Real-time monitoring of cell viability and cell density on the basis of a three dimensional optical reflectance method (3D-ORM): investigation of the effect of sub-lethal and lethal injuries

Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed and a scale-down bioreactor configured in order to reproduce mixing deficiencies during a fed-batch culture of Escherichia coli. These differences have been observed both for the obscuration factor (OBF) and the coincidence probability delivered by the probe. These parameters are correlated to flow cytometry measurement based on the PI-uptake test and cell density based on optical density measurement. This first set of results has pointed out the fact that the 3D-ORM probe is sensitive to sub-lethal injuries encountered by microbial cells in process-related conditions. The effect of lethal injuries has been further investigated on the basis of additional experiments involving heat stress and a sharp increase of the OBF has been observed indicating that cells are effectively injured by the increase of temperature. However, further improvement of the probe are needed in order to give access to single-cell measurements.     

Association mapping and meta-analysis: two complementary approaches for the detection of reliable Septoria tritici blotch quantitative resistance in bread wheat (Triticum aestivum L.)

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most ubiquitous and important diseases of bread wheat worldwide. The aim of this study was to identify markers linked to loci conferring resistance to STB from seven biparental populations. Linkage analysis, meta-analysis and association mapping were combined to identify robust quantitative trait loci (QTLs) for resistance. Linkage analysis led to the detection of 115 QTLs for resistance to STB and 66 QTLs linked to plant height and/or earliness. Meta-analysis clustered these 115 QTLs into 27 Meta-QTLs (MQTLs) of pathogen resistance, of which 14 were found to be linked to plant height and/or earliness. Both the relationship between dwarfing and susceptibility to STB and the significant negative correlation between earliness and STB symptoms were confirmed. Eleven loci were linked to STB resistance by association mapping using a general linear model and/or a mixed linear model, of which eight co-located with STB MQTLs and two co-located with individual QTLs. Associated markers located in MQTL regions enhanced the relevance of the results and validated the potential of an association mapping approach. With several biparental populations, meta-analysis is the most relevant form of genetic analysis study, but association mapping can be used as a validation method. Regions linked to resistance in both methods should be relevant for use in breeding programs for improving resistance to STB in wheat varieties. The main interest in comparing both approaches is to detect robust loci that will be functional in many genetic backgrounds rather than just in one or a few specific backgrounds.     

Next-generation sequencing for identifying new genes in rare genetic diseases: many challenges and a pinch of luck

A report on the European Society of Human Genetics conference, held in Paris, France, June 8-11, 2013.     

Stability-Based Comparison of Class Discovery Methods for DNA Copy Number Profiles

Motivation

Array-CGH can be used to determine DNA copy number, imbalances in which are a fundamental factor in the genesis and progression of tumors. The discovery of classes with similar patterns of array-CGH profiles therefore adds to our understanding of cancer and the treatment of patients. Various input data representations for array-CGH, dissimilarity measures between tumor samples and clustering algorithms may be used for this purpose. The choice between procedures is often difficult. An evaluation procedure is therefore required to select the best class discovery method (combination of one input data representation, one dissimilarity measure and one clustering algorithm) for array-CGH. Robustness of the resulting classes is a common requirement, but no stability-based comparison of class discovery methods for array-CGH profiles has ever been reported.

Results

We applied several class discovery methods and evaluated the stability of their solutions, with a modified version of Bertoni''s -based test [1]. Our version relaxes the assumption of independency required by original Bertoni''s -based test. We conclude that Minimal Regions of alteration (a concept introduced by [2]) for input data representation, sim [3] or agree [4] for dissimilarity measure and the use of average group distance in the clustering algorithm produce the most robust classes of array-CGH profiles.

Availability

The software is available from http://bioinfo.curie.fr/projects/cgh-clustering. It has also been partly integrated into "Visualization and analysis of array-CGH"(VAMP)[5]. The data sets used are publicly available from ACTuDB [6].     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.     

