Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL‐1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.     

Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

Aggregation of α‐synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca²⁺ and α‐synuclein aggregation. Analyses of cell lines and primary culture models of α‐synuclein cytopathology reveal an early phase with reduced cytosolic Ca²⁺ levels followed by a later Ca²⁺ increase. Aggregated but not monomeric α‐synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca²⁺. CPA protects the cells against α‐synuclein‐aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo. Proximity ligation assays also reveal an increased interaction between α‐synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α‐synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down‐stream processes may be therapeutic targets for treating α‐synucleinopathies.     

Volatiles and Nonvolatiles in Flourensia campestris Griseb. (Asteraceae), How Much Do Capitate Glandular Trichomes Matter?

The distribution and ultrastructure of capitate glandular trichomes (GTs) in Flourensia species (Asteraceae) have been recently elucidated, but their metabolic activity and potential biological function remain unexplored. Selective nonvolatile metabolites from isolated GTs were strikingly similar to those found on leaf surfaces. The phytotoxic allelochemical sesquiterpene (–)‐hamanasic acid A ((–)‐HAA) was the major constituent (ca. 40%) in GTs. Although GTs are quaternary ammonium compounds (QACs)‐accumulating species, glycine betaine was not found in GTs; it was only present in the leaf mesophyll. Two (–)‐HAA accompanying surface secreted products: compounds 4‐hydroxyacetophenone (piceol; 1 ) and 2‐hydroxy‐5‐methoxyacetophenone ( 2 ), which were isolated and fully characterized (GC/MS, NMR), were present in the volatiles found in GTs. The essential oils of fresh leaves revealed ca. 33% monoterpenes, 26% hydrocarbon‐ and 30% oxygenated sesquiterpenes, most of them related to cadinene and bisabolene derivatives. Present results suggest a main role of GTs in determining the volatile and nonvolatile composition of F. campestris leaves. Based on the known activities of the compounds identified, it can be suggested that GTs in F. campestris would play key ecological functions in plant‐pathogen and plant‐plant interactions. In addition, the strikingly high contribution of compounds derived from cadinene and bisabolene pathways, highlights the potential of this species as a source of high‐valued bioproducts.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

Akap1 Deficiency Promotes Mitochondrial Aberrations and Exacerbates Cardiac Injury Following Permanent Coronary Ligation via Enhanced Mitophagy and Apoptosis

A-kinase anchoring proteins (AKAPs) transmit signals cues from seven-transmembrane receptors to specific sub-cellular locations. Mitochondrial AKAPs encoded by the Akap1 gene have been shown to modulate mitochondrial function and reactive oxygen species (ROS) production in the heart. Under conditions of hypoxia, mitochondrial AKAP121 undergoes proteolytic degradation mediated, at least in part, by the E3 ubiquitin ligase Seven In-Absentia Homolog 2 (Siah2). In the present study we hypothesized that Akap1 might be crucial to preserve mitochondrial function and structure, and cardiac responses to myocardial ischemia. To test this, eight-week-old Akap1 knockout mice (Akap1^-/-), Siah2 knockout mice (Siah2^-/-) or their wild-type (wt) littermates underwent myocardial infarction (MI) by permanent left coronary artery ligation. Age and gender matched mice of either genotype underwent a left thoracotomy without coronary ligation and were used as controls (sham). Twenty-four hours after coronary ligation, Akap1^-/- mice displayed larger infarct size compared to Siah2^-/- or wt mice. One week after MI, cardiac function and survival were also significantly reduced in Akap1^-/- mice, while cardiac fibrosis was significantly increased. Akap1 deletion was associated with remarkable mitochondrial structural abnormalities at electron microscopy, increased ROS production and reduced mitochondrial function after MI. These alterations were associated with enhanced cardiac mitophagy and apoptosis. Autophagy inhibition by 3-methyladenine significantly reduced apoptosis and ameliorated cardiac dysfunction following MI in Akap1^-/- mice. These results demonstrate that Akap1 deficiency promotes cardiac mitochondrial aberrations and mitophagy, enhancing infarct size, pathological cardiac remodeling and mortality under ischemic conditions. Thus, mitochondrial AKAPs might represent important players in the development of post-ischemic cardiac remodeling and novel therapeutic targets.     

