The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response.

Streptococcus agalactiae is a leading cause of infections in neonates. This opportunistic pathogen colonizes the vagina, where it has to cope with acidic pH and hydrogen peroxide produced by lactobacilli. Thus, in the host, this bacterium possesses numerous adaptation mechanisms in which the pleiotropic regulators play a major role. The transcriptional regulator CcpA (catabolite control protein A) has previously been shown to be the major regulator involved in carbon catabolite repression in Gram-positive bacteria but is also involved in other functions. By transcriptomic analysis, we characterized the CcpA-dependent gene regulation in S. agalactiae. Approximately 13.5% of the genome of S. agalactiae depends on CcpA for regulation and comprises genes involved in sugar uptake and fermentation, confirming the role of CcpA in carbon metabolism. We confirmed by electrophoretic mobility shift assays (EMSAs) that the DNA binding site called cis-acting catabolite responsive element (cre) determined for other streptococci was effective in S. agalactiae. We also showed that CcpA is of capital importance for survival under acidic and oxidative stresses and is implicated in macrophage survival by regulating several genes putatively or already described as involved in stress response. Among them, we focused our study on SAK_1689, which codes a putative UspA protein. We demonstrated that SAK_1689, highly downregulated by CcpA, is overexpressed under oxidative stress conditions, this overexpression being harmful for the bacterium in a ΔccpA mutant. IMPORTANCE Streptococcus agalactiae is a major cause of disease burden leading to morbidity and mortality in neonates worldwide. Deciphering its adaptation mechanisms is essential to understand how this bacterium manages to colonize its host. Here, we determined the regulon of the pleiotropic regulator CcpA in S. agalactiae. Our findings reveal that CcpA is not only involved in carbon catabolite repression, but is also important for acidic and oxidative stress resistance and survival in macrophages.

[Organizational Impacts and Clinical Challenges of the COVID-19 Pandemic on a Swiss Tertiary Internal Medicine Department]. / Impacts organisationnels et défis cliniques de la pandémie COVID-19 pour un service hospitalier de médecine interne universitaire.

The rapid progression of COVID-19 is an organizational challenge for all hospitals. To secure the patient overflow, the Department internal medicine of the University Hospital of Lausanne increased nurse and medical workforces as well as bed capacity by 65 %, with extraordinary help from other departments. The implemented crisis management stood upon three pillars : a crisis management team, steering documents and internal communication. In this new form, the Department had already taken care of 442 COVID-19 admissions by April 16, 2020. Alongside organizational challenges, clinical issues such as rapid respiratory distress, clinical suspicions with negative PCR and treatment uncertainties in the absence of sufficient evidence were overcome. Despite the peak of the pandemic appearing to have passed, the next phase could be just as complicated.

La progression rapide du COVID-19 constitue un défi organisationnel pour tous les hôpitaux. Pour anticiper un afflux important de patients, le service de médecine interne du CHUV a ainsi augmenté ses forces de travail médico-soignantes et son nombre de lits de 65 % avec un soutien extraordinaire de toute l'institution. Pour opérer ces changements majeurs, l'organisation de crise mise en place s'est appuyée sur trois piliers : une cellule de conduite, des documents de pilotage et une communication interne. Sous cette nouvelle forme, le service a pris en charge 442 hospitalisations COVID-19 jusqu'au 16 avril 2020. Si les enjeux organisationnels ont été majeurs, la gestion des situations complexes, comme les manifestations respiratoires et les multiples incertitudes cliniques diagnostiques et thérapeutiques, ont été également une gageure. Le pic de la pandémie semble passé, mais la prochaine phase pourrait constituer un nouveau défi organisationnel.

Predicting the microbial cause of community-acquired pneumonia: can physicians or a data-driven method differentiate viral from bacterial pneumonia at patient presentation?

