An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α- and β-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca²⁺ and Mn²⁺, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.     

Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [³⁵S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.     

Real-Time State Estimation in a Flight Simulator Using fNIRS

Working memory is a key executive function for flying an aircraft. This function is particularly critical when pilots have to recall series of air traffic control instructions. However, working memory limitations may jeopardize flight safety. Since the functional near-infrared spectroscopy (fNIRS) method seems promising for assessing working memory load, our objective is to implement an on-line fNIRS-based inference system that integrates two complementary estimators. The first estimator is a real-time state estimation MACD-based algorithm dedicated to identifying the pilot’s instantaneous mental state (not-on-task vs. on-task). It does not require a calibration process to perform its estimation. The second estimator is an on-line SVM-based classifier that is able to discriminate task difficulty (low working memory load vs. high working memory load). These two estimators were tested with 19 pilots who were placed in a realistic flight simulator and were asked to recall air traffic control instructions. We found that the estimated pilot’s mental state matched significantly better than chance with the pilot’s real state (62% global accuracy, 58% specificity, and 72% sensitivity). The second estimator, dedicated to assessing single trial working memory loads, led to 80% classification accuracy, 72% specificity, and 89% sensitivity. These two estimators establish reusable blocks for further fNIRS-based passive brain computer interface development.     

Effects of the Sequence of Isocaloric Meals with Different Protein Contents on Plasma Biochemical Indexes in Pigs

Nutrient composition and pattern of food intake may play a significant role in weight gain. The aim of this study was to document the effects of a daily 3-meal pattern with isocaloric diets containing different dietary protein contents on growth performance and different plasma biochemical indexes including amino acid plasma concentration in castrated male pigs. Then, 21 DLY (Duroc×Landrace×Yorkshire) pigs aged 60 days were assigned randomly into 3 groups: a control group (crude protein, CP 18.1%), a group receiving high then basal and then low CP meals (High-Low group) and a group receiving low then basal and then high CP meal (Low-High group) for 40 days with pigs being feed-restricted. On day 40, after 12 h fasting, blood samples were obtained for analysis. The results showed that the insulin/glucagon ratio was lower in the High-Low group (P<0.05) when compared with the control group. Compared with the control group, the average daily gain of pigs from the High-Low group increased by 14.10% (P = 0.046). Compared with the control group, serum gamma-glutamyl transferase (GGT) decreased significantly (P<0.05) in both the High-Low and Low-High groups. Plasma concentrations of branched-chain amino acids (BCAA: valine, isoleucine and leucine) increased in the Low-High group (P<0.05) when compared with the control group; and plasma methionine and serine decreased in both the two experimental groups (P<0.05). Compared with the High-Low group, all the BCAA increased significantly (P<0.05) in the Low-High group. These findings suggest that the sequence and quantity of alimentary protein intake affect the insulin/glucagon ratio, as well as amino acid concentrations including BCAA, methionine and serine. It is proposed that meal pattern with pigs receiving high then basal and then low CP meals daily may help to improve the weight gain of pigs.     

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01196871","term_id":"NCT01196871"}}NCT01196871     

Phenotypic and Functional Characterization of Monoclonal Antibodies with Specificity for Rhesus Macaque CD200, CD200R and Mincle

Lectin-like molecules and their receptors are cell surface molecules that have been shown to play a role in either facilitating infection or serving as transporters of HIV/SIV in vivo. The role of these lectin-like molecules in the pathogenesis of HIV/SIV infection continues to be defined. In efforts to gain further insight on the potential role of these lectin-like molecules, our laboratory generated monoclonal antibodies (mAb) against the human analogs of rhesus macaque CD200, CD200R and Mincle, since the rhesus macaques are accepted as the most reliable animal model to study human HIV infection. The characterization of the cell lineages from the blood and various tissues of rhesus macaques that express these lectin-like molecules are described herein. Among the mononuclear cells, the cells of the myeloid lineage of rhesus macaques are the predominant cell lineages that express readily detectable levels of CD200, CD200R and Mincle that is similar to the expression of Siglec-1 and Siglec-3 reported by our laboratory earlier. Subset analysis revealed that a higher frequency of the CD14⁺/CD16^- subset from normal rhesus macaques express CD200, CD200R and Mincle. Differences in the frequencies and density of expression of these molecules by the gated population of CD14⁺ cells from various tissues are noted with PBMC and bone marrow expressing the highest and the mononuclear cells isolated from the colon and ileum expressing the lowest levels. While a significant frequency of pDCs and mDCs express Siglec-1/Siglec-3, a much lower frequency expresses CD200, CD200R and Mincle in PBMCs from rhesus macaques. The mAb against CD200 and CD200R but not Mincle appear to inhibit the infection of macrophage tropic SIV/SHIV in vitro. We conclude that these mAbs may have potential to be used as adjunctive therapeutic agents to control/inhibit SIV/HIV infection.     

FORGE Canada Consortium: Outcomes of a 2-Year National Rare-Disease Gene-Discovery Project

Inherited monogenic disease has an enormous impact on the well-being of children and their families. Over half of the children living with one of these conditions are without a molecular diagnosis because of the rarity of the disease, the marked clinical heterogeneity, and the reality that there are thousands of rare diseases for which causative mutations have yet to be identified. It is in this context that in 2010 a Canadian consortium was formed to rapidly identify mutations causing a wide spectrum of pediatric-onset rare diseases by using whole-exome sequencing. The FORGE (Finding of Rare Disease Genes) Canada Consortium brought together clinicians and scientists from 21 genetics centers and three science and technology innovation centers from across Canada. From nation-wide requests for proposals, 264 disorders were selected for study from the 371 submitted; disease-causing variants (including in 67 genes not previously associated with human disease; 41 of these have been genetically or functionally validated, and 26 are currently under study) were identified for 146 disorders over a 2-year period. Here, we present our experience with four strategies employed for gene discovery and discuss FORGE’s impact in a number of realms, from clinical diagnostics to the broadening of the phenotypic spectrum of many diseases to the biological insight gained into both disease states and normal human development. Lastly, on the basis of this experience, we discuss the way forward for rare-disease genetic discovery both in Canada and internationally.