Spot urine collection: A valid alternative to total urine collection for metabolomic studies in dairy cattle.

Urine is a highly suitable biological matrix for metabolomics studies. Total collection for 24-h periods is the gold standard as it ensures the presence of all metabolites excreted throughout the day. However, in animal studies, it presents limitations related to animal welfare and also due to alterations of the metabolome originating from the use of acid for preventing microbial growth or microbial contamination. In this study, we investigated whether spot urine collection is a practical alternative to total collection for metabolomic studies in lactating cows. For this purpose, we collected urine samples from 4 lactating Holstein cows fed 4 diets in a 4 × 4 Latin square design. Urine was collected for 24 h using a collecting device (i.e., total collection) or collected once per day 4 h after the morning feeding (i.e., spot urine collection). Dietary treatments differed by the amount of nitrogen content (high vs. low) and by the nature of the energy (starch vs. fiber). Urine metabolome was analyzed by 2 untargeted complementary methods, nuclear magnetic resonance and hydrophilic-interaction liquid chromatography (HILIC) coupled to a time-of-flight mass spectrometer, and by 1 targeted method, HILIC-tandem mass spectrometry. Although sampling technique had an effect on the abundance of metabolites detected, spot urine samples were equally capable of showing differences in urine metabolome than samples from total collection. When considering nitrogen levels in the diet, the robustness and precision for discriminating high- and low-nitrogen diets was equally achieved with both sampling techniques. A total of 22 discriminant metabolites associated with the N level of diets were identified from untargeted HILIC coupled to a time-of-flight mass spectrometer (n = 9) and nuclear magnetic resonance (n = 11), and 2 from targeted HILIC-tandem mass spectrometry. Alternatively, starch or fiber in the diet induced less changes in the metabolome that were not clearly discriminated independently of the sampling technique. We concluded that spot urine collection can successfully reveal differences in the urine metabolome elicited by dietary N levels and be used as a substitute of total urinary 24-h collection for metabolomic studies.

Biological control of mites by xerophile Eurotium species isolated from the surface of dry cured ham and dry beef cecina.

Some meat dry products, including dry cured ham and dry beef cecina, are cured in cellars at moderately cold temperature allowing the growth of a lawn of fungi on their surface. During the curing process, frequently these products became contaminated with fungivore mites of the Acaridae family that feed on fungal mycelium and spores. AIMS: The aim of this article is to study the possible biological control of mites by fungi that form part of the normal microbiota of these meat products. METHODS AND RESULTS: Some yellow/orange pigmented fungi growing on the ham surface decreased the proliferation of mites; therefore, we isolated from ham and cecina xerophilic yellow/orange coloured fungal strains that were identified as members of the genus Eurotium (recently reclassified as Aspergillus section Aspergillus). Using molecular genetic tools, we have identified 158 strains as Eurotium rubrum (Aspergillus ruber), Eurotium repens (Aspergillus pseudoglaucus) and Eurotium chevalieri (Aspergillus chevalieri). Two strains, E. rubrum C47 and E. rubrum C49, showed strong miticidal activity. The toxic compound(s) are associated with the formation of cleistothecia. In synchronized mite development experiments, we observed that all stages of the mite lifecycle were inhibited by the E. rubrum C47 strain. In addition, we searched for miticidal activity in 13 culture collection Eurotium strains isolated from different habitats, and found that only one, Eurotium cristatum NRRL 4222 (Aspergillus cristatus) has a strong miticidal activity. CONCLUSIONS: These fungal strains have proliferated on the surface of ham and cecina for decades, and possibly have acquired miticidal activity as a resistance mechanism against fungivores. SIGNIFICANCE AND IMPACT OF THE STUDY: Biological control of infecting mites by favouring growth of E. rubrun C47, in place of the normal mixed population of Aspergillus and Penicillium, is an attractive approach to control mite infestations.

Evidence for a New Compact Symmetric Fission Mode in Light Thorium Isotopes.

Taking benefit of the R3B/SOFIA setup to measure the mass and the nuclear charge of both fission fragments in coincidence with the total prompt-neutron multiplicity, the scission configurations are inferred along the thorium chain, from the asymmetric fission in the heavier isotopes to the symmetric fission in the neutron-deficient thorium. Against all expectations, the symmetric scission in the light thorium isotopes shows a compact configuration, which is in total contrast to what is known in the fission of the heavier thorium isotopes and heavier actinides. This new main symmetric scission mode is characterized by a significant drop in deformation energy of the fission fragments of about 19 MeV, compared to the well-known symmetric scission in the uranium-plutonium region.

Synergy between pyroelectric and photovoltaic effects for optoelectronic nanoparticle manipulation.

The combined action of the pyroelectric (PY) and photovoltaic (PV) effects, exhibited by z-cut LiNbO3:Fe substrates, has been investigated for particle trapping and patterning applications. The novel hybrid procedure provides new possibilities and versatility to optoelectronic manipulation on LiNbO3 substrates. It has allowed obtaining periodic and arbitrary 2D patterns whose particle density distribution is correlated with the light intensity profile but can be tuned through ΔT according to the relative strength of the PV and PY effects. A relevant result is that the PY and PV contributions compete for a ΔT range of 1-20 °C, very accessible for experiments. Moreover, the synergy of the PY and PV has provided two additional remarkable applications: i) A method to measure the PV field, key magnitude for photovoltaic optoelectronic tweezers. Using this method, the minimum field needed to obtain a particle pattern has been determined, resulting relatively high, E~60 kV/cm, and so, requiring highly doped crystals when only using the PV effect. ii) An strategy combining the PY and PV to get particle patterning in samples inactive for PV trapping when the PV field value is under that threshold.

Transcriptional Studies on a Streptomyces clavuligerus oppA2 Deletion Mutant: N-Acetylglycyl-Clavaminic Acid Is an Intermediate of Clavulanic Acid Biosynthesis.

The oppA2 gene encodes an oligopeptide-binding protein similar to the periplasmic substrate-binding proteins of the ABC transport systems. However, oppA2 is an orphan gene, not included in an ABC operon. This gene is located in the clavulanic acid (CA) gene cluster of Streptomyces clavuligerus and is essential for CA production. A transcriptomic study of the oppA2-null mutant S. clavuligerus ΔoppA2::aac showed changes in the expression levels of 233 genes from those in the parental strain. These include genes for ABC transport systems, secreted proteins, peptidases, and proteases. Expression of the clavulanic acid, clavam, and cephamycin C biosynthesis gene clusters was not significantly affected in the oppA2 deletion mutant. The genes for holomycin biosynthesis were upregulated 2-fold on average, and the level of upregulation increased to 43-fold in a double mutant lacking oppA2 and the pSCL4 plasmid. Strains in which oppA2 was mutated secreted into the culture the compound N-acetylglycyl-clavaminic acid (AGCA), a putative intermediate of CA biosynthesis. A culture broth containing AGCA, or AGCA purified by liquid chromatography-mass spectrometry (LC-MS), was added to the cultures of various non-CA-producing mutants. Mutants blocked in the early steps of the pathway restored CA production, whereas mutants altered in late steps did not, establishing that AGCA is a late intermediate of the biosynthetic pathway, which is released from the cells when the oligopeptide-binding protein OppA2 is not available.IMPORTANCE The oppa2 gene encodes an oligopeptide permease essential for the production of clavulanic acid. A transcriptomic analysis of S. clavuligerus ΔoppA2::aac in comparison to the parental strain S. clavuligerus ATCC 27064 is reported. The lack of OppA2 results in different expression of 233 genes, including genes for proteases and genes for transport systems. The expression of the clavulanic acid genes in the oppA2 mutant is not significantly affected, but the genes for holomycin biosynthesis are strongly upregulated, in agreement with the higher holomycin production by this strain. The oppA2-mutant is known to release N-acetylglycyl-clavaminic acid to the broth. Cosynthesis assays using non-clavulanic acid-producing mutants showed that the addition of pure N-acetylglycyl-clavaminic acid to mutants in which clavulanic acid formation was blocked resulted in the recovery of clavulanic acid production, but only in mutants blocked in the early steps of the pathway. This suggests that N-acetylglycyl-clavaminic acid is a previously unknown late intermediate of the clavulanic acid pathway.

Controlled Versus Uncontrolled Resistant Hypertension: Are They in the Same Bag?

