Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1

Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization.     

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.     

Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma,Canary Islands)

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.     

Fluctuations in Neuronal Synchronization in Brain Activity Correlate with the Subjective Experience of Visual Recognition

The scientific study of subjective experience is a current major research area in the neurosciences. Coordination patterns of brain activity are being studied to address the question of how brain function relates to behaviour, and particularly methods to estimate neuronal synchronization can unravel the spatio-temporal dynamics of the transient formation of neuronal assemblies. We report here a biophysical correlate of subjective experience. Subjects visualised figures with different levels of noise, while their brain activity was recorded using magnetoencephalography (MEG), and reported the moment in time (corresponding to a noise level) of figure recognition, which varied between individuals, as well as the moment when they saw the figure more clearly, which was mostly common among the participants (thus less subjective). This latter moment is considered to represent psychophysical stochastic resonance (PSR). Fluctuations in neuronal synchronization, quantified using a diffusion coefficient, were lower in occipital cortex when subjects recognised the figure, for a certain noise level, but did not correlate with the moment of PSR. A different pattern was observed in frontal cortex, where lower values of the diffusion coefficient in neuronal synchronization was maintained from the moment of recognition to the moment of PSR. No specific pattern was found analysing signals from temporal or parietal cortical areas. These observations provide support for distinct synchronization patterns in different cortical areas, and represent another demonstration that the subjective, first-person perspective is accessible to scientific methods.      

Pulmonary blood flow distribution after lobar oleic acid injury: a PET study

We evaluated the importance of hypoxic vasoconstriction as a mechanism for pulmonary blood flow reduction during unilobar oleic acid lung injury in dogs. Pulmonary blood flow (PBF) and lung water were measured with positron emission tomography. Data from the injured left (LCL) and right (RCL) caudal lobes were compared in 23 dogs. Six dogs were used to demonstrate that endotoxin (15 micrograms/kg) prevents changes in regional PBF during selective hypoxic ventilation of the LCL. In 17 dogs, oleic acid (OA, 0.015 ml/kg) was injected into the LCL through a balloon-wedged pulmonary arterial catheter. Five dogs received OA only (control group), eight received endotoxin (15 mcg/kg) before OA was administered (endotoxin group), and four were treated with prostaglandin E1 (PGE1) after OA (PGE1 group). The base-line left-to-right PBF ratio (LCL/RCL PBF) was 1.01 +/- 0.11 (SD) for the control group and 1.07 +/- 0.16 for the PGE1 group. One minute after OA, LCL/RCL PBF feel significantly (0.32 +/- 0.15 and 0.32 +/- 0.13 for the control and PGE1 groups, respectively) before any significant increase in lung water was detected. In all 17 dogs that received OA, the LCL/RCL PBF remained severely reduced 60 min after OA compared with base-line values (0.41 +/- 0.15, 0.49 +/- 0.06, and 0.26 +/- 0.13 for the control, PGF1, and endotoxin groups, respectively) despite treatment with endotoxin or PGE1. Lung water measurements obtained 60 min after OA increased significantly (P less than 0.05) in the injured lobe (LCL) but not in the normal lobe (RCL) in all dog groups, whereas PBF to the LCL remained significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inflammation and oxygen free radical formation during pulmonary ischemia-reperfusion injury.

In a companion study, we showed that 2 h of warm unilateral lung ischemia followed by reperfusion resulted in bilateral tissue injury, indicated by increases in extravascular density (EVD) and permeability, measured as the pulmonary transcapillary escape rate (PTCER) for radiolabeled transferrin. EVD and PTCER measurements were obtained with the quantitative imaging technique of positron emission tomography (PET). In the current study, we evaluated this increase in EVD histologically and correlated EVD and PTCER with measurements of oxidant-reactive sulfhydryls (RSH) in plasma as a marker of oxygen free radical (OFR) formation. Histologically edema, leukocyte infiltration, and hemorrhage were all present on the ischemic side, but only after reperfusion, whereas only neutrophil infiltration was observed on the nonischemic side. Histology scores correlated with EVD (r = 0.81) and PTCER (r = 0.75), but permeability was abnormal at times even in the absence of neutrophil infiltration. Plasma RSH concentration from the ischemic lung decreased significantly (P less than 0.05) during pulmonary ischemia (i.e., before reperfusion) and returned to baseline on reperfusion. The degree of RSH oxidation did not correlate with the severity of injury as measured by PET or histology. Thus pulmonary ischemia-reperfusion injury is characterized by inflammation, hemorrhage, edema, and OFR formation. Injury occurred after reperfusion, not after ischemia alone. In addition, injury to the contralateral nonischemic lung suggests a neutrophil-independent circulating mediator of injury.     

Carriage of antibiotic-resistant bacteria in urban versus rural wild boars

The Western European population of wild boar (Sus scrofa) has increased its distribution over the past several decades, and some populations have colonized areas strongly influenced by human activity. Wild boars are known carriers of antibiotic-resistant bacteria acquired from the environment, and urban populations of wild boars may be more exposed than their rural counterparts. In this work, we compared the frequency of antibiotic resistance in indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium) isolated from urban wild boars with that from rural wild boars in NE Spain. We further assessed whether bacterial isolates from the urban wild boars had a higher probability of showing antibiotic resistance when their host was highly associated to urban features. Seventy-two and 100 bacterial isolates from urban and rural habitat, respectively, were screened for antibiotic resistance against a set of antibiotics (13 per bacterial species). We found a significantly higher frequency of E. faecium showing resistance to tetracycline (70.0% vs 36.4%) and high-level resistance to streptomycin (30.0% vs 4.5%) in urban wild boars compared to rural wild boars (p?<?0.05). E. faecalis was more frequently resistant to trimethoprim in urban than rural wild boars (33.3% vs 0.0%, p?<?0.05). In isolates from urban origin, 55.6% of the likelihood of detecting antibiotic resistance depended only on the bacterial species, being more likely in the enterococci than in E. coli. These results suggest that urban wild boars may be more exposed to certain antibiotic-resistant bacteria or antibiotic resistance genes that they may acquire from the urban environment, although implications are uncertain.     

Measures of entropy and complexity in altered states of consciousness

Quantification of complexity in neurophysiological signals has been studied using different methods, especially those from information or dynamical system theory. These studies have revealed a dependence on different states of consciousness, and in particular that wakefulness is characterized by a greater complexity of brain signals, perhaps due to the necessity for the brain to handle varied sensorimotor information. Thus, these frameworks are very useful in attempts to quantify cognitive states. We set out to analyze different types of signals obtained from scalp electroencephalography (EEG), intracranial EEG and magnetoencephalography recording in subjects during different states of consciousness: resting wakefulness, different sleep stages and epileptic seizures. The signals were analyzed using a statistical (permutation entropy) and a deterministic (permutation Lempel–Ziv complexity) analytical method. The results are presented in complexity versus entropy graphs, showing that the values of entropy and complexity of the signals tend to be greatest when the subjects are in fully alert states, falling in states with loss of awareness or consciousness. These findings were robust for all three types of recordings. We propose that the investigation of the structure of cognition using the frameworks of complexity will reveal mechanistic aspects of brain dynamics associated not only with altered states of consciousness but also with normal and pathological conditions.     

Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life

Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin + N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin + N-bcaa, N-insulin + L-bcaa, and L-insulin + N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice.