Differences in Wear and Material Integrity of NAO and Low-Steel Brake Pads under Severe Conditions.

In this study, through severe reduced-scale braking tests, we investigate the wear and integrity of organic matrix brake pads against gray cast iron (GCI) discs. Two prototype pad materials are designed with the aim of representing a typical non-metal NAO and a low-steel (LS) formulation. The worn surfaces are observed with SEM. The toughness of the pad materials is tested at the raw state and after a heat treatment. During braking, the LS-GCI disc configuration produces heavy wear. The friction parts both keep their macroscopic integrity and wear appears to be homogeneous. The LS pad is mostly covered by a layer of solid oxidized steel. The NAO-GCI disc configuration wears dramatically and cannot reach the end of the test program. The NAO pad suffers many deep cracks. Compacted third body plateaus are scarce and the corresponding disc surface appears to be very heterogeneous. The pad materials both show similar strength at the raw state and similar weakening after heat treatment. However, the NAO material is much more brittle than the LS material in both states, which seems to favor the growth of cracks. The observations of crack faces suggest that long steel fibers in the LS material palliate the brittleness of the matrix, even after heat damage.

Higher-Order Interactions Dampen Pairwise Competition in the Zebrafish Gut Microbiome.

The microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health, and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multispecies consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germfree, to allow the introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, interbacterial interactions become weaker, suggesting that higher-order interactions in the vertebrate gut stabilize complex communities.IMPORTANCE Understanding the rules governing the composition of the diverse microbial communities that reside in the vertebrate gut environment will enhance our ability to manipulate such communities for therapeutic ends. Synthetic microbial communities, assembled from specific combinations of microbial species in germfree animals, allow investigation of the fundamental question of whether multispecies community composition can be predicted solely based on the combined effects of interactions between pairs of species. If so, such predictability would enable the construction of communities with desired species from the bottom up. If not, the apparent higher-order interactions imply that emergent community-level characteristics are crucial. Our findings using up to five coexisting native bacterial species in larval zebrafish, a model vertebrate, provide experimental evidence for higher-order interactions and, moreover, show that these interactions promote the coexistence of microbial species in the gut.

Usage of histological methods in determining the prescription of spleen injuries in forensic medical practice.

OBJECTIVE: Introduction: The article presents data from literary sources and a statistical analysis of one's own research on the nature, mechanism and prescription of spleen injury in the case of mechanical trauma and the absence of alcohol intoxication. The aim: To study the dynamics of changes in the histological parameters of the spleen injured tissues in case of mechanical trauma depending on the prescription of injury. PATIENTS AND METHODS: Materials and methods: The material of the study was the spleen tissue of 56 males and females aged from 20-60 who died at known and unknown time in the presence and absence of alcohol in the blood. We used histological, histochemical methods, and carried out a statistical analysis of the results. RESULTS: Results: The obtained results showed that during the mechanical injury of spleen there often developed a capsule and a parenchyma with hematoma in the area of injury. Our records showed that during the first 6 hours after injury, there appeared a hematoma in the center of the injury. Hemolysis of the erythrocyte particles was observed in the center of the hematoma. There were isolated leukocytes and fibrin tissues closer to the edge of the hematoma. CONCLUSION: Conclusions: The obtained results indicate that there are several histological changes in the damaged spleen tissues area which directly depend on the time which passed from the moment of injury.

Performance of convolutional neural networks for identification of bacteria in 3D microscopy datasets.

Three-dimensional microscopy is increasingly prevalent in biology due to the development of techniques such as multiphoton, spinning disk confocal, and light sheet fluorescence microscopies. These methods enable unprecedented studies of life at the microscale, but bring with them larger and more complex datasets. New image processing techniques are therefore called for to analyze the resulting images in an accurate and efficient manner. Convolutional neural networks are becoming the standard for classification of objects within images due to their accuracy and generalizability compared to traditional techniques. Their application to data derived from 3D imaging, however, is relatively new and has mostly been in areas of magnetic resonance imaging and computer tomography. It remains unclear, for images of discrete cells in variable backgrounds as are commonly encountered in fluorescence microscopy, whether convolutional neural networks provide sufficient performance to warrant their adoption, especially given the challenges of human comprehension of their classification criteria and their requirements of large training datasets. We therefore applied a 3D convolutional neural network to distinguish bacteria and non-bacterial objects in 3D light sheet fluorescence microscopy images of larval zebrafish intestines. We find that the neural network is as accurate as human experts, outperforms random forest and support vector machine classifiers, and generalizes well to a different bacterial species through the use of transfer learning. We also discuss network design considerations, and describe the dependence of accuracy on dataset size and data augmentation. We provide source code, labeled data, and descriptions of our analysis pipeline to facilitate adoption of convolutional neural network analysis for three-dimensional microscopy data.

