Absorptive transport of amino acids by the rat colon.

The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption

Electrocortical Evidence for Impaired Affective Picture Processing after Long-Term Immobilization

The neurobehavioral risks associated with spaceflight are not well understood. In particular, little attention has been paid on the role of resilience, social processes and emotion regulation during long-duration spaceflight. Bed rest is a well-established spaceflight analogue that combines the adaptations associated with physical inactivity and semi-isolation and confinement. We here investigated the effects of 30 days of 6 degrees head-down tilt bed rest on affective picture processing using event-related potentials (ERP) in healthy men. Compared to a control group, bed rest participants showed significantly decreased P300 and LPP amplitudes to pleasant and unpleasant stimuli, especially in centroparietal regions, after 30 days of bed rest. Source localization revealed a bilateral lower activity in the posterior cingulate gyrus, insula and precuneus in the bed rest group in both ERP time frames for emotional, but not neutral stimuli

Circadian Preference Modulates the Neural Substrate of Conflict Processing across the Day

Human morning and evening chronotypes differ in their preferred timing for sleep and wakefulness, as well as in optimal daytime periods to cope with cognitive challenges. Recent evidence suggests that these preferences are not a simple by-product of socio-professional timing constraints, but can be driven by inter-individual differences in the expression of circadian and homeostatic sleep-wake promoting signals. Chronotypes thus constitute a unique tool to access the interplay between those processes under normally entrained day-night conditions, and to investigate how they impinge onto higher cognitive control processes. Using functional magnetic resonance imaging (fMRI), we assessed the influence of chronotype and time-of-day on conflict processing-related cerebral activity throughout a normal waking day. Sixteen morning and 15 evening types were recorded at two individually adapted time points (1.5 versus 10.5 hours spent awake) while performing the Stroop paradigm. Results show that interference-related hemodynamic responses are maintained or even increased in evening types from the subjective morning to the subjective evening in a set of brain areas playing a pivotal role in successful inhibitory functioning, whereas they decreased in morning types under the same conditions. Furthermore, during the evening hours, activity in a posterior hypothalamic region putatively involved in sleep-wake regulation correlated in a chronotype-specific manner with slow wave activity at the beginning of the night, an index of accumulated homeostatic sleep pressure. These results shed light into the cerebral mechanisms underlying inter-individual differences of higher-order cognitive state maintenance under normally entrained day-night conditions

Non-Visual Effects of Light on Melatonin, Alertness and Cognitive Performance: Can Blue-Enriched Light Keep Us Alert?

Light exposure can cascade numerous effects on the human circadian process via the non-imaging forming system, whose spectral relevance is highest in the short-wavelength range. Here we investigated if commercially available compact fluorescent lamps with different colour temperatures can impact on alertness and cognitive performance

Hypertrophic cardiomyopathy is characterized by alterations of the mitochondrial calcium uniporter complex proteins: insights from patients with aortic valve stenosis versus hypertrophic obstructive cardiomyopathy

Introduction: Hypertrophies of the cardiac septum are caused either by aortic valve stenosis (AVS) or by congenital hypertrophic obstructive cardiomyopathy (HOCM). As they induce cardiac remodeling, these cardiac pathologies may promote an arrhythmogenic substrate with associated malignant ventricular arrhythmias and may lead to heart failure. While altered calcium (Ca2+) handling seems to be a key player in the pathogenesis, the role of mitochondrial calcium handling was not investigated in these patients to date.Methods: To investigate this issue, cardiac septal samples were collected from patients undergoing myectomy during cardiac surgery for excessive septal hypertrophy and/or aortic valve replacement, caused by AVS and HOCM. Septal specimens were matched with cardiac tissue obtained from post-mortem controls without cardiac diseases (Ctrl).Results and discussion: Patient characteristics and most of the echocardiographic parameters did not differ between AVS and HOCM. Most notably, the interventricular septum thickness, diastolic (IVSd), was the greatest in HOCM patients. Histological and molecular analyses showed a trend towards higher fibrotic burden in both pathologies, when compared to Ctrl. Most notably, the mitochondrial Ca2+ uniporter (MCU) complex associated proteins were altered in both pathologies of left ventricular hypertrophy (LVH). On the one hand, the expression pattern of the MCU complex subunits MCU and MICU1 were shown to be markedly increased, especially in AVS. On the other hand, PRMT-1, UCP-2, and UCP-3 declined with hypertrophy. These conditions were associated with an increase in the expression patterns of the Ca2+ uptaking ion channel SERCA2a in AVS (p = 0.0013), though not in HOCM, compared to healthy tissue. Our data obtained from human specimen from AVS or HOCM indicates major alterations in the expression of the mitochondrial calcium uniporter complex and associated proteins. Thus, in cardiac septal hypertrophies, besides modifications of cytosolic calcium handling, impaired mitochondrial uptake might be a key player in disease progression

