The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52^−/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.     

Procalcitonin Identifies Cell Injury,Not Bacterial Infection,in Acute Liver Failure

Background

Because acute liver failure (ALF) patients share many clinical features with severe sepsis and septic shock, identifying bacterial infection clinically in ALF patients is challenging. Procalcitonin (PCT) has proven to be a useful marker in detecting bacterial infection. We sought to determine whether PCT discriminated between presence and absence of infection in patients with ALF.

Method

Retrospective analysis of data and samples of 115 ALF patients from the United States Acute Liver Failure Study Group randomly selected from 1863 patients were classified for disease severity and ALF etiology. Twenty uninfected chronic liver disease (CLD) subjects served as controls.

Results

Procalcitonin concentrations in most samples were elevated, with median values for all ALF groups near or above a 2.0 ng/mL cut-off that generally indicates severe sepsis. While PCT concentrations increased somewhat with apparent liver injury severity, there were no differences in PCT levels between the pre-defined severity groups–non-SIRS and SIRS groups with no documented infections and Severe Sepsis and Septic Shock groups with documented infections, (p = 0.169). PCT values from CLD patients differed from all ALF groups (median CLD PCT value 0.104 ng/mL, (p ≤0.001)). Subjects with acetaminophen (APAP) toxicity, many without evidence of infection, demonstrated median PCT >2.0 ng/mL, regardless of SIRS features, while some culture positive subjects had PCT values <2.0 ng/mL.

Summary/Conclusions

While PCT appears to be a robust assay for detecting bacterial infection in the general population, there was poor discrimination between ALF patients with or without bacterial infection presumably because of the massive inflammation observed. Severe hepatocyte necrosis with inflammation results in elevated PCT levels, rendering this biomarker unreliable in the ALF setting.     

A Randomized,Double-Blind,Placebo-Controlled Trial of Adjunctive Metformin Therapy in Overweight/Obese Youth with Type 1 Diabetes

Context

Insulin resistance has been proposed as one of the causes of poor glycemic control in overweight/obese youth with type 1 diabetes (T1D). However, the role of adjunctive metformin, an insulin sensitizer, on glycemic control in these patients is unclear.

Objective

To compare the effect of metformin vs. placebo on hemoglobin A1c (HbA1c), total daily dose (TDD) of insulin, and other parameters in overweight/obese youth with T1D.

Hypothesis

Adjunctive metformin therapy will improve glycemic control in overweight/obese youth with T1D.

Design, Setting, and Participants

A 9-mo randomized, double-blind, placebo controlled trial of metformin and placebo in 28 subjects (13m/15f) of ages 10-20years (y), with HbA1c >8% (64 mmol/mol), BMI >85%, and T1D > 12 months was conducted at a university outpatient facility. The metformin group consisted of 15 subjects (8 m/ 7f), of age 15.0 ± 2.5 y; while the control group was made up of 13 subjects (5m/ 8f), of age 14.5 ± 3.1y. All participants employed a self-directed treat-to-target insulin regimen based on a titration algorithm of (-2)-0-(+2) units to adjust their long-acting insulin dose every 3rd day from -3 mo through +9 mo to maintain fasting plasma glucose (FPG) between 90–120 mg/dL (5.0–6.7 mmol/L). Pubertal maturation was determined by Tanner stage.

Results

Over the course of the 9 months of observation, the between-treatment differences in HbA1c of 0.4% (9.85% [8.82 to 10.88] for placebo versus 9.46% [8.47 to 10.46] for metformin) was not significant (p = 0.903). There were non-significant reduction in fasting plasma glucose (189.4 mg/dL [133.2 to 245.6] for placebo versus 170.5 mg/dL [114.3 to 226.7] for metformin), (p = 0.927); total daily dose (TDD) of short-acting insulin per kg body weight/day(p = 0.936); and the TDD of long-acting insulin per kg body weight per day (1.15 units/kg/day [0.89 to 1.41] for placebo versus 0.90 units/kg/day [0.64 to 1.16] for metformin) (p = 0.221). There was no difference in the occurrence of hypoglycemia between the groups.

Conclusions

This 9-month RCT of adjunctive metformin therapy in overweight and obese youth with T1D resulted in a 0.4% lower HbA1c value in the metformin group compared to the placebo group.

