Improved statistical analysis of low abundance phenomena in bimodal bacterial populations.

Accurate detection of subpopulation size determinations in bimodal populations remains problematic yet it represents a powerful way by which cellular heterogeneity under different environmental conditions can be compared. So far, most studies have relied on qualitative descriptions of population distribution patterns, on population-independent descriptors, or on arbitrary placement of thresholds distinguishing biological ON from OFF states. We found that all these methods fall short of accurately describing small population sizes in bimodal populations. Here we propose a simple, statistics-based method for the analysis of small subpopulation sizes for use in the free software environment R and test this method on real as well as simulated data. Four so-called population splitting methods were designed with different algorithms that can estimate subpopulation sizes from bimodal populations. All four methods proved more precise than previously used methods when analyzing subpopulation sizes of transfer competent cells arising in populations of the bacterium Pseudomonas knackmussii B13. The methods' resolving powers were further explored by bootstrapping and simulations. Two of the methods were not severely limited by the proportions of subpopulations they could estimate correctly, but the two others only allowed accurate subpopulation quantification when this amounted to less than 25% of the total population. In contrast, only one method was still sufficiently accurate with subpopulations smaller than 1% of the total population. This study proposes a number of rational approximations to quantifying small subpopulations and offers an easy-to-use protocol for their implementation in the open source statistical software environment R.

Myocardial impairment in chronic hypoxia is abolished by short aeration episodes: involvement of K+ATP channels.

In vivo exposure to chronic hypoxia is considered to be a cause of myocardial dysfunction, thereby representing a deleterious condition, but repeated aeration episodes may exert some cardioprotection. We investigated the possible role of ATP-sensitive potassium channels in these mechanisms. First, rats (n = 8/group) were exposed for 14 days to either chronic hypoxia (CH; 10% O(2)) or chronic hypoxia with one episode/day of 1-hr normoxic aeration (CH+A), with normoxia (N) as the control. Second, isolated hearts were Langendorff perfused under hypoxia (10% O(2), 30 min) and reoxygenated (94% O(2), 30 min) with or without 3 microM glibenclamide (nonselective K(+)(ATP) channel-blocker) or 100 microM diazoxide (selective mitochondrial K(+)(ATP) channel-opener). Blood gasses, hemoglobin concentration, and plasma malondialdehyde were similar in CH and CH+A and in both different from normoxic (P < 0.01), body weight gain and plasma nitrate/nitrite were higher in CH+A than CH (P < 0.01), whereas apoptosis (number of TUNEL-positive nuclei) was less in CH+A than CH (P < 0.05). During in vitro hypoxia, the efficiency (ratio of ATP production/pressure x rate product) was the same in all groups and diazoxide had no measurable effects on myocardial performance, whereas glibenclamide increased end-diastolic pressure more in N and CH than in CH+A hearts (P < 0.05). During reoxgenation, efficiency was markedly less in CH with respect to N and CH+A (P < 0.0001), and ratex pressure product remained lower in CH than N and CH+A hearts (P < 0.001), but glibenclamide or diazoxide abolished this difference. Glibenclamide, but not diazoxide, decreased vascular resistance in N and CH (P < 0.005 and < 0.001) without changes in CH+A. We hypothesize that cardioprotection in chronically hypoxic hearts derive from cell depolarization by sarcolemmal K(+)(ATP) blockade or from preservation of oxidative phosphorylation efficiency (ATP turnover/myocardial performance) by mitochondrial K(+)(ATP) opening. Therefore K(+)(ATP) channels are involved in the deleterious effects of chronic hypoxia and in the cardioprotection elicited when chronic hypoxia is interrupted with short normoxic aeration episodes.

Differential transgene expression profiles in rat brain, using rAAV2/1 vectors with tetracycline-inducible and cytomegalovirus promoters.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13:1 and 2:1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

Time constant of the cerebral arterial bed in normal subjects.

The time constant of cerebral arterial bed (in brief time constant) is a product of brain arterial compliance (C(a)) and resistance (CVR). We tested the hypothesis that in normal subjects, changes in end-tidal CO(2) (EtCO(2)) affect the value of the time constant. C(a) and CVR were estimated using mathematical transformations of arterial pressure (ABP) and transcranial Doppler (TCD) cerebral blood flow velocity waveforms. Responses of the time constant to controlled changes in EtCO(2) were compared in 34 young volunteers. Hypercapnia shortened the time constant (0.22 s [0.17, 0.26] vs. 0.16 s [0.13, 0.20]; p = 0.000001), while hypocapnia lengthened the time constant (0.22 s [0.17, 0.26] vs. 0.23 s [0.19, 0.32]; p < 0.0032). The time constant was negatively correlated with changes in EtCO(2) (R(partial) = -0.68, p < 0.000001). This was associated with a decrease in CVR when EtCO(2) increased (R(partial) = -0.80, p < 0.000001) and C(a) remained independent of changes in EtCO(2). C(a) was negatively correlated with mean ABP (R(partial) = -0.68, p < 0.000001). In summary, the time constant shortens with increasing EtCO(2). Its potential role in cerebrovascular investigations needs further studies.

SH3TC2, a protein mutant in Charcot-Marie-Tooth neuropathy, links peripheral nerve myelination to endosomal recycling

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

Pharmacologic profile of UR-7247, an orally active angiotensin II AT1 receptor antagonist, in healthy volunteers. p6.

This study was conducted to assess the pharmacologic properties of the new orally active angiotensin II subtype I (AT1) antagonist UR-7247, a product with a half-life >100 h in humans. The experiment was designed as an open-label, single-dose administration study with four parallel groups of four healthy men receiving increasing single oral doses (2.5, 5, and 10 mg) of UR-7247 or losartan, 100 mg. Angiotensin II receptor blockade was investigated < or =96 h after drug intake, with three independent methods [i.e., the inhibition of blood pressure (BP) response to exogenous Ang II, an in vitro Ang II-receptor assay (RRA), and the reactive increase in plasma angiotensin II. Plasma drug levels also were measured. The degree of blockade observed in vivo was statistically significant < or = 96 h with all UR-7247 doses for diastolic BP (p < 0.05) and < or =48 h for systolic BP. The maximal inhibition achieved with 10 mg UR-7247 was measured 6-24 h after drug intake and reached 54 +/- 17% and 48 +/- 20% for diastolic and systolic responses, respectively. Losartan, 100 mg, induced a greater short-term AT1-receptor blockade than 2.5- and 5.0-mg doses of UR-7247 (p < 0.001 for diastolic BP), but the UR-7247 effect was longer lasting. In vivo, no significant difference was observed between 10 mg UR-7247 and 100 mg losartan 4 h after drug intake, but in vitro, the blockade achieved with 100 mg losartan was higher than that seen with UR-7247. Finally, the results confirm that UR-7247 has a very long plasma elimination half-life, which may be due to a high but also tight binding to protein binding sites. In conclusion, UR-7247 is a long-lasting, well-tolerated AT1 receptor in healthy subjects.

Understanding and Fighting the Medicine Counterfeit Market

Medicine counterfeiting is a serious worldwide issue, involving networks of manufacture and distribution that are an integral part of industrialized organized crime. Despite the potentially devastating health repercussions involved, legal sanctions are often inappropriate or simply not applied. The difficulty in agreeing on a definition of counterfeiting, the huge profits made by the counterfeiters and the complexity of the market are the other main reasons for the extent of the phenomenon. Above all, international cooperation is needed to thwart the spread of counterfeiting. Moreover effort is urgently required on the legal, enforcement and scientific levels. Pharmaceutical companies and agencies have developed measures to protect the medicines and allow fast and reliable analysis of the suspect products. Several means, essentially based on chromatography and spectroscopy, are now at the disposal of the analysts to enable the distinction between genuine and counterfeit products. However the determination of the components and the use of analytical data for forensic purposes still constitute a challenge. The aim of this review article is therefore to point out the intricacy of medicine counterfeiting so that a better understanding can provide solutions to fight more efficiently against it.