BACKGROUND: Community-acquired pneumonia (CAP) requires urgent and specific antimicrobial therapy. However, the causal pathogen is typically unknown at the point when anti-infective therapeutics must be initiated. Physicians synthesize information from diverse data streams to make appropriate decisions. Artificial intelligence (AI) excels at finding complex relationships in large volumes of data. We aimed to evaluate the abilities of experienced physicians and AI to answer this question at patient admission: is it a viral or a bacterial pneumonia? METHODS: We included patients hospitalized for CAP and recorded all data available in the first 3-h period of care (clinical, biological and radiological information). For this proof-of-concept investigation, we decided to study only CAP caused by a singular and identified pathogen. We built a machine learning model prediction using all collected data. Finally, an independent validation set of samples was used to test the pathogen prediction performance of: (i) a panel of three experts and (ii) the AI algorithm. Both were blinded regarding the final microbial diagnosis. Positive likelihood ratio (LR) values > 10 and negative LR values < 0.1 were considered clinically relevant. RESULTS: We included 153 patients with CAP (70.6% men; 62 [51-73] years old; mean SAPSII, 37 [27-47]), 37% had viral pneumonia, 24% had bacterial pneumonia, 20% had a co-infection and 19% had no identified respiratory pathogen. We performed the analysis on 93 patients as co-pathogen and no-pathogen cases were excluded. The discriminant abilities of the AI approach were low to moderate (LR+ = 2.12 for viral and 6.29 for bacterial pneumonia), and the discriminant abilities of the experts were very low to low (LR+ = 3.81 for viral and 1.89 for bacterial pneumonia). CONCLUSION: Neither experts nor an AI algorithm can predict the microbial etiology of CAP within the first hours of hospitalization when there is an urgent need to define the anti-infective therapeutic strategy.

Epitaxial growth of TbFe2 on piezoelectric LiNbO3 Z-cut.

The strongly magnetostrictive TbFe2 compound has been epitaxially grown on Z-cut Lithium Niobate (LiNbO3) substrates after the deposition of various buffer layers (Mo, Ti and Ti/Mo). Detailed and combined RHEED and x-ray analysis permitted to unravel the in-plane and relative orientation relationships (OR) of the different materials in the system. Despite the use of different templates with different structural orders, similar final OR are eventually found between the piezoelectric substrate and the magnetic layer. The structural and magnetic properties are analyzed in order to get a TbFe2 layer of optimum quality to build a magnetostrictive/piezoelectric hybrid system with efficient strain mediated coupling. Such systems are of interest for the development of magnetic sensors as well as for the electric control of magnetization.

Epitaxial growth of magnetostrictive TbFe2 films on piezoelectric LiNbO3.

The TbFe2 compound has been deposited by molecular beam epitaxy on lithium niobate (LN) substrates with different orientations (LN Z-, 128 Y- and 41 Y-cuts). Despite the challenging growth on these unconventional substrates, crystalline TbFe2 films (as a single orientated domain or with a limited number of orientations) of reasonable structural quality could be obtained after the deposition of a Mo buffer layer. Detailed and combined RHEED and x-ray analysis permitted to unravel the complex Mo and TbFe2 crystal orientations and to reveal common 3D orientation relationships between the different lattices, whatever the initial LN cut. The magnetic properties and especially the magnetic anisotropy have been investigated in taking magnetocrystalline, magnetoelastic and magnetostatic contributions into account. These promising results on the epitaxial growth of hybrid piezoelectric/magnetostrictive crystalline system constitute an important step towards the control of magnetization via electrically generated static and/or dynamic strains, and towards the development of magnetic sensors based on surface acoustic wave devices.

Diagnostic performance of multiplex PCR on pulmonary samples versus nasopharyngeal aspirates in community-acquired severe lower respiratory tract infections.

BACKGROUND: PCR-based techniques for the diagnosis of community- acquired severe lower respiratory tract infections are becoming the standard of care. However, their relative ability to identify either atypical bacteria or viruses that cause LRTI from clinical samples from various sources is yet to be determined. OBJECTIVES AND STUDY DESIGN: The aim of our study was to compare the diagnostic yield of nasopharyngeal aspirates with that of pulmonary samples for the etiological diagnosis of severe acute lower respiratory tract infections by multiplex PCR. Patients were adults with community-acquired pneumonia or acute exacerbation of chronic obstructive pulmonary disease. RESULTS: We obtained concordant results for 81 (79%) of the 103 pairs of samples. In 14 of the 22 discordant results, more pathogens were evidenced in the lower respiratory tract samples. CONCLUSIONS: Pulmonary samples had a similar diagnostic sensitivity for virus detection by multiplex PCR as nasopharyngeal aspirates. In contrast, in our study, the diagnostic efficacy of pulmonary samples for Legionella pneumophila over simple aspirates was clearly superior.

IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 1. Discriminative recognition of mixed micelles versus liposomes.