CTL Responses of High Functional Avidity and Broad Variant Cross-Reactivity Are Associated with HIV Control

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication in vivo. However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct ex-vivo samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control

Validierung eines Protokolls zur manuellen, mutationsspezifischen BRAF V600E Immunhistochemie

BRAF ist die B-Isoform der RAF Kinase, welche Teil des MAP-Kinase-Signalwegs ist und die Zellproliferation reguliert. Sie ist als Protoonkogen anfällig für Mutationen, die zur Entstehung von Malignomen führen können. Über 95% der Mutationen des BRAF Gens macht die BRAF V600E Mutation aus. Sie lässt sich in verschiedensten humanen Krebsarten nachweisen und ist eine der häufigsten Mutationen in Malig-nem Melanom (44%), papillärem Schilddrüsenkarzinom (52%) und Klassischer Haarzellleukämie (100%). Wird sie nachgewiesen, muss man davon ausgehen, dass sie teil- oder möglicherweise sogar hauptverantwortlich für die Entstehung des Malignoms ist, und eine zielgerichtete Therapie (targeted therapy) mit einem Inhi-bitor gegen das mutierte Protein ist zu erwägen. Häufig stoppt eine solche Behand-lung den Tumorprogress und es gibt eindeutige Evidenz, dass dadurch die Mittlere, sowie die Gesamtüberlebensrate signifikant erhöht werden können. Im Focus der Diskussion um die geeignete BRAF Diagnostik steht neben sequenzanalytischen Verfahren auch der Mutationsnachweis auf Zellebene mittels Immunhistochemie. In der hier vorgelegten Arbeit wurde erstmals gezeigt, wie man BRAF V600E manuell mittels Immunhistochemie nachweisen kann, und es wurde anhand einer Kohorte aus 33 Fällen erörtert, wie sensitiv und spezifisch dieses Verfahren im Vergleich zu bereits etablierten immunhistochemischen Protokollen abeitet, die allerdings nur für automatisierte Färbesysteme publiziert sind. Ferner wurde die Methode mit Pyrose-quenzergebnissen der Kohorte verglichen, die aus der Klinikroutine der Uniklinik Ulm stammen. Die empirische Protokollentwicklung ergab, dass sich der diagnosti-sche BRAF V600E spezifische Antikörper am besten nach einer hitzeinduzierten Epitopdemaskierung in EDTA Puffer pH 8,0 im Dampfgarer und einer Detektion mit-tels Peroxidase sichtbar machen lässt. Dies gelingt auch bei formalinfixierten, in Paraffin eingebetteten Proben, die zuvor dekalzifiziert wurden. Zur Primärantikör-perinkubation sind Verdünnungen bis 1:100 möglich, ohne Verluste bei der Fär-bungsintensität hinnehmen zu müssen. In der anschließenden Validierung des Pro-tokolls mittels Kohortenstudie zeigte sich, dass sowohl die manuelle, als auch die in Heidelberg von der Arbeitsgruppe des Erstbeschreibers des Antikörpers durchge-führte automatisierte Färbung insgesamt mit 95%iger Sensitivität und 100%iger Spezifität sehr gut funktionierten, jedoch pro Methode einen mutierten Fall nicht kor-rekt klassifizieren konnten. Interessanterweise konnte aber auch die Pyrosequen-zierung in einem Fall keine Aussage zum Mutationsstatus der Probe machen und erreichte somit auch nur eine Sensitivität von 95% in der Studienkohorte. Da jede der Methoden einen anderen mutierten Fall nicht erkannt hat, und vor dem Hinter-grund der klinischen Relevanz einer möglichst sensitiven Diagnostik der Mutation, lautet die Schlussfolgerung dieser Arbeit, dass beide Verfahren (Immunhistochemie und Pyrosequenzierung) sequentiell und sich gegenseitig ergänzend eingesetzt werden sollten. Trotz guter Ergebnisse bei der BRAF V600E Detektion, muss man allerdings die Limitation der Immunhistochemie auf die V600E Mutation berücksich-tigen. Der zwar insgesamt geringe Anteil von unter 5% bzw. je nach Malignom auch bis zu 10% (Malignes Melanom) betragende Anteil anderer BRAF Mutationen als V600E, beispielsweise V600K, konnte mit dem hier verwendeten Antikörper nicht detektiert werden. Obwohl Fälle beschrieben sind, in denen eine Färbung anders mutierter BRAF Proteine auch mit dem V600E spezifischen Antikörper gelungen ist, können diese Mutationen, wie auch in dieser Studie, in der Regel nur mittels Se-quenzanalyse nachgewiesen werden. Und da Malignompatienten mit anderen BRAF Mutationen als V600E ebenfalls von den Inhibitoren Vemurafenib und Da-brafenib profitieren können, bleibt für die sichere Identifikation in diesen seltenen Fällen bisher nur die Sequenzanalyse übrig