Profiling of counterfeit medicines by vibrational spectroscopy

Counterfeit pharmaceutical products have become a widespread problem in the last decade. Various analytical techniques have been applied to discriminate between genuine and counterfeit products. Among these, Near-infrared (NIR) and Raman spectroscopy provided promising results.The present study offers a methodology allowing to provide more valuable information fororganisations engaged in the fight against counterfeiting of medicines.A database was established by analyzing counterfeits of a particular pharmaceutical product using Near-infrared (NIR) and Raman spectroscopy. Unsupervised chemometric techniques (i.e. principal component analysis - PCA and hierarchical cluster analysis - HCA) were implemented to identify the classes within the datasets. Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FT-IR) were used to determine the number of different chemical profiles within the counterfeits. A comparison with the classes established by NIR and Raman spectroscopy allowed to evaluate the discriminating power provided by these techniques. Supervised classifiers (i.e. k-Nearest Neighbors, Partial Least Squares Discriminant Analysis, Probabilistic Neural Networks and Counterpropagation Artificial Neural Networks) were applied on the acquired NIR and Raman spectra and the results were compared to the ones provided by the unsupervised classifiers.The retained strategy for routine applications, founded on the classes identified by NIR and Raman spectroscopy, uses a classification algorithm based on distance measures and Receiver Operating Characteristics (ROC) curves. The model is able to compare the spectrum of a new counterfeit with that of previously analyzed products and to determine if a new specimen belongs to one of the existing classes, consequently allowing to establish a link with other counterfeits of the database.

The Gene Ontology: enhancements for 2011.

The Gene Ontology (GO) (http://www.geneontology.org) is a community bioinformatics resource that represents gene product function through the use of structured, controlled vocabularies. The number of GO annotations of gene products has increased due to curation efforts among GO Consortium (GOC) groups, including focused literature-based annotation and ortholog-based functional inference. The GO ontologies continue to expand and improve as a result of targeted ontology development, including the introduction of computable logical definitions and development of new tools for the streamlined addition of terms to the ontology. The GOC continues to support its user community through the use of e-mail lists, social media and web-based resources.

Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit.

A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes. In astrocytes, all substances increased GSH levels and the activity of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). In neurons, curcumin and to a lesser extent tBHQ increased GCL activity and GSH levels, while quercetin decreased GSH and led to cell death. In the two cell types, the gene that showed the greatest increase in its expression was the one coding for the modifier subunit of GCL (GCLM). The increase in mRNA levels of GCLM was 3 to 7-fold higher than that of the catalytic subunit. In astrocytes from GCLM-knock-out mice showing low GSH (-80%) and low GCL activity (-50%), none of the substances succeeded in increasing GSH synthesis. Our results indicate that GCLM is essential for the up-regulation of GCL activity induced by curcumin, quercetin and tBHQ.

Ecological assembly rules in plant communities--approaches, patterns and prospects.

Understanding how communities of living organisms assemble has been a central question in ecology since the early days of the discipline. Disentangling the different processes involved in community assembly is not only interesting in itself but also crucial for an understanding of how communities will behave under future environmental scenarios. The traditional concept of assembly rules reflects the notion that species do not co-occur randomly but are restricted in their co-occurrence by interspecific competition. This concept can be redefined in a more general framework where the co-occurrence of species is a product of chance, historical patterns of speciation and migration, dispersal, abiotic environmental factors, and biotic interactions, with none of these processes being mutually exclusive. Here we present a survey and meta-analyses of 59 papers that compare observed patterns in plant communities with null models simulating random patterns of species assembly. According to the type of data under study and the different methods that are applied to detect community assembly, we distinguish four main types of approach in the published literature: species co-occurrence, niche limitation, guild proportionality and limiting similarity. Results from our meta-analyses suggest that non-random co-occurrence of plant species is not a widespread phenomenon. However, whether this finding reflects the individualistic nature of plant communities or is caused by methodological shortcomings associated with the studies considered cannot be discerned from the available metadata. We advocate that more thorough surveys be conducted using a set of standardized methods to test for the existence of assembly rules in data sets spanning larger biological and geographical scales than have been considered until now. We underpin this general advice with guidelines that should be considered in future assembly rules research. This will enable us to draw more accurate and general conclusions about the non-random aspect of assembly in plant communities.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Role of the glyoxalase system in astrocyte-mediated neuroprotection.

