Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

Contribution of the genes of the renin-angiotensin system to interindividual differences in blood pressure levels in Caucasians from Rochester, Minnesota

The Renin-Angiotensin system (RAS) regulates blood pressure through its effects on vascular tone, renal hemodynamics, and renal sodium and fluid balance. The genes encoding the four major components of the RAS, angiotensinogen, renin, angiotensin I-converting enzyme (ACE), and angiotensin II receptor type 1 (AT1), have been investigated as candidate genes in the pathogenesis of essential hypertension. However, studies have primarily focused on small samples of diseased individuals, and, therefore, have provided little information about the determinants of interindividual variation in blood pressure (BP) in the general population.^ Using data from a large population-based sample from Rochester, MN, I have evaluated the contribution of variation in the region of the RAS genes to interindividual variation in systolic, diastolic, and mean arterial pressure in the population-at-large. Marker genotype data from four polymorphisms located within or very near these genes were first collected on 3,974 individuals from 583 randomly ascertained three-generation pedigrees. Haseman-Elston regression and variance component methods of linkage analysis were then carried out to estimate the proportion of interindividual variance in BP attributable to the effects of variation at these four measured loci.^ A significant effect of the ACE locus on interindividual variation in mean arterial pressure (MAP) was detected in a sample of siblings belonging to the youngest generation. After allowing for measured covariates, this effect accounted for 15-25% of the interindividual variance in MAP, and was even greater in a subset with a positive family history of hypertension. When gender-specific analyses were carried out, this effect was significant in males but not in females. Extended pedigree analyses also provided evidence for an effect of the ACE locus on interindividual variation in MAP, but no difference between males and females was observed. Circumstantial evidence suggests that the ACE gene itself may be responsible for the observed effects on BP, although the possibility that other genes in the region may be at play cannot be excluded.^ No definitive evidence for an effect of the renin, angiotensinogen, or AT1 loci on interindividual variation in BP was obtained in this study, suggesting that the impact of these genes on BP may not be great in the Caucasian population-at-large. However, this does not preclude a larger effect of these genes in some subsets of individuals, especially among those with clinically manifest hypertension or coronary heart disease, or in other populations. ^

A Food Recognition System for Diabetic Patients based on an Optimized Bag of Features Model

Computer vision-based food recognition could be used to estimate a meal's carbohydrate content for diabetic patients. This study proposes a methodology for automatic food recognition, based on the Bag of Features (BoF) model. An extensive technical investigation was conducted for the identification and optimization of the best performing components involved in the BoF architecture, as well as the estimation of the corresponding parameters. For the design and evaluation of the prototype system, a visual dataset with nearly 5,000 food images was created and organized into 11 classes. The optimized system computes dense local features, using the scale-invariant feature transform on the HSV color space, builds a visual dictionary of 10,000 visual words by using the hierarchical k-means clustering and finally classifies the food images with a linear support vector machine classifier. The system achieved classification accuracy of the order of 78%, thus proving the feasibility of the proposed approach in a very challenging image dataset.

No evidence for a local renin-angiotensin system in liver mitochondria

The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.

A web-based non-intrusive ambient system to measure and classify activities of daily living.

BACKGROUND The number of older adults in the global population is increasing. This demographic shift leads to an increasing prevalence of age-associated disorders, such as Alzheimer's disease and other types of dementia. With the progression of the disease, the risk for institutional care increases, which contrasts with the desire of most patients to stay in their home environment. Despite doctors' and caregivers' awareness of the patient's cognitive status, they are often uncertain about its consequences on activities of daily living (ADL). To provide effective care, they need to know how patients cope with ADL, in particular, the estimation of risks associated with the cognitive decline. The occurrence, performance, and duration of different ADL are important indicators of functional ability. The patient's ability to cope with these activities is traditionally assessed with questionnaires, which has disadvantages (eg, lack of reliability and sensitivity). Several groups have proposed sensor-based systems to recognize and quantify these activities in the patient's home. Combined with Web technology, these systems can inform caregivers about their patients in real-time (e.g., via smartphone). OBJECTIVE We hypothesize that a non-intrusive system, which does not use body-mounted sensors, video-based imaging, and microphone recordings would be better suited for use in dementia patients. Since it does not require patient's attention and compliance, such a system might be well accepted by patients. We present a passive, Web-based, non-intrusive, assistive technology system that recognizes and classifies ADL. METHODS The components of this novel assistive technology system were wireless sensors distributed in every room of the participant's home and a central computer unit (CCU). The environmental data were acquired for 20 days (per participant) and then stored and processed on the CCU. In consultation with medical experts, eight ADL were classified. RESULTS In this study, 10 healthy participants (6 women, 4 men; mean age 48.8 years; SD 20.0 years; age range 28-79 years) were included. For explorative purposes, one female Alzheimer patient (Montreal Cognitive Assessment score=23, Timed Up and Go=19.8 seconds, Trail Making Test A=84.3 seconds, Trail Making Test B=146 seconds) was measured in parallel with the healthy subjects. In total, 1317 ADL were performed by the participants, 1211 ADL were classified correctly, and 106 ADL were missed. This led to an overall sensitivity of 91.27% and a specificity of 92.52%. Each subject performed an average of 134.8 ADL (SD 75). CONCLUSIONS The non-intrusive wireless sensor system can acquire environmental data essential for the classification of activities of daily living. By analyzing retrieved data, it is possible to distinguish and assign data patterns to subjects' specific activities and to identify eight different activities in daily living. The Web-based technology allows the system to improve care and provides valuable information about the patient in real-time.