PURPOSE OF REVIEW: Resistant hypertension (RHTN) is a condition in which besides the antihypertensive therapy using at least three different drugs (including a diuretics), brachial blood pressure does not reach the target (e.g., 140/90 mmHg). RECENT FINDINGS: Despite the diversity of clinical presentations, we divide RHTN in two major groups according to blood pressure and number of drugs taken: controlled (C-RHTN) and uncontrolled (UC-RHTN) resistant hypertension, with refractory hypertension (RfHTN) included in the latter subgroup. Both C-RHTN and UC-RHTN are heterogenic and complex syndromes. To better approach this matter, the some pathophysiological mechanisms (increased volemia, hyperactivity, plasma cortisol, adipocitokines, and other pro-inflammatory factors), have a pivotal clinical role. Some features (African ethnic, obesity, age > 60, LV hypertrophy, and vascular stiffness) increase the risk of refractoriness as well as worst prognosis. Based on increased target organ damage, cardiovascular risk and events will be addressed in this review. Our conclusion is that although both C-RHTN and UC-RHTN are extreme phenotypes of hard-to-control BP, some mechanisms of the disease and clinical expressions are distinct. According to these differences, "UC-RHTN and C-RHTN are not in the same bag."

Constructed wetlands for water quality improvements: Benefit transfer analysis from Ohio.

Water resources provide many benefits that generate value for residents and recreation users alike but run-off from agricultural and impervious surfaces can impair water quality, reducing any generated value. A possible solution to this problem is the construction of treatment wetlands to remove excessive nutrients from water bodies. This study uses environmental and economic data to approximate the costs of constructing and operating free surface water wetlands to remove phosphorus and estimates the amenity and recreational benefits of the resulting improvements in water quality for 24 lakes in Ohio. A ten percent improvement in water quality from a decrease in phosphorus loadings generates positive net benefits for all lakes in the sample with a lifetime cost benefit ratio of 2.92. The study also examines the potential use of constructed wetlands as the sole strategy to achieve a reduction goal for phosphorus loadings and find that the costs of doing so are prohibitive. Constructed wetlands can be a cost-effective component of a comprehensive strategy for small-scale nutrient reduction and water quality improvements for surface water bodies, but other treatment methods would be required to achieve any proposed targeted improvements.

Enhancing a sedation score to include truly noxious stimulation: the Extended Observer's Assessment of Alertness and Sedation (EOAA/S).

BACKGROUND: Although the Modified Observer's Assessment of Alertness and Sedation (MOAA/S) is frequently used in sedation-related drug and device studies, a major shortcoming is that it does not differentiate between lighter and deeper levels of general anaesthesia because the only noxious stimulus of the MOAA/S is a trapezius squeeze. The primary aim of this investigation was to expand the MOAA/S score to include truly noxious stimulation, thereby extending the dynamic range of the assessment to include sedation states consistent with deeper levels of general anaesthesia. METHODS: Twenty healthy volunteers received target controlled infusions of fentanyl (target=0.8 ng ml(-1)) and propofol (starting at 0.5 µg ml(-1) and gradually increasing to 5 µg ml(-1)). At each propofol concentration, a MOAA/S score was obtained before and after tetanic electrical stimulation. The tetanic electrical stimulation current was gradually increased until the subject responded or until 50 mA was delivered without a response. A pharmacodynamic model was constructed to characterize the concentration-effect relationship between propofol and the MOAA/S scores. RESULTS: All subjects required a significantly higher propofol concentration to produce unresponsiveness to tetanic electrical stimulation at 50 mA compared with a standardized trapezius squeeze. The pharmacodynamic model adequately characterized the concentration-effect relationship. CONCLUSIONS: The Extended Observer's Assessment of Alertness and Sedation (or EOAA/S) extends the range of the widely used MOAA/S score to include truly noxious stimulation, thereby enabling the identification of drug-induced central nervous system depression representative of surgical anaesthesia.

A natural short pathway synthesizes roquefortine C but not meleagrin in three different Penicillium roqueforti strains.

The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.

Immunoglobulins IgG1, IgM and IgA: a synergistic team influencing survival in sepsis.