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Atypical Human Infections by Animal Trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Key Learning Points

• The classical human trypanosomoses are human African trypanosomosis (HAT) or sleeping sickness, and Chagas disease, the Latin American human trypanosomosis.
• Atypical human infections caused by Trypanosoma species that normally are restricted to animals have been reported. These cases of atypical human trypanosomoses (a-HT) are mostly transient, but some require treatment and can be fatal.
• Only a few cases of a-HT have been fully confirmed, especially in Asia, leading to the hypothesis that the actual prevalence is probably underestimated.
• The detection of a case of a-HT should be based on observation of the parasite by direct microscopy. Evaluating/improving the diagnoses through serological and PCR assays would help in detecting and identifying atypical trypanosomosis infections in humans. These laboratory research and field activities are needed to evaluate the actual occurrence of atypical cases.

Top Five Papers

1. Verma A, Manchanda S, Kumar N, Sharma A, Goel M, et al. (2011) Trypanosoma lewisi or Trypanosoma lewisi-like infection in a 37-day-old infant. Am J Trop Med Hyg 85: 221–224.
2. Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, et al. (2008) Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 61: 428–433.
3. Vanhollebeke B, Truc P, Poelvoorde P, Pays A, Joshi PP, et al. (2006) Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I. N Engl J Med 355: 2752–2756.
4. Joshi PP, Shegokar V, Powar S, Herder S, Katti R, et al. (2005) Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. Am J Trop Med Hyg 73: 491–495.
5. Howie S, Guy M, Fleming L, Bailey W, Noyes H, et al. (2006) A Gambian infant with fever and an unexpected blood film. PLoS Med 3: e355. doi:10.1371/journal.pmed.0030355.

     

IL-17A Mediates Early Post-Transplant Lesions after Heterotopic Trachea Allotransplantation in Mice

Primary graft dysfunction (PGD) and bronchiolitis obliterans (BO) are the leading causes of morbidity and mortality after lung transplantation. Reports from clinical and rodent models suggest the implication of IL-17A in either PGD or BO. We took advantage of the heterotopic trachea transplantation model in mice to study the direct role of IL-17A in post-transplant airway lesions. Across full MHC barrier, early lesions were controlled in IL-17A^-/- or anti-IL17 treated recipients. In contrast, IL-17A deficiency did not prevent subsequent obliterative airway disease (OAD). Interestingly, this early protection occurred also in syngeneic grafts and was accompanied by a decrease in cellular stress, as attested by lower HSP70 mRNA levels, suggesting the involvement of IL-17A in ischemia-reperfusion injury (IRI). Furthermore, persistence of multipotent CK14⁺ epithelial stem cells underlined allograft protection afforded by IL-17A deficiency or neutralisation. Recipient-derived γδ⁺ and CD4⁺ T cells were the major source of IL-17A. However, lesions still occurred in the absence of each subset, suggesting a high redundancy between the innate and adaptive IL-17A producing cells. Notably, a double depletion significantly diminished lesions. In conclusion, this work implicated IL-17A as mediator of early post-transplant airway lesions and could be considered as a potential therapeutic target in clinical transplantation.     

Male Gender,Increased Blood Viscosity,Body Mass Index and Triglyceride Levels Are Independently Associated with Systemic Relative Hypertension in Sickle Cell Anemia

Patients with sickle cell anemia (SCA) have usually lower diastolic, systolic and mean blood pressure (BP) than the general population. However, BP values ≥120/70 mmHg considerably increase the risk for acute and chronic complications in SCA. The aim of this study was to identify biological factors associated with relative hypertension in adults with SCA. We compared the hematological, lipid and hemolytic profiles, as well as blood viscosity, between SCA patients with normal BP (<120/70 mmHg, n = 54) and those with relative hypertension (BP≥120/70 mmHg, n = 43). Our results demonstrated that male gender (OR: 3.49; 95%CI 1.20 to 10.16, p<0.05), triglycerides (OR: 9.19; 95% CI 2.29 to 36.95, p<0.01), blood viscosity (OR: 1.35; 95% CI 1.01 to 1.81, p<0.05) and body mass index (OR: 1.37; 95% CI 1.14 to 1.64, p<0.01) were independent risks factors for relative hypertension in SCA. No association was found between the BP status and the positive history of painful vaso-occlusive crisis or acute chest syndrome. An association between triglycerides level and the occurrence of these two major acute complications was detected. Our study suggests that male gender, increased triglycerides level, BMI and blood viscosity could increase the risk for developing relative hypertension in SCA. In addition, our results support a role of moderately elevated triglycerides in the pathophysiology of vaso-occlusive events.