Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.     

A method for equilibration chamber sampling and gas chromatographic analysis of the soil atmosphere

The method includes sampling of gases from an equilibration chamber permanently installed in the soil, transferring the sample to laboratory and subsequent measurement by gas chromatography. The equilibration chamber allows sampling of the gas phase both above and below the groundwater level, which is a major advantage. After significant concentration changes in non-saturated soils, gases in chambers regain equilibrium with the surrounding soils within 1–2 days. In the most unfavourable equilibration situations,i.e. in mineral subsoils with stagnant groundwater and very low biological activity, 90% equilibrium is attained in about 15 days. N₂, O₂+Ar, CO₂, CH₄, N₂O, H₂ and Ne, are measured on a series/bypass multi-column system, followed by a thermal conductivity detector.     

Occupancy versus colonization–extinction models for projecting population trends at different spatial scales

Understanding spatiotemporal population trends and their drivers is a key aim in population ecology. We further need to be able to predict how the dynamics and sizes of populations are affected in the long term by changing landscapes and climate. However, predictions of future population trends are sensitive to a range of modeling assumptions. Deadwood‐dependent fungi are an excellent system for testing the performance of different predictive models of sessile species as these species have different rarity and spatial population dynamics, the populations are structured at different spatial scales, and they utilize distinct substrates. We tested how the projected large‐scale occupancies of species with differing landscape‐scale occupancies are affected over the coming century by different modeling assumptions. We compared projections based on occupancy models against colonization–extinction models, conducting the modeling at alternative spatial scales and using fine‐ or coarse‐resolution deadwood data. We also tested effects of key explanatory variables on species occurrence and colonization–extinction dynamics. The hierarchical Bayesian models applied were fitted to an extensive repeated survey of deadwood and fungi at 174 patches. We projected higher occurrence probabilities and more positive trends using the occupancy models compared to the colonization–extinction models, with greater difference for the species with lower occupancy, colonization rate, and colonization:extinction ratio than for the species with higher estimates of these statistics. The magnitude of future increase in occupancy depended strongly on the spatial modeling scale and resource resolution. We encourage using colonization–extinction models over occupancy models, modeling the process at the finest resource‐unit resolution that is utilizable by the species, and conducting projections for the same spatial scale and resource resolution at which the model fitting is conducted. Further, the models applied should include key variables driving the metapopulation dynamics, such as the availability of suitable resource units, habitat quality, and spatial connectivity.     

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K^trans and v_e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K^trans was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v_e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K^trans is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K^trans may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.     

Combined observational and experimental data provide limited support for facilitation in lichens

It is increasingly recognized that facilitative interactions can shape communities. One of the mechanisms through which facilitation may operate is when one species facilitates the colonization of another through the exchange of shared symbionts. Lichens are symbiotic associations composed of a mycobiont (lichenised‐fungus) and one or two photobionts (algae or cyanobacteria). Different lichen species may have overlapping specificity for photobionts, creating the possibility that facilitation drives lichen community assembly. To investigate whether facilitation occurs in lichens, we combined an observational study (a) with a manipulative field experiment (b). For (a), we quantified the effect of local patch conditions, facilitation and the size of the surrounding metapopulation on colonizations of an epixylic lichen species (Cladonia botrytes) in an area of managed boreal forest. This was done by twice surveying lichens on 293 stumps, located in stands of three age classes. For (b), we treated unoccupied surfaces of 56 cut stumps with algal mixtures of an Asterochloris photobiont and recorded C. botrytes colonizations over three years. In (a), colonization rates of C. botrytes increased with increasing abundance of other lichen species with specificity for Asterochloris photobionts, consistent with an effect of facilitation. However, in the field experiment (b), colonizations of the focal species did not provide support for facilitation. We conclude that our study provides limited support for facilitation in green‐algal lichens, underscoring the importance of combining observational studies with experiments when studying species interactions.     

Methods for the detection and analysis of protein-protein interactions

A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.     

Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.     

The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium

Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations. In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene. This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo(5)U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm(5)s(2)U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site. We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS. Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm(5)s(2)U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S. enterica were reduced in the iscS51 mutant. In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level. After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm(5)s(2)U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm(5)s(2)U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm(5)s(2)U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm(5)s(2)U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant.