Quantitative TMT-based proteomics revealing host, dietary and microbial proteins in bovine faeces including barley serpin Z4, a prominent component in the head of beer.

There has been little information about the proteome of bovine faeces or about the contribution to the faecal proteome of proteins from the host, the feed or the intestinal microbiome. Here, the bovine faecal proteome and the origin of its component proteins was assessed, while also determining the effect of treating barley, the major carbohydrate in the feed, with either ammonia (ATB) or sodium propionate (PTB) preservative. Healthy continental crossbreed steers were allocated to two groups and fed on either of the barley-based diets. Five faecal samples from each group were collected on Day 81 of the trial and analysed by quantitative proteomics using nLC-ESI-MS/MS after tandem mass tag labelling. In total, 281 bovine proteins, 199 barley proteins, 176 bacterial proteins and 190 archaeal proteins were identified in the faeces. Mucosal pentraxin, albumin and digestive enzymes were among bovine proteins identified. Serpin Z4 a protease inhibitor was the most abundant barley protein identified which is also found in barley-based beer, while numerous microbial proteins were identified, many originating bacteria from Clostridium, while Methanobrevibacter was the dominant archaeal genus. Thirty-nine proteins were differentially abundant between groups, the majority being more abundant in the PTB group compared to the ATB group. SIGNIFICANCE: Proteomic examination of faeces is becoming a valuable means to assess the health of the gastro-intestinal tract in several species, but knowledge on the proteins present in bovine faeces is limited. This investigation aimed to characterise the proteome of bovine faecal extracts in order to evaluate the potential for investigations of the proteome as a means to assess the health, disease and welfare of cattle in the future. The investigation was able to identify proteins in bovine faeces that had been (i) produced by the individual cattle, (ii) present in the barley-based feed eaten by the cattle or (iii) produced by bacteria and other microbes in the rumen or intestines. Bovine proteins identified included mucosal pentraxin, serum albumin and a variety of digestive enzymes. Barley proteins found in the faeces included serpin Z4, a protease inhibitor that is also found in beer having survived the brewing process. Bacterial and archaeal proteins in the faecal extracts were related to several pathways related to the metabolism of carbohydrates. The recognition of the range of proteins that can be identified in bovine faeces raises the possibility that non-invasive sample collection of this material could provide a novel diagnostic approach to cattle health and welfare.

Modulating Oncolytic Adenovirus Immunotherapy by Driving Two Axes of the Immune System by Expressing 4-1BBL and CD40L.

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8+ T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4+ T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.

Early outcomes in youth with psychogenic nonsyncopal collapse.

OBJECTIVE: To evaluate several early outcome measures following diagnosis of psychogenic nonsyncopal collapse (PNSC). METHODS: Over a 34-month period, a prospective cohort study was conducted of patients referred for tilt-table evaluation of fainting and orthostatic intolerance. Clinical histories were obtained and anxiety and depressive symptom questionnaires were completed prior to testing. Among 539 patients referred, 100 (18.6%) were diagnosed with PNSC. Outcome data were collected by telephone or during routine follow-up a median of 572 days postdiagnosis. RESULTS: Eighty-four patients (84%) provided outcome data. Following communication of the diagnosis, 32 patients (38%) had immediate PNSC resolution. Attack resolution occurred in 44% by 1 month, 51% by 6 months, 52% by 12 months, 69% after 12 months, and 31% continued to have PNSC at the time of follow-up. Patients with continued PNSC had higher anxiety scores than patients with immediate resolution (p = 0.047). Following diagnosis, emergency department visits for fainting decreased from 78.6% to 20.2% (p = 0.017), and management by psychology or psychiatry increased from 26.2% to 76.2% (p < 0.001). During the follow-up period, 8 patients (9.5%) were hospitalized for suicidal ideation, a median of 253 (range 33-470) days postdiagnosis; 12 patients (14.3%) developed new (non-PNSC) conversion disorders, a median of 86 (range 9-504) days postdiagnosis. Suicidal ideation was associated with higher anxiety (p = 0.007) but not higher depression scores. CONCLUSIONS: The diagnostic rate of PNSC parallels that of PNES among patients referred for tertiary care evaluations. The improvements in attack frequency following PNSC diagnosis must be tempered by the potential risks of self-harm and the development of new conversion disorders.

