1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)₂D₃ and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)₂D₃ altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)₂D₃-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)₂D₃. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)₂D₃ in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.     

Design of a potent, soluble glucokinase activator with increased pharmacokinetic half-life

The continued optimization of a series of glucokinase activators is described, including attempts to understand the interplay between molecular structure and the composite parameter of unbound clearance. These studies resulted in the discovery of a new scaffold for glucokinase activators and further exploration of this scaffold led to the identification of GKA60. GKA60 maintains an excellent balance of potency and physical properties whilst possessing a significantly different, but complimentary, pre-clinical pharmacokinetic profile compared with the previously disclosed compound GKA50.     

Does maternal care evolve through egg recognition or directed territoriality?

The mechanism that facilitates the evolution of maternal care is ambiguous in egg‐laying terrestrial vertebrates: does the ability of mothers to recognize their own eggs lead them under some circumstances to begin providing care or can maternal care evolve from simply being in close proximity to the eggs (e.g. through territorial behaviour)? This question is difficult to answer because in most species, parental care is either absent altogether or present; in only a few species we have the opportunity to observe intraspecific variation in the expression of parental care. We studied a population of long‐tailed skinks (Eutropis longicaudata) in which females have recently evolved maternal care from a noncaring state. Females on Orchid Island, Taiwan, remain with their eggs during incubation and when doing so, actively deter egg predation by egg‐eating snakes (Oligodon formosanus); in all other populations, females lack post‐ovipositional maternal care. Nest‐guarding females on Orchid Island (i) showed antipredator behaviours only in the original nest site in which they laid eggs, even after we removed all of the eggs or substituted them with those of a conspecific; (ii) protect any eggs present inside the original nest site (even when the eggs belong to a conspecific); and (iii) develop this behaviour while gravid (i.e. prior to laying eggs). This supports the hypothesis that long‐tailed skinks cannot recognize their own eggs, suggesting that maternal care is a directed form of territoriality only expressed towards egg‐eating snakes and only during reproduction. Nest guarding is among the most primitive forms of parental care, and the recent evolution of this behaviour in a single population provides insight into one of the mechanisms by which parental care can originate in terrestrial vertebrates.     

Accreditation and the use of validated/recognised methods to analyse human semen

Accreditation of laboratories who perform diagnostic semen analysis in Australia and New Zealand is a requirement of the healthcare system. Within the accreditation process laboratories are required to set ISO standards within their policies and procedures. In order to achieve their aims, laboratories need to be able to measure a number of defined semen parameters both accurately and repetitively, especially around the lower limit of the reference intervals. The methods documented in the WHO-manual are used almost universal as the laboratory standard. Some laboratories incorporate minor method variations into their procedures. As part of the ISO requirements all variations require validation using internally approved processes that are documented and that incorporate appropriate statistical analysis and comparison of results. Validation is an ongoing process and regular review is essential. Evidence of the validation must be available for review by external auditors during accreditation. Where any validated variant method returns results that are significantly different to any method within the WHO-manual, the laboratory needs to develop its own, in-house reference interval for that method.     

Human glycolipid transfer protein (GLTP) expression modulates cell shape

Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ～70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member.     

Human biodistribution and dosimetry of ¹¹C-CUMI-101, an agonist radioligand for serotonin-1a receptors in brain

As a reported agonist,¹¹C-CUMI-101 is believed to selectively bind the G-protein-coupled state of the serotonin-1A (5-HT_1A) receptor, thereby providing a measure of the active subset of all 5-HT_1A receptors in brain. Although ¹¹C-CUMI-101 has been successfully used to quantify 5-HT_1A receptors in human and monkey brain, its radiation exposure has not previously been reported. The purpose of this study was to calculate the radiation exposure to organs of the body based on serial whole-body imaging with positron emission tomography (PET) in human subjects.

Methods

Nine healthy volunteers were injected with 428±84 MBq (mean ± SD) ¹¹C-CUMI-101 and then imaged with a PET-only device for two hours from head to mid-thigh. Eleven source organs (brain, heart, liver, pancreas, stomach, spleen, lungs, kidneys, lumbar spine L1-5, thyroid, and urinary bladder) were identified on whole body images and used to calculate radiation doses using the software program OLINDA/EXM 1.1. To confirm that we had correctly identified the pancreas, a tenth subject was imaged on a PET/CT device.

Results

Brain had high uptake (∼11% of injected activity (IA)) at 10 min. Although liver had the highest uptake (∼35% IA at 120 min), excretion of this activity was not visible in gall bladder or intestine during the scanning session. Organs which received the highest doses (microSv/MBq) were pancreas (32.0), liver (18.4), and spleen (14.5). The effective dose of ¹¹C-CUMI-101 was 5.3±0.5 microSv/MBq.

Conclusion

The peak brain uptake (∼11% IA) of ¹¹C-CUMI-101 is the highest among more than twenty ¹¹C-labeled ligands reported in the literature and provides good counting statistics from relatively low injected activities. Similar to that of other ¹¹C-labeled ligands for brain imaging, the effective dose of ¹¹C-CUMI-101 is 5.3±0.5 microSv/MBq, a value that can now be used to estimate the radiation risks in future research studies.     

Cardiac pathology associated with the infection of Oncorhynchus mykiss Walbaum with Apatemon gracilis Rud. 1819

The chronological development of gross pathological and histopathological changes associated with the infection of rainbow trout, Oncorhynchus mykiss Walbaum, with metacercariae of Apatemon gracilis Rudolphi was investigated.