves [0.89 (95%CI 0.84–0.93) vs 0.64 (0.55–0.71) vs 0.53 (0.46–0.60) all P<0.001], survival curves and multivariate Cox model [regression coefficient 5.2 (3.5–6.9; P<0.001) vs 0.53 (0.15–0.92; P = 0.007) vs −0.001 (−0.003−0.000;P = 0.052)] respectively. A new definition of inactive carriers was proposed with an algorithm combining “zero” scores for FT-AT (F0 and A0) and viral load classes. This new algorithm provides a 100% negative predictive value for the prediction of liver related complications or death. Among the 275 patients with the classic definition of inactive carrier, 62 (23%) had fibrosis presumed with FT, and 3 died or had complications at 4 year.

Conclusion

In patients with chronic hepatitis B, a combination of FibroTest-ActiTest and viral load testing accurately defined the prognosis and the inactive carrier status.