Pointing perception is precise.

The spontaneity and ease with which we point understates the gesture's significance to understanding cognition. Onset of pointing in infancy predicts early word acquisition and signals a capacity for shared intentionality. Yet, notwithstanding its importance, there is little research on the perception of pointing and its referents. Here we show that perceptual acuity for discerning where another person is pointing is remarkably accurate. Thresholds, as low as 0.5° of visual angle across an interpersonal distance of ∼2 m, are modulated by the referent's location in space and the hand used to point and remain constant when the pointer's eyes are occluded from view and when 'embodiment' cues are enhanced or minimized. Pointing with the index finger not only directs attention toward a general region of space but the morphology of arm, hand and finger can be used to discern the location of the pointer's attention with precision.

Identifying Aß-specific pathogenic mechanisms using a nematode model of Alzheimer's disease.

Multiple gene expression alterations have been linked to Alzheimer's disease (AD), implicating multiple metabolic pathways in its pathogenesis. However, a clear distinction between AD-specific gene expression changes and those resulting from nonspecific responses to toxic aggregating proteins has not been made. We investigated alterations in gene expression induced by human beta-amyloid peptide (Aß) in a Caenorhabditis elegans AD model. Aß-induced gene expression alterations were compared with those caused by a synthetic aggregating protein to identify Aß-specific effects. Both Aß-specific and nonspecific alterations were observed. Among Aß-specific genes were those involved in aging, proteasome function, and mitochondrial function. An intriguing observation was the significant overlap between gene expression changes induced by Aß and those induced by Cry5B, a bacterial pore-forming toxin. This led us to hypothesize that Aß exerts its toxic effect, at least in part, by causing damage to biological membranes. We provide in vivo evidence consistent with this hypothesis. This study distinguishes between Aß-specific and nonspecific mechanisms and provides potential targets for therapeutics discovery.

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.

Surface kinetics for cooperative fungal cellulase digestion of cellulose from quartz crystal microgravimetry.

The kinetic behavior of aqueous cellulase on insoluble cellulose is best quantified through surface-based assays on a well-defined cellulose substrate of known area. We use a quartz crystal microbalance (QCM) to measure the activity of binary mixtures of Trichoderma longibrachiatum cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) on spin-coated cellulose films. By extending a previous surface kinetic model for cellulase activity, we obtain rate constants for competitive adsorption of Cel7A and Cel7B, their irreversible binding, their complexation with the cellulose surface, and their cooperative cellulolytic activity. The activity of the two cellulases is linked through the formation of cellulose chain ends by Cel7B that provide complexation sites from which Cel7A effects cellulose chain scission. Although the rate-limiting step in Cel7A activity is complexation, Cel7B activity is limited by adsorption to the cellulose surface. A 2:1 bulk mass ratio of aqueous Cel7A:Cel7B, corresponding to a 4:1 surface mass ratio, effects the greatest rate of cellulose degradation across a range of cellulase concentrations at 25 °C. We find that surface chain-end concentration is a major predictor of Cel7A activity. Disruption of the hydrogen-bonding structure of cellulose by Cel7B enhances the activity of Cel7A on the cellulose surface.

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

Risk factors for preterm delivery in patients demonstrating sonographic evidence of premature dilation of the internal os, prolapse of the membranes in the endocervical canal and shortening of the distal cervical segment by second trimester ultrasound.