Diagnosis of Midwestern Endemic Mycoses

Histoplasmosis and blastomycosis are the two most common midwestern endemic mycoses. A history of exposure to the geographic areas in which these organisms occur is central to raising suspicion for an endemic fungal infection. For infection with these organisms, the diagnosis is definitively established by recovery of the organism in tissue or body fluids, which may take weeks. A rapid diagnosis can be made by finding the distinctive yeasts in tissues or body fluids. Antigen testing allows a presumptive diagnosis of these endemic fungal infections while awaiting culture results, but cross-reactivity between assays for Histoplasma and Blastomyces is routinely seen. Antibody tests provide supportive evidence for infection with either of these endemic mycoses but are less useful in immunosuppressed patients.     

Decompressive Hemicraniectomy in a South American Population – Morbidity and Outcomes Analysis

Background

Malignant cerebral artery strokes have a poor prognosis, with nearly 80% of mortality in some series despite intensive care. After a large randomized trial, decompressive hemicraniectomy has been performed more often in stroke patients. Here, we describe patients in a tertiary teaching hospital in Brazil, emphasizing the impact of age on outcomes.

Methods

A retrospective cohort of patients, with malignant strokes which received a decompressive hemicraniectomy, from paper and electronic medical records, from January 2010 to December 2013 was divided into two groups according to age.

Results

The final analysis included 60 patients. The overall mortality was higher among patients older than 60 yrs (67% vs. 41%; p = 0.039), whose group also had a worse outcome (76% with mRS 5 or 6) at 90 days (OR 3.91 CI95% 1.30–11.74), whereas only 24% had mRS of 0–4 (p = 0.015). All patients who presented with sepsis died (p = 0.003). The incidence of pulmonary infection was very high in the elderly group (76%) with significant intergroup differences (p = 0.027, OR 8.32 CI95% 0.70–98.48).

Conclusions

Older patients present more commonly with infections, more disabilities and a higher mortality, highlighting very poor results in elderly population. These results should be proved with a South American trial, and if confirmed, it can impact on future decisions regarding decompressive craniectomy for acute ischemic stroke in our region.     

Major Histocompatibility Complex (MHC) class II sequence polymorphism in long-finned pilot whale (Globicephala melas) from the North Atlantic

Determining how intra-specific genetic diversity is apportioned among natural populations is essential for detecting local adaptation and identifying populations with inherently low levels of extant diversity which may become a conservation concern. Sequence polymorphism at two adaptive loci (MHC DRA and DQB) was investigated in long-finned pilot whales (Globicephala melas) from four regions in the North Atlantic and compared with previous data from New Zealand (South Pacific). Three alleles were resolved at each locus, with trans-species allele sharing and higher levels of non-synonymous to synonymous substitution, especially in the DQB locus. Overall nucleotide diversities of 0.49?±?0.38% and 4.60?±?2.39% were identified for the DRA and DQB loci, respectively, which are relatively low for MHC loci in the North Atlantic, but comparable to levels previously described in New Zealand (South Pacific). There were significant differences in allele frequencies within the North Atlantic and between the North Atlantic and New Zealand. Patterns of diversity and divergence are consistent with the long-term effects of balancing selection operating on the MHC loci, potentially mediated through the effects of host-parasite coevolution. Differences in allele frequency may reflect variation in pathogen communities, coupled with the effects of differential drift and gene flow.     

The Role of Community and Individuals in the Formation of Social Capital

Because social capital shapes many desirable socioeconomic outcomes, we ask what incentives drive private investments in social capital. We estimate the association between private investments in social capital (outcome variable) and the following explanatory variables: (a) individual-level variables from an optimal investment model, (b) spillovers from group social capital, (c) village income inequality, and (d) market openness. We draw on information from Tsimane’, a native Amazonian society of foragers and farmers in Bolivia, and equate social capital with gifts, help given, and communal labor offered by the household. Age bore an inverted U-shaped and income bore a positive association with social capital, but geographic mobility, wealth, and schooling bore no significant association with social capital. We found strong group-level associations even after instrumenting social capital; the association probably stems from strong kinship ties which tend to blur the line between the group and the individual. Village measures of social capital were positively and significantly associated with private investments in social capital. We found some evidence that village income inequality and market openness were negatively associated with private investments in social capital.