Trial Registration

ClinicalTrial.gov NCT01334125     

Philornis downsi parasitism is the primary cause of nestling mortality in the critically endangered Darwin’s medium tree finch (Camarhynchus pauper)

Darwin’s medium tree finch (Camarhynchus pauper) meets the 2009 International Union for Conservation of Nature Red List criteria for a critically endangered species because it has “a very small range on a single island” and is “declining rapidly owing to the effects of the parasite Philornis downsi”, habitat degradation, and introduced predators. The medium tree finch is only found in patches of remnant highland forest on Floreana Island, where it co-exists with breeding populations of small and large tree finches (C. parvulus and C. psittacula). Here, we examine the intensity of P. downsi in nests of small, medium, and large tree finches on Floreana. We expected that parasite intensity would increase with finch body size, and with greater rainfall, and would also correlate with increased nestling mortality. We found a trend in the expected direction for parasite intensity and rainfall. Combined meta-analytically with data from a previous study, the overall trend for the two studies was significant. We also found a significant linear trend in parasite intensity with finch body size. In addition, the medium tree finch exhibited a somewhat higher parasite intensity than would be expected based on body mass alone. Of 63 active medium tree finch nests, 17 nests had nestlings: all of which were infested with P. downsi. Only 25% of medium tree finch nestlings fledged, 28% were depredated, 41% died due to P. downsi parasitism, and 6% died for other reasons.     

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.     

Beta-Actin Is Involved in Modulating Erythropoiesis during Development by Fine-Tuning Gata2 Expression Levels

The functions of actin family members during development are poorly understood. To investigate the role of beta-actin in mammalian development, a beta-actin knockout mouse model was used. Homozygous beta-actin knockout mice are lethal at embryonic day (E)10.5. At E10.25 beta-actin knockout embryos are growth retarded and display a pale yolk sac and embryo proper that is suggestive of altered erythropoiesis. Here we report that lack of beta-actin resulted in a block of primitive and definitive hematopoietic development. Reduced levels of Gata2, were associated to this phenotype. Consistently, ChIP analysis revealed multiple binding sites for beta-actin in the Gata2 promoter. Gata2 mRNA levels were almost completely rescued by expression of an erythroid lineage restricted ROSA26-promotor based GATA2 transgene. As a result, erythroid differentiation was restored and the knockout embryos showed significant improvement in yolk sac and embryo vascularization. These results provide new molecular insights for a novel function of beta-actin in erythropoiesis by modulating the expression levels of Gata2 in vivo.     

Maternal Microchimerism: Increased in the Insulin Positive Compartment of Type 1 Diabetes Pancreas but Not in Infiltrating Immune Cells or Replicating Islet Cells

Background

Maternal microchimeric cells (MMc) transfer across the placenta during pregnancy. Increased levels of MMc have been observed in several autoimmune diseases including type 1 diabetes but their role is unknown. It has been suggested that MMc are 1) effector cells of the immune response, 2) targets of the autoimmune response or 3) play a role in tissue repair. The aim of this study was to define the cellular phenotype of MMc in control (n = 14) and type 1 diabetes pancreas (n = 8).

Methods

Using sex chromosome-based fluorescence in-situ hybridization, MMc were identified in male pancreas and their phenotype determined by concomitant immunofluorescence.

Results

In normal pancreas, MMc positive for endocrine, exocrine, duct and acinar markers were identified suggesting that these cells are derived from maternal progenitors. Increased frequencies of MMc were observed in type 1 diabetes pancreas (p = 0.03) with particular enrichment in the insulin positive fraction (p = 0.01). MMc did not contribute to infiltrating immune cells or Ki67+ islet cell populations in type 1 diabetes.

Conclusion

These studies provide support for the hypothesis that MMc in human pancreas are derived from pancreatic precursors. Increased frequencies of MMc beta cells may contribute to the initiation of autoimmunity or to tissue repair but do not infiltrate islets in type 1 diabetes.     

Adenosine receptor expression in rheumatoid synovium: a basis for methotrexate action

Introduction

Methotrexate (MTX) exerts at least part of its anti-inflammatory effects through adenosine receptors (ADOR). The aims of this study were to determine the expression of all four adenosine receptor genes (ADORA₁, ADORA_2A, ADORA_2B, ADORA₃ and ADORA_3variant) in rheumatoid synovial tissue and any influence of MTX exposure on this expression. Furthermore, we investigated whether polymorphisms within ADORA₃ were associated with response and/or adverse effects associated with MTX.

Methods

Adenosine receptor gene expression was undertaken using PCR in 20 rheumatoid arthritis (RA) synovial samples. A separate cohort of 225 RA patients receiving MTX was genotyped for SNPs in the ADORA₃ receptor gene. Double immunofluorescence was used to identify cells expressing ADOR protein.

Results

All ADOR genes were expressed in all synovial samples. ADORA₃ and A_3variant were the dominant subtypes expressed irrespective of MTX therapy. Expression of ADORA_2A and ADORA_2B was increased in patients receiving MTX compared to those not receiving MTX. There was no association between the ADORA₃ rs1544224 SNP and high and low disease activity or MTX-associated adverse effects. ADORA_2B protein expression was most obvious in vascular endothelial cells whereas ADORA₃ protein was more abundant and expressed by synovial fibroblasts.

Conclusions

We have shown that adenosine receptors are expressed in RA synovium. There is differential expression of receptors such that ADORA₃ is expressed at significantly higher levels. This evidence demonstrates the potential for MTX to exert its anti-inflammatory effects at the primary site of pathology within the joints of patients with RA.