Nominal logistic regression analysis of variables determining needle visibility in ultrasound images – a full factorial cadaver study

Needle visualization is essential to avoid vascular puncture and nerve injury in ultrasound-guided regional anesthesia. Several factors that statistically influence needle visibility have been described but the dimensions of their individual impact remain unclear. This study aimed to quantify the impact of various independent factors on ultrasound needle visibility. total of 1500 ultrasound videos of in-plane needle insertions were obtained in embalmed cadavers with ten different commercially available echogenic and non-echogenic needles at different insertion angles and bevel orientations in a full factorial study design. The visibility of needle tip and shaft were rated as “good” or “poor” visibility. Nominal logistic regression analyses were calculated for the visibility of the needle tip and shaft. SonoPlex Stim Sprotte, SonoTAP Facet (needle tip and shaft) and Spinostar PencilPoint (needle tip)), insertion angle and bevel orientation were associated with good ultrasound visibility, reaching statistical significance (p [ 0.05). The range of the effect on the log-odds scale for needle tip visibility was largest for the insertion angle with 6.33, followed by the tissue condition (3.76), bevel orientation (1.45) and the needle types (1.25). Regarding the needle shaft visibility, the largest effect range was observed with the insertion angle (7.36), followed by the tissue conditions with 3.96, needle type (1.86) and bevel orientation (0.95). In-plane needle visibility in ultrasound images depends mainly on the insertion angle, as expected. This is closely followed by the tissue condition, which is a factor related to the patient, thus cannot be altered to improve needle visibility. In the dimensions of the log-odds scale, the choice of a specific needle is far less important towards achieving a good visualization, whereas optimizing the bevel orientation can have a larger impact than the needle choice. Concluding from the relative dimensions of factors that determine needle visibility in this model, the importance of needles with echogenic features may be overrated

1H NMR-Based Identification of Intestinally Absorbed Metabolites by Ussing Chamber Analysis of the Rat Cecum.

The large intestine (cecum and colon) is a complex biochemical factory of vital importance to human health. It plays a major role in digestion and absorption by salvaging nutrients from polysaccharides via fermentation initiated by the bacteria that comprise the gut microbiome. We hypothesize that the intestinal epithelium absorbs a limited number of luminal metabolites with bioactive potential while actively excluding those with toxic effects. To explore this concept, we combined 1H NMR detection with Ussing chamber measurements of absorptive transport by rat cecum. Numerous metabolites transported across the epithelium can be measured simultaneously by 1H NMR, a universal detector of organic compounds, alleviating the need for fluorescent or radiolabeled compounds. Our results demonstrate the utility of this approach to delineate the repertoire of fecal solutes that are selectively absorbed by the cecum and to determine their transport rates