Reverse engineering field-derived vertical distribution profiles to infer larval swimming behaviors.

Biophysical models are well-used tools for predicting the dispersal of marine larvae. Larval behavior has been shown to influence dispersal, but how to incorporate behavior effectively within dispersal models remains a challenge. Mechanisms of behavior are often derived from laboratory-based studies and therefore, may not reflect behavior in situ. Here, using state-of-the-art models, we explore the movements that larvae must undertake to achieve the vertical distribution patterns observed in nature. Results suggest that behaviors are not consistent with those described under the tidally synchronized vertical migration (TVM) hypothesis. Instead, we show (i) a need for swimming speed and direction to vary over the tidal cycle and (ii) that, in some instances, larval swimming cannot explain observed vertical patterns. We argue that current methods of behavioral parameterization are limited in their capacity to replicate in situ observations of vertical distribution, which may cause dispersal error to propagate over time, due to advective differences over depth and demonstrate an alternative to laboratory-based behavioral parameterization that encompasses the range of environmental cues that may be acting on planktic organisms.

Designing a survey to monitor multi-scale impacts of agri-environment schemes on mobile taxa.

Agri-environment schemes (AES) are key mechanisms to deliver conservation policy, and include management to provide resources for target taxa. Mobile species may move to areas where resources are increased, without this necessarily having an effect across the wider countryside or on populations over time. Most assessments of AES efficacy have been at small spatial scales, over short timescales, and shown varying results. We developed a survey design based on orthogonal gradients of AES management at local and landscape scales, which will enable the response of several taxa to be monitored. An evidence review of management effects on butterflies, birds and pollinating insects provided data to score AES options. Predicted gradients were calculated using AES uptake, weighted by the evidence scores. Predicted AES gradients for each taxon correlated strongly, and with the average gradient across taxa, supporting the co-location of surveys across different taxa. Nine 1 × 1 km survey squares were selected in each of four regional blocks with broadly homogenous background habitat characteristics. Squares in each block covered orthogonal contrasts across the range of AES gradients at local and landscape scales. This allows the effects of AES on species at each scale, and the interaction between scales, to be tested. AES options and broad habitats were mapped in field surveys, to verify predicted gradients which were based on AES option uptake data. The verified AES gradient had a strong positive relationship with the predicted gradient. AES gradients were broadly independent of background habitat within each block, likely allowing AES effects to be distinguished from potential effects of other habitat variables. Surveys of several mobile taxa are ongoing. This design will allow mobile taxa responses to AES to be tested in the surrounding countryside, as well as on land under AES management, and potentially in terms of population change over time. The design developed here provides a novel, pseudo-experimental approach for assessing the response of mobile species to gradients of management at two spatial scales. A similar design process could be applied in other regions that require a standardized approach to monitoring the impacts of management interventions on target taxa at landscape scales, if equivalent spatial data are available.

Prospective evaluation of treatment response and disease reversibility of paediatric localized scleroderma (morphoea) to steroids and methotrexate using multi-modal imaging.

BACKGROUND: Paediatric localized scleroderma is a severe inflammatory disorder associated with tissue atrophy, often leading to disability. Assessing disease activity and response to treatment has always been challenging and remains an important difficulty in clinical practice. OBJECTIVES: To investigate prospectively the efficacy of systemic treatment with corticosteroids and methotrexate in children with localized scleroderma and the validity of infrared thermography, laser Doppler flowmetry and high-frequency ultrasound in assessing disease activity. METHODS: Children with localized scleroderma were prospectively treated with corticosteroids (initially pulsed IV methylprednisolone 30 mg/kg/day, maximum 500 mg/day and/or oral prednisolone 0.5-1 mg/kg/day) and methotrexate (15 mg/m2 weekly). Treatment response was evaluated using a clinical activity score. Skin temperature, blood flow, dermal thickness and dermal echogenicity of clinically active skin lesions were determined in relation to the unaffected contralateral site at baseline and after 3, 6, 12 and 18 months. Patient charts were later reviewed for long-term follow-up. RESULTS: Twenty-two patients were included [age 6.0 (0.2-14.4] years; female-to-male ratio 3.4 : 1) All responded well to therapy. Disease reversibility was demonstrated in the majority of children with partial resolution of skin sclerosis and regrowth of hair. Laser Doppler flowmetry and high-frequency ultrasound findings correlated with disease activity at baseline. Thermography had no added value in this cohort. The recurrence rate was 36% in the follow-up period. CONCLUSIONS: Corticosteroids and methotrexate are highly effective as first-line therapy in paediatric localized scleroderma, leading to partial reversal of skin manifestations. However, the recurrence rate is substantial and affected children require long-term follow-up. Laser Doppler flowmetry and high-frequency ultrasound correlate with disease activity in the acute phase and may assist decision-making in these patients.

Defined co-cultures of yeast and bacteria modify the aroma, crumb and sensory properties of bread.

AIMS: Yeast and bacterial communities inhabit a sourdough starter to make artisanal bread. This study shows whether the interactions of micro-organisms derived from Australian sourdough starters provide some of the positive flavour, and aroma properties to bread by using defined sourdough cultures as the sole leaven in bread production. METHODS AND RESULTS: An investigation of Australian sourdough starters found that they contained Saccharomyces cerevisiae and Kazachstania exigua yeasts. When these yeasts were inoculated alone to ferment wheat flour in an extended fermentation, the bread had a heterogeneous crumb structure, a deeper colour and a distinctive chemical aroma profile than those made with commercial baker's yeast. When bread was made combining these yeasts individually and in combinations with lactic acid bacteria also isolated from these sourdough starters, including Lactobacillus plantarum, L. brevis, L. rossiae, L. casei, the bread aroma profiles and crumb structure were more distinctive, with compounds associated with sour aromas produced, and preferred by sensory panels. CONCLUSIONS: The use of defined mixed cultures as the leaven in bread making, by exploiting the microbial diversity of artisanal Australian starters, can produce bread with distinctive and attractive aromas. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding and identifying the community ecosystems found in sourdough cultures and using them as the sole leaven in bread production provide novel insights into microbial interactions and how they affect food quality by removing the effects of commercial yeast strains.

Association of serum VEGF levels with prefrontal cortex volume in schizophrenia.

A large body of evidence indicates alterations in brain regional cellular energy metabolism and blood flow in schizophrenia. Among the different molecules regulating blood flow, vascular endothelial growth factor (VEGF) is generally accepted as the major factor involved in the process of angiogenesis. In the present study, we examined whether peripheral VEGF levels correlate with changes in the prefrontal cortex (PFC) volume in patients with schizophrenia and in healthy controls. Whole-blood samples were obtained from 96 people with schizophrenia or schizoaffective disorder and 83 healthy controls. Serum VEGF protein levels were analyzed by enzyme-linked immunosorbent assay, whereas quantitative PCR was performed to measure interleukin-6 (IL-6, a pro-inflammatory marker implicated in schizophrenia) mRNA levels in the blood samples. Structural magnetic resonance imaging scans were obtained using a 3T Achieva scanner on a subset of 59 people with schizophrenia or schizoaffective disorder and 65 healthy controls, and prefrontal volumes were obtained using FreeSurfer software. As compared with healthy controls, individuals with schizophrenia had a significant increase in log-transformed mean serum VEGF levels (t(177)=2.9, P=0.005). A significant inverse correlation (r=-0.40, P=0.002) between serum VEGF and total frontal pole volume was found in patients with schizophrenia/schizoaffective disorder. Moreover, we observed a significant positive association (r=0.24, P=0.03) between serum VEGF and IL-6 mRNA levels in patients with schizophrenia. These findings suggest an association between serum VEGF and inflammation, and that serum VEGF levels are related to structural abnormalities in the PFC of people with schizophrenia.

Complexity and health functionality of plant cell wall fibers from fruits and vegetables.

The prevalence of lifestyle-related diseases is increasing in developing countries with the causes for death starting to follow the same pattern in the developed world. Lifestyle factors including inadequate dietary intake of fruits and vegetables and over consumption of nutrient-poor processed foods, are considered to be major causal risk factors associated with increased susceptibility to developing certain diseases (Alldrick, 1998 ; Kiani, 2007 ). Recent epidemiological evidence confirms a strong association between dietary fiber and reduced all-cause mortality risk, as well as a risk reduction for a number of non-communicable diseases (Chuang et al., 2012 ). The relationship between dietary fiber and mortality has been described as "convincing observations that call for mechanistic investigations" (Landberg, 2012 ). In particular, the health protective roles played by dietary fibers of different origin are not well understood. Whilst Hippocrates was the earliest known physician to study the health benefits of fiber derived from grains (Burkitt, 1987 ), the functionality of fruit and vegetable fiber, especially in association with other compounds such as polyphenols and carotenoids, is an area of more recent interest. Hence the objective of this review is to assess the complexity and health-related functional role of plant cell wall (PCW) fibers from fruits and vegetables with a particular emphasis on interactions between cell walls and phytonutrients.

The effect of training on infrared thermographic images of the forelimb and hindlimb joints of healthy racehorses.

The aim of this study was to evaluate the influence of training on body surface temperature over the joints in racehorses, measured by infrared thermography. The study involved monitoring of 14 Thoroughbred racehorses in 6 imaging sessions over a period of 3 months. Temperature measurements of the forelimb and hindlimb joints were made before and just after training. Joint temperature of limbs increased significantly after training. Environmental temperature had a statistically significant influence on surface temperature over the joints. The lowest surface temperatures were recorded over the metacarpophalangeal and metatarsophalangeal joint and the highest temperatures in the shoulder, elbow, hip and stifle joint. The metacarpophalangeal and metatarsophalangeal joints warmed the least during training, but were influenced the most by differences in environmental temperature. The surface temperature difference before and after training is an important indicator of the thermoregulatory response to exercise in racing horses. Understanding surface temperature changes in response to regular training is necessary for future studies on diagnosing injuries of joints.

Prevalence of iatrogenic heart murmurs in a population of apparently healthy cats.

OBJECTIVES: To determine the prevalence of iatrogenic heart murmurs in a population of apparently healthy cats and to investigate factors that may predispose cats to iatrogenic murmurs. MATERIALS AND METHODS: Apparently healthy cats presenting for routine examinations were prospectively enrolled. Following a physical examination to confirm there were no abnormalities, a cardiac examination was performed before and during a provocative manoeuvre performed independently by two clinicians. The provocative manoeuvre was performed on the right side of the chest wall by applying gentle pressure with a stethoscope to ascertain whether a murmur became audible, graded using a modified descriptive scale (soft/moderate). The cat's heart rate before and during the provocative manoeuvre and their age, sex, body condition score and bodyweight were recorded. RESULTS: One hundred and six cats were prospectively enrolled. The average prevalence of iatrogenic murmurs was 28.2% with good interobserver agreement recorded on both the presence (kappa 0.67 and 0.62) and grade of the murmur (kappa 0.6 and 0.53). A significant association was noted between age and body condition score, with iatrogenic murmurs more likely in cats with increasing age and decreasing body condition score. There was no association between heart rate and the presence of an iatrogenic murmur. CLINICAL SIGNIFICANCE: Iatrogenic murmurs are a common finding in cats with increasing prevalence noted in older and lean cats. Iatrogenic murmurs should therefore be considered as a differential diagnosis in a cat when an audible soft systolic right-sided murmur occurs with gentle pressure application to the chest wall during auscultation.

Relationship between maximum eye temperature and plasma cortisol concentration in racehorses during intensive training.

The aim of the study was to determine the utility of maximum eye temperature measured by infrared thermography (IRT) as a stress indicator compared with plasma cortisol concentration in Thoroughbred and Arabian racehorses. The study included thirty racehorses undergoing standard training for racing. Measurements of maximum eye temperature and blood collection for plasma cortisol concentration were carried out before training (BT), and within 5 (5AT) and 120 minutes (120AT) after the end of the each training session in three repetitions, with a monthly interval. Both parameters were elevated at 5AT compared to BT (p⟨0.001). Compared to BT, at 120AT the maximum eye temperature remained elevated (p⟨0.001) and plasma cortisol concentration decreased (p⟨0.001). The study indicated significant weak correlations (r=0.220; p⟨0.001) between both measurements at all time points. The results support the use of IRT technique to monitor the response of horses to stress, potentially improving animal management and welfare.

Heterocyclic acetamide and benzamide derivatives as potent and selective beta3-adrenergic receptor agonists with improved rodent pharmacokinetic profiles.

A series of amide derived beta(3)-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation beta(3)-AR agonists for the treatment of overactive bladder.

Vesicle budding: insights from cell-free assays.

Transfer of proteins and lipids between the various membrane-bound subcellular compartments of the eukaryotic cell is mediated by transport vesicles. The development of cell-free assays has allowed rapid progress towards a molecular description of the formation, or budding, of these vesicles. This article reviews and integrates data obtained from various yeast and mammalian systems on molecules involved in the budding reaction.

