Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal.Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

Background

Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297).

Methodology/Principal Findings

We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway.

Conclusions

Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.     

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.     

Frequency and Pathophysiology of Acute Liver Failure in Ornithine Transcarbamylase Deficiency (OTCD)

BackgroundAcute liver failure (ALF) has been reported in ornithine transcarbamylase deficiency (OTCD) and other urea cycle disorders (UCD). The frequency of ALF in OTCD is not well-defined and the pathogenesis is not known.AimTo evaluate the prevalence of ALF in OTCD, we analyzed the Swiss patient cohort. Laboratory data from 37 individuals, 27 females and 10 males, diagnosed between 12/1991 and 03/2015, were reviewed for evidence of ALF. In parallel, we performed cell culture studies using human primary hepatocytes from a single patient treated with ammonium chloride in order to investigate the inhibitory potential of ammonia on hepatic protein synthesis.ResultsMore than 50% of Swiss patients with OTCD had liver involvement with ALF at least once in the course of disease. Elevated levels of ammonia often correlated with (laboratory) coagulopathy as reflected by increased values for international normalized ratio (INR) and low levels of hepatic coagulation factors which did not respond to vitamin K. In contrast, liver transaminases remained normal in several cases despite massive hyperammonemia and liver involvement as assessed by pathological INR values. In our in vitro studies, treatment of human primary hepatocytes with ammonium chloride for 48 hours resulted in a reduction of albumin synthesis and secretion by approximately 40%.ConclusionIn conclusion, ALF is a common complication of OTCD, which may not always lead to severe symptoms and may therefore be underdiagnosed. Cell culture experiments suggest an ammonia-induced inhibition of hepatic protein synthesis, thus providing a possible pathophysiological explanation for hyperammonemia-associated ALF.     

From septicemia to sepsis 3.0 – from Ignaz Semmelweis to Louis Pasteur

Sepsis remains a contemporary threat, and its frequency remains high amongst an aging population. Its definition has been regularly revisited, but the impact of the translational research studying it remains very modest compared to the results seen after the introduction of hygiene and the use of antibiotics. In the past, the main forms of sepsis were hospital gangrene (also known as nosocomial fever or putrid fever) that affected the wounded, and puerperal fever that affected women shortly after delivery. In 1858, Armand Trousseau stated that these two pathologies were identical. Lucrezia Borgia, who died in 1519, is undoubtedly the most famous woman to die from puerperal fever. The notion of sepsis as a real epidemic was deplored. For decades doctors remained deaf to the recommendations of their clairvoyant colleagues who advocated for the use of hygienic measures. It was as early as 1795 that Alexander Gordon (UK) and later in 1843, Oliver Holmes (USA), called for the use of hygienic practices. In 1847, Ignaz Semmelweis, a Hungarian physician, provided an irrefutable demonstration of the importance of hygiene in the prevention of contamination by the hands of the practitioners. But Ignaz Semmelweis' life was a tragedy, his fight against the medical nomenklatura was a tragedy, and his death was a tragedy! Nowadays, Ignaz Semmelweis is receiving the honor that he deserves, but never received during his life. Carl Mayrhofer, Victor Feltz, and Léon Coze were the first to associate the presence of bacteria with sepsis. These observations were confirmed by Louis Pasteur who, thanks to his prestige, had a great influence on how to undertake measures to prevent infections. He inspired Joseph Lister who reduced mortality associated with surgery, particularly amputation, by utilizing antiseptic methods.     

Contribution of Flooded Soils to Sediment and Nutrient Fluxes in a Hydropower Reservoir (Sarrans,Central France)

Ecosystems - When a water reservoir is created, the pre-existing soils and vegetation are flooded. Here, we took advantage of the complete emptying of the Sarrans Reservoir, which was flooded...     

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.     

An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.