Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls

The labeling of targeting peptides with (99m)Tc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac-[(99m)Tc(CO)(3)](+) core for introducing (99m)Tc in biomolecules in general and in peptides in particular. The three coordination sites available in [(99m)Tc(OH(2))(3)(CO)(3)](+) can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed-ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.     

On the morphology of antennular sensory and attachment organs in cypris larvae of the deep‐sea vent/seep barnacles,Ashinkailepas and Neoverruca

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep‐sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution. J. Morphol. 277:594–602, 2016. © 2016 Wiley Periodicals, Inc.     

GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.     

Experimental acute hypoxia in healthy subjects: evaluation of systolic and diastolic function of the left ventricle at rest and during exercise using echocardiography

To clarify whether or not systolic and diastolic function of the human left ventricle (LV) were decreased during acute hypoxia, at rest and with exercise, 14 healthy male volunteers [age 25.9 (SD 3.0) years, height 182.9 (SD 7.1) cm, body mass 75.9 (SD 6.9)kg] were examined using M-mode and 2D-mode echocardiography to determine the systolic LV function as well as Doppler-echocardiography for the assessment of diastolic LV function on 2 separate test days. In random order, the subjects breathed either air on 1 day (N) or a gas mixture with reduced oxygen content on the other (H; oxygen fraction in inspired gas 0.14). Measurements on either day were made at rest, several times during incremental cycle exercise in a supine position (6-min increments of 50 W, maximal load 150 W) and in 6th min of recovery. Corresponding measurements during N and H were compared statistically. Arterial O₂ tension (P _aO₂) was normal on N-day. All subjects showed a marked acute hypoxia at rest [P _aO₂, 54.5 (SD 4.6) mmHg], during exercise and recovery on H-day. The latter was associated with tachycardia compared to N-day. All echocardiographic measurements at rest were within the limits of normal values on both test days. Ejection time, end-systolic and end-diastolic left ventricular dimensions as well as the thickness of left posterior wall and of interventricular septum showed no statistically significant influence of H either at rest or during exercise. Stroke volume and cardiac output were always higher on H-day, which could be attributed to a slight reduction in end-systolic volume with unaffected end-diastolic volume as well as to increased heart rates. Among the indices of systolic LV function the fractions of thickening in the left ventricular posterior wall and interventricular septum showed no differences between H and N at rest or during exercise. However, fibre shortening, ejection fraction and mean circumferential fibre shortening were increased on H-day on all occasions. The mitral-valve-Doppler ratio, the index of diastolic LV function, was decreased with H at rest, showed a more pronounced reduction during exercise and was still lower in 6th min of recovery compared to N-day. It was concluded that with acute hypoxia of the severity applied in this study left ventricular systolic function in our healthy subjects showed a pronounced improvement and left ventricular diastolic function was reduced, both at rest and with exercise.     

Directional Migration of Recirculating Lymphocytes through Lymph Nodes via Random Walks

Naive T lymphocytes exhibit extensive antigen-independent recirculation between blood and lymph nodes, where they may encounter dendritic cells carrying cognate antigen. We examine how long different T cells may spend in an individual lymph node by examining data from long term cannulation of blood and efferent lymphatics of a single lymph node in the sheep. We determine empirically the distribution of transit times of migrating T cells by applying the Least Absolute Shrinkage & Selection Operator () or regularised to fit experimental data describing the proportion of labelled infused cells in blood and efferent lymphatics over time. The optimal inferred solution reveals a distribution with high variance and strong skew. The mode transit time is typically between 10 and 20 hours, but a significant number of cells spend more than 70 hours before exiting. We complement the empirical machine learning based approach by modelling lymphocyte passage through the lymph node . On the basis of previous two photon analysis of lymphocyte movement, we optimised distributions which describe the transit times (first passage times) of discrete one dimensional and continuous (Brownian) three dimensional random walks with drift. The optimal fit is obtained when drift is small, i.e. the ratio of probabilities of migrating forward and backward within the node is close to one. These distributions are qualitatively similar to the inferred empirical distribution, with high variance and strong skew. In contrast, an optimised normal distribution of transit times (symmetrical around mean) fitted the data poorly. The results demonstrate that the rapid recirculation of lymphocytes observed at a macro level is compatible with predominantly randomised movement within lymph nodes, and significant probabilities of long transit times. We discuss how this pattern of migration may contribute to facilitating interactions between low frequency T cells and antigen presenting cells carrying cognate antigen.     

Nonlinear scaling of foraging contacts with rodent population density

Density‐dependent shifts in population processes like territoriality, reproduction, dispersal, and parasite transmission are driven by changes in contacts between individuals. Despite this, surprisingly little is known about how contacts change with density, and thus the mechanisms driving density‐dependent processes. A simple linear contact–density function is often assumed, but this is not based on a sound basis of empirical data. We addressed this question using a replicated, semi‐natural experiment in which we measured contacts at feeding stations between multimammate mice, Mastomys natalensis, across ten distinct, linearly increasing densities between 10 and 272 animals/ha. Unexpectedly, unique contacts increased not linearly but sigmoidally with density, which we attribute to the species’ scramble competition mating system, small‐scale dominance/avoidance and absence of territoriality. These results provide new insights into how species’ characteristics can relate to density‐dependent changes in contacts, and the unexpected shape of the contact–density function warrants that density‐dependence in ecological models, such as parasite transmission models, must be parameterized with care.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Discrete emotion effects on lexical decision response times

Our knowledge about affective processes, especially concerning effects on cognitive demands like word processing, is increasing steadily. Several studies consistently document valence and arousal effects, and although there is some debate on possible interactions and different notions of valence, broad agreement on a two dimensional model of affective space has been achieved. Alternative models like the discrete emotion theory have received little interest in word recognition research so far. Using backward elimination and multiple regression analyses, we show that five discrete emotions (i.e., happiness, disgust, fear, anger and sadness) explain as much variance as two published dimensional models assuming continuous or categorical valence, with the variables happiness, disgust and fear significantly contributing to this account. Moreover, these effects even persist in an experiment with discrete emotion conditions when the stimuli are controlled for emotional valence and arousal levels. We interpret this result as evidence for discrete emotion effects in visual word recognition that cannot be explained by the two dimensional affective space account.