OBJECTIVE: The impact of endogenous immunoglobulin isotypes on the prognosis of patients with severe sepsis has not been sufficiently explored. The aim of this study was to evaluate the association between immunoglobulin levels in plasma and survival in patients with this condition. DESIGN AND PATIENTS: A prospective multicentre cohort study was conducted. A total of 172 adult patients admitted to the intensive care unit (ICU) with severe sepsis or septic shock were recruited. Patients were classified based on deciles of immunoglobulin concentrations at diagnosis of sepsis. Categorical variables were created and tested for their association with survival during hospitalization in the ICU. RESULTS: Overall, 42 patients died in the ICU during the study. Kaplan-Meier analysis showed that immunoglobulin concentrations below 300 mg dL(-1) for IgG1, 35 mg dL(-1) for IgM and 150 mg dL(-1) for IgA were associated with shorter survival times. Multivariate regression analysis showed that IgG1 < 300 mg dL(-1) was a risk factor for mortality [odds ratio (OR) 2.50, 95% confidence interval (CI) 1.04-6.03; P = 0.042]. The combined presence of IgG1, IgM and IgA levels below the described thresholds had a synergistic impact on mortality risk (OR 5.27, 95% CI 1.41-19.69; P = 0.013). A similar effect was observed for combined low levels of IgG1 and IgA (OR 4.10, 95% CI 1.28-13.12; P = 0.018) and also of IgG1 and IgM (OR 3.10. 95% CI 1.13-8.49; P = 0.028). CONCLUSIONS: The combined presence of low levels of the endogenous immunoglobulins IgG1, IgM and IgA in plasma is associated with reduced survival in patients with severe sepsis or septic shock. Assessment of the concentrations of these immunoglobulins could improve the results of treatment with exogenous immunoglobulins in patients with sepsis.

New insights into the isopenicillin N transport in Penicillium chrysogenum.

In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane.

A 1.8-Mb-reduced Streptomyces clavuligerus genome: relevance for secondary metabolism and differentiation.

A large part (21%) of the wild-type Streptomyces clavuligerus genome is located in a 1.8-Mb megaplasmid that greatly influences secondary metabolites biosynthesis even if the secondary metabolites are chromosomally encoded. The megaplasmid copy number may change depending on the nutritional and environmental conditions. The S. clavuligerus oppA2::aph mutant described by Lorenzana et al. (2004) does not form aerial mycelium, spores, and clavulanic acid, but overproduces holomycin. Transcriptomic studies, polymerase chain reactions (PCR), qPCR, and RT-qPCR analysis showed that S. clavuligerus oppA2::aph has a drastically reduced number of copies (about 25,000-fold lower than the parental strain) of plasmids pSCL1 (10.5 kb), pSCL2 (149.4 kb), and the megaplasmid pSCL4 (1.8 Mb). To clarify the role of the linear plasmids and the function of OppA2 in S. clavuligerus oppA2::aph we constructed oppA2 mutants which contained: (1) a normal copy number of the linear plasmids, (2) completely lack of the linear plasmids, and (3) a parA-parB pSCL4 mutant that resulted in lack of pSCL4. In addition, a strain with a functional oppA2 gene was constructed lacking the megaplasmid pSCL4. The results confirmed that the oppA2 gene is essential for clavulanic acid production, independently of the presence or absence of linear plasmids, but oppA2 has little relevance on differentiation. We demonstrated that the lack of sporulation of S. clavuligerus oppA2::aph is due to the absence of linear plasmids (particularly pSCL4) and the holomycin overproduction is largely due to the lack of pSCL4 and is stimulated by the oppA2 mutation.

Expression of the endogenous and heterologous clavulanic acid cluster in Streptomyces flavogriseus: why a silent cluster is sleeping.

Clusters for clavulanic acid (CA) biosynthesis are present in the actinomycetes Streptomyces flavogriseus ATCC 33331 and Saccharomonospora viridis DSM 43017. These clusters, which are silent, contain blocks of conserved genes in the same order as those of the Streptomyces clavuligerus CA cluster but assembled in a different organization. S. flavogriseus was grown in nine different media, but clavulanic acid production was undetectable using bioassays or by high-performance liquid chromatography analyses. Reverse-transcriptase polymerase chain reaction (RT-PCR) of S. flavogriseus CA biosynthesis genes showed that the regulatory genes ccaR and claR and some biosynthetic genes were expressed whereas expression of cyp, orf12, orf13, and oppA2 was undetectable. The ccaR gene of S. clavuligerus was unable to switch on CA production in S. flavogriseus::[Pfur-ccaR C], but insertion of a cosmid carrying the S. clavuligerus CA cluster (not including the ccaR gene) conferred clavulanic acid production on S. flavogriseus::[SCos-CA] particularly in TBO and YEME media; these results suggests that some of the S. flavogriseus CA genes are inactive. The known heptameric sequences recognized by CcaR in S. clavuligerus are poorly or not conserved in S. flavogriseus. Quantitative RT-PCR analysis of the CA gene clusters of S. clavuligerus and S. flavogriseus showed that the average expression value of the expressed genes in the former strain was in the order of 1.68-fold higher than in the later. The absence of CA production by S. flavogriseus can be traced to the lack of expression of the essential genes cyp, orf12, orf13, orf14, and oppA2. Heterologous expression of S. clavuligerus CA gene cluster in S. flavogriseus::[SCos-CA] was 11- to 14-fold lower than in the parental strain, suggesting that the genetic background of the host strain is important for optimal production of CA in Streptomyces.

Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

Quantitative MAS NMR characterization of the LiMn(1/2)Ni(1/2)O(2) electrode/electrolyte interphase.

The conditions in which degradation processes at the positive electrode/electrolyte interface occur are still incompletely understood and traditional surface analytical techniques struggle to characterize and depict accurately interfacial films. In the present work, information on the growth and evolution of the interphases upon storage and cycling as well as their electrochemical consequences are gathered in the case of LiNi(1/2)Mn(1/2)O(2) with commonly used LiPF(6) (1M in EC/DMC) electrolyte. The use of (7)Li, (19)F and (31)P MAS NMR, made quantitative through the implementation of empirical calibration, is combined with transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) to probe the elements involved in surface species and to unravel the inhomogenous architecture of the interphase. At room temperature, contact with the electrolyte leads to a covering of the oxide surface first by LiF and lithiated organic species are found on the outer part of the interphase. At 55°C, not only the interphase proceeds in further covering of the surface but also thickens resulting in an increase of 240% of lithiated species and the presence of -POF(2) fluorophosphates. The composition gradient within the interphase depth is also strongly affected by the temperature. In agreement with the electrochemical performance, quantitative NMR surface analyses show that the use of LiBOB-modified electrolyte results in a Li-enriched interphase, intrinsically less resistive than the standard LiPF(6)-based interphase, comprised of a mixture of resistive LiF with non lithiated species.

GABAergic and glutamatergic identities of developing midbrain Pitx2 neurons.

Pitx2, a paired-like homeodomain transcription factor, is expressed in post-mitotic neurons within highly restricted domains of the embryonic mouse brain. Previous reports identified critical roles for PITX2 in histogenesis of the hypothalamus and midbrain, but the cellular identities of PITX2-positive neurons in these regions were not fully explored. This study characterizes Pitx2 expression with respect to midbrain transcription factor and neurotransmitter phenotypes in mid-to-late mouse gestation. In the dorsal midbrain, we identified Pitx2-positive neurons in the stratum griseum intermedium (SGI) as GABAergic and observed a requirement for PITX2 in GABAergic differentiation. We also identified two Pitx2-positive neuronal populations in the ventral midbrain, the red nucleus, and a ventromedial population, both of which contain glutamatergic precursors. Our data suggest that PITX2 is present in regionally restricted subpopulations of midbrain neurons and may have unique functions that promote GABAergic and glutamatergic differentiation.

Intubated patients developing tracheobronchitis or pneumonia have distinctive complement system gene expression signatures in the pre-infection period: a pilot study.

INTRODUCTION: It remains unknown why some intubated patients remain infection-free while others develop tracheobronchitis (VAT) or pneumonia (VAP). OBJECTIVE: To identify and compare VAP/VAT gene expression "signatures" using genome-wide oligonucleotide microarrays. MATERIAL AND METHODS: A prospective translational study of gene expression profiles of VAP and VAT groups was carried out, establishing comparisons in both pre-infection and infection phases. Pathway and functional analyses were performed with Ingenuity Pathway Analysis (IPA). Data analysis and hierarchical clustering of the genes involved in the signalling pathways expressed differentially in the two groups were performed with GeneSpring GX 11.0. RESULTS: Eight patients developing respiratory infections (3 VAP and 5 VAT) after 4 days of mechanical ventilation were assessed. Comparison of gene expression profiles in the pre-infection period revealed 5595 genes expressed differentially between VAP and VAT (p<0.01, fold change >2). Comparative IPA analysis identified a significant depression of the complement system signalling pathway in the VAP group, affecting the classical pathway along with the final common pathway (p<0.05). In addition, the cAMP and calcium signalling pathways were also significantly depressed in the VAP group during the pre-infection phase also. CONCLUSION: Intubated patients complicated with pneumonia developed immune impairment in the pre-infection period, manifesting as a relatively lower expression of genes involved in the complement system that differed from patients developing tracheobronchitis. These findings suggest that a significant proportion of VAP episodes cannot be prevented, but might be treatable through pre-emptive therapy.

Breaking the reproductive barrier of divergent species to explore the genomic landscape.

Background: Climate change will have significant consequences for species. Species range shifts induce the emergence of new hybrid zones or the spatial displacement of pre-existing ones. These hybrid zones may become more porous as alleles are passed from one species to another. Currently, hybridization between highly divergent species living in sympatry seems extremely limited. Indeed, this phenomenon involves breaking two barriers. The first is the pre-mating barrier, related to the reproductive phenology of the two species. The second is the post-zygotic barrier, related to the genetic divergence between these species. Here, we were interested in identifying new hybridization patterns and potential implications, especially in the context of environmental modifications. Methods: We sampled Telestes souffia and Parachondrostoma toxostoma wild specimens from different locations across France and genotyped them for SNP markers. We identified discriminant loci using F1-hybrid specimens and parental species and performed principal component analysis and Bayesian model-based clustering to analyze phylogenetic information. Furthermore, we assessed deviation in allele frequency from F1 to F2 and for Hardy-Weinberg equilibrium for F2 and assessed gene function associated with two F2 cohorts. Results: We demonstrate that by breaking the ecological barrier, massive introgressive hybridization is possible between two endemic lineages of Cyprinidae belonging to two distinct genera. For both cohorts studied (=2 cm and >2 cm), a large majority of loci (>88%) presented no deviation in allele frequency and no departure from the Hardy-Weinberg equilibrium. For individuals beyond the 2 cm stage, two phenomena were observed. The first was an allelic imbalance in favor of P. toxostoma, for some genomic regions, with genes involved in developmental regulatory processes, cytoskeletal organization, and chromosome organization. The second was an excess of heterozygous loci coupled with an equilibrium of allelic frequencies for genes involved in immune response and kidney/liver development. Moreover, the 2 cm-sized specimens with high mortality yielded a particular genomic signature. Conclusion: Our study displayed important results for understanding the early stages of hybridization between divergent lineages and predicting the emergence of future hybrid zones in the wild. Moreover, this hybridization generates a wide spectrum of hybrids that are a potential source of important evolutionary novelties.

Alterations in pro- and anti-inflammatory mediators are involved in microvascular dysfunction in postmenopausal women with type 2 diabetes mellitus.

Evidence has shown that women with type 2 diabetes mellitus (T2DM) have a greater risk of cardiovascular complications compared with men, but this sex difference is not clearly understood. This study assessed the microvascular function and circulatory biomarkers in postmenopausal women (PMW) with T2DM compared with diabetic men and their non-diabetic counterparts. Sixty participants were divided into nondiabetic PMW, PMW with T2DM, non-diabetic men, and diabetic men. Microvascular function was assessed using non-invasive equipment (EndoPAT®) and reported as reactive hyperemia index (RHI). Anthropometric and cardiovascular parameters were also measured. Two-way ANOVA was performed using sex (women or men) and T2DM (non-diabetic and diabetic) as the two factors. RHI impairment (1.97±0.14) was detected in diabetic PMW compared with women without T2DM (2.5±0.13) accompanied by lower adiponectin levels (T2DM: 9.3±1.2 and CTL: 13.8±1.8 ug/mL, P<0.05). An increase in the Nε-carboxymethyllysine (CML), nitrate/nitrite, and C-reactive protein (CRP) levels were observed in diabetic PMW compared to the other groups. Although a poor glycemia control was seen in diabetic men, neither RHI nor circulatory biomarkers were affected by T2DM. Multiple linear regression stratified by sex and T2DM identified some variables with RHI only in PMW with T2DM: HbA1c (P=0.003), body mass index (P=0.029), CML (P=0.032), and CRP (P=0.006). Diabetic PMW were more susceptible to the deleterious effects of hyperglycemia than men, showing microvascular dysfunction with high levels of pro-inflammatory mediators (CML and CRP) and a lower adiponectin concentration.

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum.

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the "early" cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a "Fungal_trans" domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.