Automated high-throughput light-sheet fluorescence microscopy of larval zebrafish.

Light sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.

Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages.

Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

Passive and Active Microrheology of the Intestinal Fluid of the Larval Zebrafish.

The fluids of the intestine serve as a physical barrier to pathogens, a medium for the diffusion of nutrients and metabolites, and an environment for commensal microbes. The rheological properties of intestinal mucus have therefore been the subject of many investigations, thus far limited to in vitro studies due to the difficulty of measurement in the natural context of the gut. This limitation especially hinders our understanding of how the gut microbiota interact with the intestinal space, since examination of this calls not only for in vivo measurement techniques, but for techniques that can be applied to model organisms in which the microbial state of the gut can be controlled. We have addressed this challenge with two complementary approaches. We performed passive microrheological measurements using thermally driven nanoparticles and active microrheology using micron-scale ellipsoidal magnetic microparticles, in both cases using light-sheet fluorescence microscopy to optically access the intestinal bulb of the larval zebrafish, a model vertebrate. We present viscosity measurements in germ-free animals (devoid of gut microbes), animals colonized by a single bacterial species, and conventionally reared animals, and find that in all cases, the mucin-rich intestinal liquid is well described as a Newtonian fluid. Surprisingly, despite known differences in the number of secretory cells in germ-free zebrafish and their conventional counterparts, the fluid viscosity for these two groups is very similar, as measured with either technique. Our study provides, to our knowledge, the first in vivo microrheological measurements of the intestinal space in living animals, and we comment on its implications for timescales of host-microbe interactions in the gut.

Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells.

It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.

Automated inference procedure for the determination of cell growth parameters.

The growth rate and carrying capacity of a cell population are key to the characterization of the population's viability and to the quantification of its responses to perturbations such as drug treatments. Accurate estimation of these parameters necessitates careful analysis. Here, we present a rigorous mathematical approach for the robust analysis of cell count data, in which all the experimental stages of the cell counting process are investigated in detail with the machinery of Bayesian probability theory. We advance a flexible theoretical framework that permits accurate estimates of the growth parameters of cell populations and of the logical correlations between them. Moreover, our approach naturally produces an objective metric of avoidable experimental error, which may be tracked over time in a laboratory to detect instrumentation failures or lapses in protocol. We apply our method to the analysis of cell count data in the context of a logistic growth model by means of a user-friendly computer program that automates this analysis, and present some samples of its output. Finally, we note that a traditional least squares fit can provide misleading estimates of parameter values, because it ignores available information with regard to the way in which the data have actually been collected.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/ß cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Effect of environmental contaminants on mammalian testis.

Exposure of humans and wildlife to pollutants released in the environment is a centre of attention nowadays. Many of these chemicals (generally referred to as environmental pollutants) have been shown to interfere with normal hormonal signalling and biological functions, leading to reproductive disorders or infertility, which has been a matter of concern within the recent decades. The present paper reviews adverse effects of these toxicants on mammalian testes, with emphasis on alteration of steroidogenesis, spermatogenesis, and histopathological effects. From the publications reviewed, it appears that environmental toxicants, especially heavy metals and organic chemicals of synthetic and microbiological origins, disrupt hormone production and action in the mammalian testes. Endocrine disruption leads to disorders of testicular function and thereby compromises the normal phenotypic development of male sexual characteristics, initiation and maintenance of spermatogenesis. The toxicants also induce impairment of testicular cells function, testicular histology, and sperm cells function directly. The release of the toxicants in the environment is still ongoing, despite alarming quantities that already exist in the atmosphere. If appropriate measures are not taken, their impact on the male reproductive function and especially on testicular function will be more serious.

[Differential modulation of TLR2 and TLR4-induced TNF production by murin haemorrhagic shock]. / Modulation différentielle des effets de la stimulation des récepteurs Toll-like 2 et 4 par l'état de choc hémorragique.