Guinea pig pancreatic lipase-related protein 2 (GPLRP2) is an interesting model enzyme that can hydrolyze a large set of acylglycerols in vitro but displays however some selectivity depending on the supramolecular structure of substrate and the presence of surfactants like bile salts. We showed that GPLRP2 hydrolyzes 1,2-dipalmitoyl phosphatidylcholine (DPPC) present in mixed micelles with sodium taurodeoxycholate (NaTDC) but not in multilamellar (MLV) and large unilamellar (LUV) vesicles of DPPC. After characterization of these lipid aggregates by dynamic light scattering (DLS), the discriminative recognition of DPPC in DPPC/NaTDC micelles versus MLV and LUV by an inactive variant (S152G) of GPLRP2 to avoid the effect of substrate hydrolysis was investigated using Fourier transform infrared spectroscopy (FTIR). IR spectra were recorded after hydrogen/deuterium exchange, at pD 6 and various temperatures to study phase transitions. We analyzed the methylene asymmetric stretching (ν(CH2)as), the carbonyl stretching (ν(CO)) and the composite polar head-group vibration bands, first to characterized differences in DPPC micelles and vesicles, and second to estimate the degree of interaction of GPLRP2 S152G with phospholipid. Our results indicate that a significant interaction between GPLRP2 S152G and DPPC is only observed when NaTDC is added to the system to form micelles and this can be explained by the different organization of DPPC in mixed micelles compared to lamellar vesicles (higher hydration of polar head, higher mobility of alkyl chains) that favors GPLRP2 penetration into the phospholipid layer.

Molecular characterization of Leptospira sp by multilocus variable number tandem repeat analysis (MLVA) from clinical samples: a case report.

Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA) to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative.

In vitro digestion of citric acid esters of mono- and diglycerides (CITREM) and CITREM-containing infant formula/emulsions.

CITREM is an emulsifier used in the food industry and contains citric acid esters of mono- and diglycerides (GCFE). It is generally recognized as safe but no publication on its digestibility under gastrointestinal conditions and impact on fat digestion was available. It was shown here that fatty acids are released from CITREM by gastric lipase, pancreatic lipase, pancreatic-lipase-related protein 2 and carboxyl ester hydrolase. A two-step in vitro digestion model mimicking lipolysis in the stomach and upper small intestine of term and preterm infants was then used to evaluate the digestibility of CITREM alone, CITREM-containing infant formula and fat emulsions, and isolated GCFE fractions. Overall, it was shown that fat digestion is not significantly changed by the presence of CITREM, and fatty acids contained in CITREM compounds are released to a large extent by lipases. Nevertheless, undigestible water-soluble compounds containing glycerol and citric acid units were identified, indicating that the ester bond between citric acid and glycerol is not fully hydrolyzed throughout the proposed digestion.

Effect of environmental conditions on various enzyme activities and triacylglycerol contents in cultures of the freshwater diatom, Asterionella formosa (Bacillariophyceae).

A detailed analysis of triacylglycerols (TAGs) contents, fatty acid patterns and key enzyme activities in the freshwater diatom Asterionella formosa was performed under various conditions, including nitrate, iron and silicon limitation (stress conditions), or bicarbonate and phytohormones supplementation (stimulation conditions). Of all the conditions tested, the addition of bicarbonate produced the greatest increase (5-fold) in TAGs contents compared to the control while the biomass increased. The addition of phytohormones also allowed a significant increase in TAGs of about 3-fold while the biomass increased. Silicon, unlike iron and nitrate limitation, also triggered a significant increase in TAGs contents of 3.5-fold but negatively affected the biomass. Analysis of fatty acid profiles showed that the mono-unsaturated C16:1 fatty acid was the most abundant in A. formosa, followed by C16:0, C14:0 and eicosapentaenoic acid (EPA; C20:5 n-3). EPA levels were found to increase under nitrate and iron limitation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) activities differed with growth conditions. Most enzymes were up-regulated in stimulated cells while in the case of stressed cells, the pattern of activities was more variable. Detailed analysis of all enzyme activities showed that the most important enzyme among those tested was GAPDH which could be a good candidate for genetic engineering of high lipid-producing algae. This study provides a better understanding of key enzymes and biochemical pathways involved in lipid accumulation processes in diatoms.

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans.

Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.

Prenatal exposure to tobacco and risk for schizophrenia: a retrospective epidemiological study.

INTRODUCTION: In animal studies, long-term prenatal nicotinic exposure alters the development of dopaminergic neurons. To determine whether prenatal smoking exposure was associated with schizophrenia, using a retrospective design study, we compared the prevalence of tobacco use during pregnancy in mothers of subjects with and without schizophrenia. METHODS: One hundred patients with schizophrenia, 100 nonschizophrenic-matched subjects, and their respective mothers were interviewed. The prevalence of smoking was measured in these individuals as well as in their respective mothers during the pregnancy. RESULTS: Patients with schizophrenia smoked more often compared with controls (73% vs 57%). In contrast, the prevalence of smoking during pregnancy did not differ between the groups of mothers. Indeed, the amount of tobacco used was significantly lower in mothers of patients with schizophrenia vs mothers of nonpsychotic subjects. CONCLUSION: This study did not show any association between prenatal tobacco exposure and further development of schizophrenia.

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.