The glyoxalase system is the most important pathway for the detoxification of methylglyoxal (MG), a highly reactive dicarbonyl compound mainly formed as a by-product of glycolysis. MG is a major precursor of advanced glycation end products (AGEs), which are associated with several neurodegenerative disorders. Although the neurotoxic effects of MG and AGEs are well characterized, little is known about the glyoxalase system in the brain, in particular with regards to its activity in different neural cell types. Results of the present study reveal that both enzymes composing the glyoxalase system [glyoxalase-1 (Glo-1) and Glo-2] were highly expressed in primary mouse astrocytes compared with neurons, which translated into higher enzymatic activity rates in astrocytes (9.9- and 2.5-fold, respectively). The presence of a highly efficient glyoxalase system in astrocytes was associated with lower accumulation of AGEs compared with neurons (as assessed by Western blotting), a sixfold greater resistance to MG toxicity, and the capacity to protect neurons against MG in a coculture system. In addition, Glo-1 downregulation using RNA interference strategies resulted in a loss of viability in neurons, but not in astrocytes. Finally, stimulation of neuronal glycolysis via lentiviral-mediated overexpression of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase-3 resulted in increased MG levels and MG-modified proteins. Since MG is largely produced through glycolysis, this suggests that the poor capacity of neurons to upregulate their glycolytic flux as compared with astrocytes may be related to weaker defense mechanisms against MG toxicity. Accordingly, the neuroenergetic specialization taking place between these two cell types may serve as a protective mechanism against MG-induced neurotoxicity.

Growth Arrest-Specific Gene 6 product modulates innate immunity in sepsis

Background: Growth Arrest-Specific Gene 6 product (Gas6) is, like anticoagulant protein C, a vitamin K-dependent protein. Our aim was to determine whether Gas6 plays a role in sepsis. Materials and methods: We submitted mice lacking Gas6 (Gas6)/)) or one of its receptors (Axl)/), Tyro3)/) or Mertk)/)) to LPS-induced endotoxemia and peritonitis (cecal ligation and puncture (CLP) and inoculation of E. coli). In addition, we measured Gas6 or its soluble receptors in plasma of eight volunteers that received LPS, 13 healthy subjects, 28 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Results: Gas6 and its soluble receptor sAxl raised in mice models and TNF-a was more elevated in Gas6)/) mice than in wild-type (WT). Protein array showed that before and after LPS injection, titers of 62 cytokines were more elevated in plasma of Gas6)/) than WT mice. Endotoxemia-induced mortality was higher in Gas6)/), Axl)/), Tyro3)/) and Mertk)/) compared to WT mice and mortality subsequent to CLP was amplified in Gas6)/) mice. LPS-stimulated Gas6)/) macrophages produced more cytokines than WT macrophages. This production was dampened by recombinant Gas6. Phosphorylation of Akt in Gas6)/) macrophages was reduced, but p38 phosphorylation and NF-jB translocation were increased. In human, Gas6 raised in plasma after LPS (2 ng/kg). Gas6 and sAxl were higher in patients with severe sepsis than in healthy subjects or control patients, and there was a non-significant trend for higher Gas6 in the survival group. Conclusions: Our data point to Gas6 as a major modulator of innate immunity and provide thereby novel insights into the mechanism of sepsis. Thus Gas6 and its receptors might constitute potential therapeutic targets for the development of new immunomodulating drugs.

The role of laparoscopic biopsies in lumbar spondylodiscitis.

The infection of an intervertebral disk is a serious condition. The diagnosis often is elusive and difficult to make. It is imperative to have appropriate microbiologic specimens before the initiation of treatment. We report the case of a 51-year-old woman with lumbar spondylodiscitis caused by infection after the placement of an epidural catheter for postoperative analgesia. A spinal magnetic resonance imaging (MRI) scan confirmed the diagnosis, but computed tomography (CT)-guided fine-needle biopsy did not yield adequate material for a microbiologic diagnosis. Laparoscopic biopsies of the involved disk provided good specimens and a diagnosis of Propionibacterium acnes infection. We believe that this minimally invasive procedure should be performed when CT-guided fine-needle biopsy fails to yield a microbiologic diagnosis in spondylodiscitis.