Digital multitext editions from scratch to electronic performance. Transcription and collation routines transformed in a flexible database system

The poster demonstrates the preparatory steps of a digital multi-text edition that are abstracted from the experiences made in the Parzival Project, based at the University of Bern, the Berlin-Brandenburg Academy of Sciences and the University of Erlangen. This edition of Wolfram von Eschenbach’s German Grail novel, written shortly after 1200 and transmitted during several centuries in ca. hundred witnesses, has now been completed by more than a half of the textual corpus. As the text is transmitted in medieval manuscripts the witnesses have to be transcribed according to specific encoding rules. The transcriptions then are collated following certain ideas and concepts of how the transmission process could have developed. The transcriptions and collations finally have to be transferred to a digital edition that allows the users to explore the characteristics of single witnesses as well as the history of a text, which is delivered in variants and in different versions. A dynamically organized database offering various components and adapted to the needs of diverse user-profiles is nowadays the right tool for this purpose.

Investigation of young water inflow in karst aquifers using SF6–CFC–3H/He–85Kr–39Ar and stable isotope components

Karst aquifers are known for their wide distribution of water transfer velocities. From this observation, a multiple geochemical tracer approach seems to be particularly well suited to provide a significant assessment of groundwater flows, but the choice of adapted tracers is essential. In this study, several common tracers in karst aquifers such as physicochemical parameters, major ions, stable isotopes, and d13C to more specific tracers such as dating tracers – 14C, 3H, 3H–3He, CFC-12, SF6 and 85Kr, and 39Ar – were used, in a fractured karstic carbonated aquifer located in Burgundy (France). The information carried by each tracer and the best sampling strategy are compared on the basis of geochemical monitoring done during several recharge events and over longer time periods (months to years). This study’s results demonstrate that at the seasonal and recharge event time scale, the variability of concentrations is low for most tracers due to the broad spectrum of groundwater mixings. The tracers used traditionally for the study of karst aquifers, i.e., physicochemical parameters and major ions, efficiently describe hydrological processes such as the direct and differed recharge, but require being monitored at short time steps during recharge events to be maximized. From stable isotopes, tritium, and Cl� contents, the proportion of the fast direct recharge by the largest porosity was estimated using a binary mixing model. The use of tracers such as CFC-12, SF6, and 85Kr in karst aquifers provides additional information, notably an estimation of apparent age, but they require good preliminary knowledge of the karst system to interpret the results suitably. The CFC-12 and SF6 methods efficiently determine the apparent age of baseflow, but it is preferable to sample the groundwater during the recharge event. Furthermore, these methods are based on different assumptions such as regional enrichment in atmospheric SF6, excess air, and flow models among others. 85Kr and 39Ar concentrations can potentially provide a more direct estimation of groundwater residence time. Conversely, the 3H–3He method is inefficient in the karst aquifer for dating due to 3He degassing.

From stellar nebula to planets: The refractory components

Context. To date, calculations of planet formation have mainly focused on dynamics, and only a few have considered the chemical composition of refractory elements and compounds in the planetary bodies. While many studies have been concentrating on the chemical composition of volatile compounds (such as H2O, CO, CO2) incorporated in planets, only a few have considered the refractory materials as well, although they are of great importance for the formation of rocky planets. Aims. We computed the abundance of refractory elements in planetary bodies formed in stellar systems with a solar chemical composition by combining models of chemical composition and planet formation. We also considered the formation of refractory organic compounds, which have been ignored in previous studies on this topic. Methods. We used the commercial software package HSC Chemistry to compute the condensation sequence and chemical composition of refractory minerals incorporated into planets. The problem of refractory organic material is approached with two distinct model calculations: the first considers that the fraction of atoms used in the formation of organic compounds is removed from the system (i.e., organic compounds are formed in the gas phase and are non-reactive); and the second assumes that organic compounds are formed by the reaction between different compounds that had previously condensed from the gas phase. Results. Results show that refractory material represents more than 50 wt % of the mass of solids accreted by the simulated planets with up to 30 wt % of the total mass composed of refractory organic compounds. Carbide and silicate abundances are consistent with C/O and Mg/Si elemental ratios of 0.5 and 1.02 for the Sun. Less than 1 wt % of carbides are present in the planets, and pyroxene and olivine are formed in similar quantities. The model predicts planets that are similar in composition to those of the solar system. Starting from a common initial nebula composition, it also shows that a wide variety of chemically different planets can form, which means that the differences in planetary compositions are due to differences in the planetary formation process. Conclusions. We show that a model in which refractory organic material is absent from the system is more compatible with observations. The use of a planet formation model is essential to form a wide diversity of planets in a consistent way.

NA61/SHINE facility at the CERN SPS: beams and detector system

NA61/SHINE (SPS Heavy Ion and Neutrino Experiment) is a multi-purpose experimental facility to study hadron production in hadron-proton, hadron-nucleus and nucleus-nucleus collisions at the CERN Super Proton Synchrotron. It recorded the first physics data with hadron beams in 2009 and with ion beams (secondary 7Be beams) in 2011. NA61/SHINE has greatly profited from the long development of the CERN proton and ion sources and the accelerator chain as well as the H2 beamline of the CERN North Area. The latter has recently been modified to also serve as a fragment separator as needed to produce the Be beams for NA61/SHINE. Numerous components of the NA61/SHINE set-up were inherited from its predecessors, in particular, the last one, the NA49 experiment. Important new detectors and upgrades of the legacy equipment were introduced by the NA61/SHINE Collaboration. This paper describes the state of the NA61/SHINE facility — the beams and the detector system — before the CERN Long Shutdown I, which started in March 2013.