Islet hypertrophy following pancreatic disruption of Smad4 signaling

To investigate the role of transforming growth factor (TGF)-beta family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-beta family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-beta family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas.     

Orchard pollination in Capitol Reef National Park,Utah, USA. Honey bees or native bees?

Capitol Reef National Park in central Utah, USA surrounds 22 managed fruit orchards started over a century ago by Mormon pioneers. Honey bees are imported for pollination, although the area in which the Park is embedded has over 700 species of native bees, many of which are potential orchard pollinators. We studied the visitation of native bees to apple, pear, apricot, and sweet cherry over 2 years. Thirty species of bees visited the flowers but, except for pear flowers, most were uncommon compared to honey bees. Evidence that honey bees prevented native bees from foraging on orchard crop flowers was equivocal: generally, honey bee and native bee visitation rates to the flowers were not negatively correlated, nor were native bee visitation rates positively correlated with distance of orchards from honey bee hives. Conversely, competition was tentatively suggested by much larger numbers of honey bees than natives on the flowers of apples, apricots and cherry; and by the large increase of native bees on pears, where honey bee numbers were low. At least one-third of the native bee species visiting the flowers are potential pollinators, including cavity-nesting species such as Osmia lignaria propinqua, currently managed for small orchard pollination in the US, plus several fossorial species, including one rosaceous flower specialist (Andrena milwaukiensis). We suggest that gradual withdrawal of honey bees from the Park would help conserve native bee populations without decreasing orchard crop productivity, and would serve as a demonstration of the commercial value of native pollinators.     

Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning

Establishment of the left-right axis is essential for normal organ morphogenesis and function. Ca(2+) signaling and cilia function in the zebrafish Kuppfer's Vesicle (KV) have been implicated in laterality. Here we describe an endogenous Ca(2+) release event in the region of the KV precursors (dorsal forerunner cells, DFCs), prior to KV and cilia formation. Manipulation of Ca(2+) release to disrupt this early flux does not impact early DFC specification, but results in altered DFC migration or cohesion in the tailbud at somite stages. This leads to disruption of KV formation followed by bilateral expression of asymmetrical genes, and randomized organ laterality. We identify beta-catenin inhibition as a Ca(2+)-signaling target and demonstrate that localized loss of Ca(2+) within the DFC region or DFC-specific activation of beta-catenin is sufficient to alter laterality in zebrafish. We identify a previously unknown DFC-like cell population in Xenopus and demonstrate a similar Ca(2+)-sensitive stage. As in zebrafish, manipulation of Ca(2+) release results in ectopic nuclear beta-catenin and altered laterality. Overall, our data support a conserved early Ca(2+) requirement in DFC-like cell function in zebrafish and Xenopus.     

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.     

Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Inhibition of platelet thromboxane synthetase by L-1-tosylamido-2-phenylethyl chloromethyl ketone

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B₂ (TxB₂) formation from exogenous 20:4 or PGH₂, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH₂, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB₂ formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.     

Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura

Environments vary stochastically, and animals need to behave in ways that best fit the conditions in which they find themselves. The social environment is particularly variable, and responding appropriately to it can be vital for an animal’s success. However, cues of social environment are not always reliable, and animals may need to balance accuracy against the risk of failing to respond if local conditions or interfering signals prevent them detecting a cue. Recent work has shown that many male Drosophila fruit flies respond to the presence of rival males, and that these responses increase their success in acquiring mates and fathering offspring. In Drosophila melanogaster males detect rivals using auditory, tactile and olfactory cues. However, males fail to respond to rivals if any two of these senses are not functioning: a single cue is not enough to produce a response. Here we examined cue use in the detection of rival males in a distantly related Drosophila species, D. pseudoobscura, where auditory, olfactory, tactile and visual cues were manipulated to assess the importance of each sensory cue singly and in combination. In contrast to D. melanogaster, male D. pseudoobscura require intact olfactory and tactile cues to respond to rivals. Visual cues were not important for detecting rival D. pseudoobscura, while results on auditory cues appeared puzzling. This difference in cue use in two species in the same genus suggests that cue use is evolutionarily labile, and may evolve in response to ecological or life history differences between species.     

Validity of Diagnostic Codes for Acute Stroke in Administrative Databases: A Systematic Review

Objective

To conduct a systematic review of studies reporting on the validity of International Classification of Diseases (ICD) codes for identifying stroke in administrative data.

Methods

MEDLINE and EMBASE were searched (inception to February 2015) for studies: (a) Using administrative data to identify stroke; or (b) Evaluating the validity of stroke codes in administrative data; and (c) Reporting validation statistics (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), or Kappa scores) for stroke, or data sufficient for their calculation. Additional articles were located by hand search (up to February 2015) of original papers. Studies solely evaluating codes for transient ischaemic attack were excluded. Data were extracted by two independent reviewers; article quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies tool.

Results

Seventy-seven studies published from 1976–2015 were included. The sensitivity of ICD-9 430-438/ICD-10 I60-I69 for any cerebrovascular disease was ≥ 82% in most [≥ 50%] studies, and specificity and NPV were both ≥ 95%. The PPV of these codes for any cerebrovascular disease was ≥ 81% in most studies, while the PPV specifically for acute stroke was ≤ 68%. In at least 50% of studies, PPVs were ≥ 93% for subarachnoid haemorrhage (ICD-9 430/ICD-10 I60), 89% for intracerebral haemorrhage (ICD-9 431/ICD-10 I61), and 82% for ischaemic stroke (ICD-9 434/ICD-10 I63 or ICD-9 434&436). For in-hospital deaths, sensitivity was 55%. For cerebrovascular disease or acute stroke as a cause-of-death on death certificates, sensitivity was ≤ 71% in most studies while PPV was ≥ 87%.

Conclusions

While most cases of prevalent cerebrovascular disease can be detected using 430-438/I60-I69 collectively, acute stroke must be defined using more specific codes. Most in-hospital deaths and death certificates with stroke as a cause-of-death correspond to true stroke deaths. Linking vital statistics and hospitalization data may improve the ascertainment of fatal stroke.     

Semaphorin 3B is a 1,25-Dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.     

Persistence of circadian variation in arterial blood pressure in beta1/beta2-adrenergic receptor-deficient mice

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.