OBJECTIVE: To investigate the outcomes and risk factors associated with the second trimester sonographic findings of premature dilation of the internal cervical os, prolapse of the membranes into the endocervical canal and shortening of the distal segment. STUDY DESIGN: Retrospective chart review to identify patients who met the following criteria: (i) gestational age between 16 and 24 weeks; (ii) a vaginal probe ultrasound documenting dilatation of the internal os, prolapsed membranes into the endocervical canal but not extending beyond the external os, and a shortened distal cervix; and (iii) no evidence of increased uterine activity METHODS: Sonographic measurements obtained included: (i) the width of the internal os dilation; (ii) the depth of membranes prolapsed into endocervix (funneling); (iii) the distal cervical length; and (iv) the total cervical length. Patients were stratified into two groups according to gestational age at delivery: the preterm group (< 34 weeks) and the near-term group (> or = 34 weeks). RESULTS: Thirty-seven patients had complete records for analysis and their pregnancies resulted in 47 live births. Eighteen patients were stratified to the near term group and 19 were stratified to the preterm group. There were no stillborn infants and 10 neonatal deaths. Ten patients received cerclage and seven patients delivered at < 34 weeks gestational age. Risk factors found to be significant for preterm delivery included a gestational age at diagnosis of < or = 20 weeks gestation (p = 0.03), dilatation of the internal os > 1.3 cm (p = 0.04), and a composite Benham score > or = 1.0 (p = 0.02). CONCLUSIONS: The sonographic findings of premature dilatation of the internal os, prolapse of the membranes into the endocervical canal and shortening of the distal cervix are associated with a high rate of delivery < 34 weeks (51%) and neonatal death (27%).

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

A double-blind, placebo-controlled crossover study investigating the effect of porcine secretin in children with autism.

OBJECTIVES: A recent patient series reported the incidental findings of improved social and language skills in 3 children with autistic spectrum disorders after the administration of secretin, a peptide hormone. However, a subsequent study did not find evidence for a drug effect. Parents are seeking treatment with secretin despite the absence of empirical investigations demonstrating amelioration in autism symptomology. In order to more precisely measure the effects of secretin, this study investigated the effect of a single intravenous dose of porcine secretin on 12 autistic children through a randomized, double-blind, placebo-controlled, crossover study. Children were assessed on objective language and on social, neuropsychological, and gastrointestinal measures to evaluate drug effects. The study was conducted over a 16-week trial. The results indicated that significant differences were not observed on the majority of the dependent variables. Statistically significant differences were observed on measures of positive affect and activity level following secretin infusion. In general, the autistic children did not demonstrate the improvements described in the initial retrospective report.

Identity matching of consonant-vowel-consonant words by prereaders.

Using an identity matching-to-sample procedure, normally developing prereaders who matched individual letters with high accuracy (e.g., m and s) did not show high accuracy in matching three-letter printed words that differed only in the first letter (e.g., mad and sad). Teachers and researchers should not assume that children who can discriminate individual letters can also discriminate minimally different words that contain those letters.

Emergent symbolic relations in speakers and nonspeakers.

Eight adults with developmental disabilities and four typically developing preschool children participated in an experiment studying emergent stimulus-stimulus relations after match-to-sample instruction. Participants were taught to match lexigrams (arbitrary graphic stimuli) to objects. Each object was functionally related to another object in the teaching set. For example, brush and comb were members of the teaching set. Most participants were able to select objects when the objects were named in pretests. Postteaching probes assessed emergent relations between spoken names and lexigrams. In addition, we included probes to detect emergent relations between related lexigrams. That is, would participants select the lexigram for comb when the experimenter displayed the lexigram for brush (without explicit teaching)? Two preschool children and five adults with developmental disabilities showed emergent relations between lexigrams and spoken words. However, only two preschool children showed the emergent lexigram to related lexigram relations.

Factors influencing single mother's employment status.