OBJECTIVE: To investigate the influence of haemorrhagic shock in mice on ex vivo TNF production by whole blood cells (WBC) stimulated through Toll-like receptors (TLR) 4 and 2. STUDY DESIGN AND ANIMALS: Experimental study using BALB/c male mice. METHODS: Haemorrhage (0,026+/-0,003 ml/g) by transparietal cardiac puncture under general anaesthesia. Measurement of left intraventricular pressure through a direct subcostal cardiac puncture. Possible restitution of shed blood volume (SBV) in retroorbital venous plexus, 60 minutes following haemorrhage. Lethal exsanguination 120 minutes following general anaesthesia (Control group), cardiac puncture (Sham group), blood sample (Haemorrhage group), or 60 minutes following SBV retransfusion (SBV group). Cultures (24 hours) of whole blood from the exsanguination, alone or with Escherichia coli endotoxin (LPS, TLR 4) or with heat-killed Staphylococcus aureus Cowan (SAC, TLR 2). Assessment of TNF levels in the cultures supernatant (Elisa). RESULTS: Hemorrhage (approximately 30% of calculated blood volume) resulted in arterial hypotension (-50%) which was reversed by SBV retransfusion. TNF production by LPS-stimulated WBC was reduced by haemorrhage (approximately -50%) with or without SBV retransfusion. TNF production by SAC-stimulated WBC remained unchanged. CONCLUSION: The reduction of proinflammatory cytokines production by WBC stimulated with pathogen-associated molecular patterns is not a generalized phenomenon following murin haemorrhagic shock. It depends on the used stimulus and studied signalling pathways.

Non-invasive assessment of cerebral perfusion pressure in brain injured patients with moderate intracranial hypertension.

BACKGROUND: A non-invasive estimation of cerebral perfusion pressure (CPP) using transcranial Doppler sonography was assessed in brain-injured patients by comparing conventional measurements of CPP (difference between mean arterial pressure and intracranial pressure) (CPPm) with the difference between AP(mean) and the critical closing pressure of the cerebral circulation (CPPe). METHODS: Twenty adults with bilateral and diffuse brain injuries were included in the study. CPPe was estimated using a formula combining the phasic values of flow velocities and arterial pressure. In group A (n=10) the comparison was repeatedly performed under stable conditions. In group B (n=10) the comparison was performed during a CO(2) reactivity test. Covariance analysis was used to assess the relationships. RESULTS: In group A, CPPe and CPPm were correlated (slope, 0.76; intercept, +10.9; 95% CI, -3.5 to +25.4). During the increase in intracranial pressure (group B) (+1.9 (sd 1.5) mm Hg per mm Hg of Pe'(co(2))) the relationship persisted (slope, 0.55; intercept, +32.6; 95% CI, +16.3 to +48.9) but the discrepancy between the two variables increased as reflected by the increase in bias and variability. CONCLUSION: Non-invasive estimation of CPP can be used for brain monitoring of head-injured patients, but the accuracy of the method may depend on the level of intracranial hypertension.

[Circulating troponin I assay: uses and misuses in medical practice]. / Le dosage de la troponine I: intérêt et limites en pratique médicale courante.

Cardiac troponin I (cTnI) contributes to the modulation of the myocardial contractile function in the troponins complex. The assay of circulating cTnI is highly specific of the cardiac isoform and allows a quantification of the myocardial injury provided that both free and combined cTnI were recognized by the antibodies. The assay is a major contribution to the diagnosis of postoperative cardiac complications through its specificity because peripheral muscle trauma is a confounding factor during this period. Circulating cTnI mirrors the magnitude of an acute circulatory failure whatever the origin (cardiac, hemorrhagic or septic). A positive cTnI assay in an "asymptomatic" patient has to be confirmed and needs some investigations to discard an occult cardiac disease; cTnI has a limited role by itself in predicting mortality and hospital admissions. Circulating cTnI confirms the severity of an acute coronary syndrome and has a level-dependent prognostic value.

Co-amoxiclav pharmacokinetics during posttraumatic hemorrhagic shock.

OBJECTIVE: To determine the effects of severe trauma with hemorrhagic shock on amoxicillin and clavulanate concentrations in plasma and their pharmacokinetics. DESIGN: A prospective, open, descriptive study. SETTING: A 12-bed, adult surgical intensive care unit in a university-affiliated hospital in France. SUBJECTS: Subjects were 12 patients (10 men, 2 women) with severe trauma: median (range) Injury Severity Score, 38 (17-48); Acute Physiology and Chronic Health Evaluation II, 16 (7-38); Simplified Acute Physiology Score II, 41 (23-77). Also enrolled were 12 healthy volunteers who were matched on age (+/-5 yrs), gender, and body-surface area (+/-20 cm2). All the trauma patients suffered hemorrhagic shock defined as the association of at least one episode of systolic blood pressure <90 mm Hg and an intravascular volume expansion >2000 mL between trauma and surgery. INTERVENTION: Prophylactic perioperative administration of 2 g of amoxicillin and 0.2 g of clavulanate in combination during the first 12 hrs posttrauma in patients, and at the start of the pharmacokinetic study in volunteers. MEASUREMENTS AND MAIN RESULTS: Serial plasma samples (n = 13) were obtained after the first antibiotic administration to measure antibiotic levels by using high-performance liquid chromatography assays. Compared with volunteers, trauma patients had higher plasma amoxicillin and clavulanate concentrations, attributed to a reduction of the volume of distribution (p =.001 and p =.06, respectively) and, to a lesser extent, of the total body clearance (p =.09 and p =.20, respectively). Consequently, amoxicillin and clavulanate elimination half-lives were similar for the two groups of subjects. The interindividual variabilities for all the amoxicillin pharmacokinetic parameters were higher in patients. CONCLUSIONS: In trauma patients with hemorrhagic shock requiring surgery, the administration of 2 g of amoxicillin and 0.2 g of clavulanate seems adequate, according to the antibiotic concentrations observed in plasma for both drugs. However, further studies exploring antibiotic concentrations in tissues are warranted.

