Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Risk factors and outcomes of candidemia caused by biofilm-forming isolates in a tertiary care hospital

Background

Very few data exist on risk factors for developing biofilm-forming Candida bloodstream infection (CBSI) or on variables associated with the outcome of patients treated for this infection.

Methods and Findings

We identified 207 patients with CBSI, from whom 84 biofilm-forming and 123 non biofilm-forming Candida isolates were recovered. A case-case-control study to identify risk factors and a cohort study to analyze outcomes were conducted. In addition, two sub-groups of case patients were analyzed after matching for age, sex, APACHE III score, and receipt of adequate antifungal therapy. Independent predictors of biofilm-forming CBSI were presence of central venous catheter (odds ratio [OR], 6.44; 95% confidence interval [95% CI], 3.21–12.92) or urinary catheter (OR, 2.40; 95% CI, 1.18–4.91), use of total parenteral nutrition (OR, 5.21; 95% CI, 2.59–10.48), and diabetes mellitus (OR, 4.47; 95% CI, 2.03–9.83). Hospital mortality, post-CBSI hospital length of stay (LOS) (calculated only among survivors), and costs of antifungal therapy were significantly greater among patients infected by biofilm-forming isolates than those infected by non-biofilm-forming isolates. Among biofilm-forming CBSI patients receiving adequate antifungal therapy, those treated with highly active anti-biofilm (HAAB) agents (e.g., caspofungin) had significantly shorter post-CBSI hospital LOS than those treated with non-HAAB antifungal agents (e.g., fluconazole); this difference was confirmed when this analysis was conducted only among survivors. After matching, all the outcomes were still favorable for patients with non-biofilm-forming CBSI. Furthermore, the biofilm-forming CBSI was significantly associated with a matched excess risk for hospital death of 1.77 compared to non-biofilm-forming CBSI.

Conclusions

Our data show that biofilm growth by Candida has an adverse impact on clinical and economic outcomes of CBSI. Of note, better outcomes were seen for those CBSI patients who received HAAB antifungal therapy.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

SIRT1 regulates macrophage self‐renewal

Mature differentiated macrophages can self‐maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self‐renewal ability in vitro and in vivo. Overexpression of SIRT1 during bone marrow‐derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self‐renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine‐induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self‐renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self‐renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self‐renewal might be a relevant parameter of ageing.     

Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression

Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.     

Plasma N-glycome composition associates with chronic low back pain

Background

Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex

Olfactory receptors (Ors) convert chemical signals--the binding of odors and pheromones--to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K(+) selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.