Changes in the welfare system limit the length of time a person can receive welfare benefits, thus mandating employment for many current welfare recipients. Single mothers with young children who do not become employed will lose financial support for housing, food, clothing, and health care and place their own and their children's health and safety at risk. The purpose of this qualitative study was to explore women's experiences of being unemployed and to examine the barriers to employment perceived by single mothers who expressed a desire to be employed. Nine mothers were recruited from a larger sample of single mothers who had participated in a quantitative study about employment conducted 1 to 2 years earlier. Using focus group interviews, mothers were asked what it was like to be a single mother, and then what barriers to their employment they perceived. Two dimensions were identified from the mothers' statements. The first, a sense of obligation, included themes of "being there" for their own and their child's benefit and doing what it takes to optimize the child's growth and development. The second, negotiating the obstacles, referred to problems regarding child care, lack of involvement of the child's father and lack of support from relatives and friends for the mother's efforts toward securing employment. These findings have important implications for welfare reform, namely, that efforts aimed at moving nonemployed single mothers into the workforce will fail if these factors are not considered.

Local contrast in natural images: normalisation and coding efficiency.

The visual system employs a gain control mechanism in the cortical coding of contrast whereby the response of each cell is normalised by the integrated activity of neighbouring cells. While restricted in space, the normalisation pool is broadly tuned for spatial frequency and orientation, so that a cell's response is adapted by stimuli which fall outside its 'classical' receptive field. Various functions have been attributed to divisive gain control: in this paper we consider whether this output nonlinearity serves to increase the information carrying capacity of the neural code. 46 natural scenes were analysed with the use of oriented, frequency-tuned filters whose bandwidths were chosen to match those of mammalian striate cortical cells. The images were logarithmically transformed so that the filters responded to a luminance ratio or contrast. In the first study, the response of each filter was calibrated relative to its response to a grating stimulus, and local image contrast was expressed in terms of the familiar Michelson metric. We found that the distribution of contrasts in natural images is highly kurtotic, peaking at low values and having a long exponential tail. There is considerable variability in local contrast, both within and between images. In the second study we compared the distribution of response activity before and after implementing contrast normalisation, and noted two major changes. Response variability, both within and between scenes, is reduced by normalisation, and the entropy of the response distribution is increased after normalisation, indicating a more efficient transfer of information.

Communication forms and functions of children and adults with severe mental retardation in community and institutional settings.

The forms and functions of expressive communication produced by 84 individuals with severe mental retardation were assessed, using a structured communication sampling procedure. Symbolic communication acts were produced by 39 participants, and 27 of these symbolic communicators produced one or more multiword/multisymbol utterances. Of the remaining participants, 38 produced intentional but nonsymbolic communication acts; 7 were not observed to produce any intentional communication. For all participants who produced intentional communication, there were significantly more imperative than declarative communication acts. Significant differences in the frequencies and functions of communication acts produced by these participants were associated with differences in their communication levels (contact gesture, distal gesture, or symbolic), age (child vs. adult), and residential status (community home vs. large facility).

Simultaneous and delayed matching to sample in gesture users and speakers with mental retardation.

Sixty-eight participants with severe mental retardation participated in a study of representational matching to sample. Participants were asked to match objects to identical objects, line drawings, miniature objects (icons), pantomimes, and spoken names. Participants who were successful in these matching tasks also experienced delayed matching tasks. Participants differed in their expressive communication and comparisons were made between symbolic (speaking) individuals, distal gesture users, and contact gesture users. Contact gesture users were significantly worse on identical matching to sample tasks than other participants. Mean scores on matching objects to line drawings were significantly better than mean scores on other matching tasks. In delayed matching, however, scores for matching objects to spoken names were significantly better than other tasks. The implications of these results for learning to use an augmentative communication device are discussed.

Is human motion detection subserved by a single or multiple channel mechanism?

Two recent versions of a single channel model of motion perception have had impressive success in explaining direction discrimination by human observers for spatially filtered noise images in two-flash apparent motion. It has been argued that the dramatic breakdown in motion perception which occurs when one image in the two-flash sequence is low-pass filtered can be explained only by a single channel model. We show that neither version of the single channel model which has been proposed can explain performance for noise images chosen to provide comparable stimulation in the spatial channels known to subserve human vision. A multi-channel model of motion perception has little difficulty in explaining these results.