Comparative effects of frovatriptan and sumatriptan on coronary and internal carotid vascular haemodynamics in conscious dogs.

The effects of frovatriptan and sumatriptan on internal carotid and coronary vascular haemodynamics were investigated and compared in conscious dogs. Frovatriptan and sumatriptan (0.1 - 100 microg kg(-1)) induced a transient increase in external coronary artery diameter (eCOD) of up to 2.9+/-1.2 and 1.8+/-0.6%, respectively (both P:<0.05). This was followed by a prolonged and dose-dependent decrease in eCOD of up to -5.2+/-1.2 and -5.3+/-0.9% (both P:<0.05), with ED(50) values of 86+/-21 and 489+/-113 micromol kg(-1), respectively. In contrast, only a decrease in the external diameter of the internal carotid artery was observed (-6.0+/-0.6 and -6.2+/-1.4%, both P:<0.05, and ED(50) values of 86+/-41 and 493+/-162 micromol kg(-1), respectively). Frovatriptan was thus 5.7 fold more potent than sumatriptan at the level of both large coronary and internal carotid arteries. After endothelium removal by balloon angioplasty in coronary arteries, the initial dilatation induced by the triptans was abolished and delayed constriction enhanced. The selective antagonist for the 5-HT(1B) receptors SB224289 dose-dependently blocked the effects of sumatriptan on large coronary and internal carotid arteries whereas the selective antagonist for the 5-HT(1D) receptors BRL15572 did not affect any of these effects. In conclusion, frovatriptan and sumatriptan initially dilate and subsequently constrict large coronary arteries in the conscious dog, whereas they directly constrict the internal carotid artery. The vascular endothelium modulates the effects of these triptans on large coronary arteries. Finally, 5-HT(1B) but not 5-HT(1D) receptors are primarily involved in canine coronary and internal carotid vasomotor responses to sumatriptan.

Gram staining of protected pulmonary specimens in the early diagnosis of ventilator-associated pneumonia.

We evaluated prospectively the use of Gram staining of protected pulmonary specimens to allow the early diagnosis of ventilator-associated pneumonia (VAP), compared with the use of 60 bronchoscopic protected specimen brushes (PSB) and 126 blinded plugged telescopic catheters (PTC) obtained from 134 patients. Gram stains were from Cytospin slides; they were studied for the presence of microorganisms in 10 and 50 fields by two independent observers and classified according to their Gram stain morphology. Quantitative cultures were performed after serial dilution and plating on appropriate culture medium. A final diagnosis of VAP, based on a culture of > or = 10(3) c.f.u. ml-1, was established after 81 (44%) samplings. When 10 fields were analysed, a strong relationship was found between the presence of bacteria on Gram staining and the final diagnosis of VAP (for PSB and PTC respectively: sensitivity 74 and 81%, specificity 94 and 100%, positive predictive value 91 and 100%, negative predictive value 82 and 88%). The correlation was less when we compared the morphology of microorganisms observed on Gram staining with those of bacteria obtained from quantitative cultures (for PSB and PTC respectively: sensitivity 54 and 69%, specificity 86 and 89%, positive predictive value 72 and 78%, negative predictive value 74 and 84%). Increasing the number of fields read to 50 was associated with a slight decrease in specificity and positive predictive value of Gram staining, but with a small increase in its sensitivity and negative predictive value. The results obtained by the two observers were similar to each other for both numbers of fields analysed. Gram staining of protected pulmonary specimens performed on 10 fields predicted the presence of VAP and partially identified (using Gram stain morphology) the microorganisms growing at significant concentrations, and could help in the early choice of the treatment of VAP. Increasing the number of fields read or having the Gram stain analysed by two independent individuals did not improve the results.