Targeting and fusion in vesicular transport.

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.

TGN38/41: a molecule on the move.

TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN.

Phosphatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the TGN.

An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro.

We have investigated two aspects of membrane traffic at early stages of endocytosis: membrane fusion and microtubule-dependent transport. As a marker, we have used the trans-membrane glycoprotein G of vesicular stomatitis virus implanted into the plasma membrane and then internalized for different times at 37 degrees C. The corresponding endosomal fractions were immunoisolated using the cytoplasmic domain of the G protein as antigen. These fractions were then used in an in vitro assay to quantify the efficiency of fusion between endosomal vesicles. To identify the vesicular partners of the fusion, these in vitro studies were combined with in vivo biochemical and morphological experiments. Internalized molecules were delivered to early endosomal elements, which corresponded to a network of tubular and tubulovesicular structures. Rapid recycling back to the plasma membrane and routing to late stages of the pathway occurred from these early endosomal elements. These elements exhibited a high and specific fusion activity with each other in vitro, suggesting that individual elements of the early endosomal compartment interact with each other in vivo. After their appearance in the early endosome, the molecules destined to be degraded were observed at the next stage of the pathway in distinct spherical vesicles (0.5 micron diam) and then in late endosomes and lysosomes. When the microtubules were depolymerized with nocodazole, endocytosis proceeded as in control cells. However, internalized molecules remained in the spherical vesicles and did not appear in late endosomes or lysosomes. These spherical vesicles had relatively little fusion activity with each other or with early endosomal elements in vitro. Our observations suggest that the spherical vesicles mediate transport between the early endosome and late endosomes and that this process requires intact microtubules.

Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean?

NADPH cytochrome c (cyt c) reductase and glucose-6-phosphatase, two enzymes thought to be restricted to the endoplasmic reticulum (ER) and widely used as ER markers, are present in isolated Golgi fractions assayed immediately after their isolation. Both enzymes are rapidly inactivated in fractions stored at 0 degrees C in 0.25 M sucrose, conditions which do not affect the activity of other enzymes in the same preparation. The inactivation process was shown to be dependent on time and protein concentration and could be prevented by EDTA and catalase. Morphological evidence shows that extensive membrane damage occurs parallel with the inactivation. Taken together with the immunological data in the companion paper, the findings indicate that the enzymes NADPH cyt c reductase and probably glucose-6-phosphate are indigenous components of Golgi membranes.

Hepatic Golgi fractions resolved into membrane and content subfractions.

Golgi fractions isolated from rat liver homogenates have been resolved into membrane and content subfractions by treatment with 100 mM Na2CO3 pH 11.3. This procedure permitted extensive extraction of content proteins and lipoproteins, presumably because it caused an alteration of Golgi membranes that minimized the reformation of closed vesicles. The type and degree of contamination of the fractions was assessed by electron microscopy and biochemical assays. The membrane subfraction retained 15% of content proteins and lipids, and these could not be removed by various washing procedures. The content subfraction was contaminated by both membrane fragments and vesicles and accounted for 5 to 10% of the membrane enzyme activities of the original Golgi fraction. The lipid compositions of the subfractions was determined, and the phospholipids of both membrane and content were found to be uniformly labeled with [33P]phosphate administered in vivo.

Heterogeneity of lipoprotein particles in hepatic Golgi fractions.

Newly synthesized phospholipids, labeled with either [14C]choline, [3H]myo-inositol, or [33P]phosphate, partioned preferentially (greater than 80% of total incorporated radioactivity) in a Golgi membrane subfraction, although the cognate content subfraction contained a relatively large amount of secretory lipoproteins. The labeling pattern was the same for all phospholipids tested in the two subfractions. An active exchange process of polar lipids between Golgi membranes and Golgi secretory lipoproteins is postulated as a plausible explanation for these findings. Less than half of all Golgi lipoprotein particles have the density of serum VLDLs and a similar, but not identical, biochemical composition. The remaining lipoprotein particles are characterized by a continuous spectrum of sizes, and (to the extent tested) by a lipid and protein composition different from that of serum VLDLs and HDLs. Results obtained in control experiments rule out the possibility that the heterogeneous population of Golgi lipoprotein particles is an artefact caused by our preparation procedures. It is assumed that these heterogeneous particles are immature precursors of both VLDLs and HDLs.

Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms.

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.

Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.