Exercise training reverses cardiac aging phenotypes associated with heart failure with preserved ejection fraction in male mice

Heart failure with preserved ejection fraction (HFpEF) is the most common type of HF in older adults. Although no pharmacological therapy has yet improved survival in HFpEF, exercise training (ExT) has emerged as the most effective intervention to improving functional outcomes in this age‐related disease. The molecular mechanisms by which ExT induces its beneficial effects in HFpEF, however, remain largely unknown. Given the strong association between aging and HFpEF, we hypothesized that ExT might reverse cardiac aging phenotypes that contribute to HFpEF pathophysiology and additionally provide a platform for novel mechanistic and therapeutic discovery. Here, we show that aged (24–30 months) C57BL/6 male mice recapitulate many of the hallmark features of HFpEF, including preserved left ventricular ejection fraction, subclinical systolic dysfunction, diastolic dysfunction, impaired cardiac reserves, exercise intolerance, and pathologic cardiac hypertrophy. Similar to older humans, ExT in old mice improved exercise capacity, diastolic function, and contractile reserves, while reducing pulmonary congestion. Interestingly, RNAseq of explanted hearts showed that ExT did not significantly modulate biological pathways targeted by conventional HF medications. However, it reversed multiple age‐related pathways, including the global downregulation of cell cycle pathways seen in aged hearts, which was associated with increased capillary density, but no effects on cardiac mass or fibrosis. Taken together, these data demonstrate that the aged C57BL/6 male mouse is a valuable model for studying the role of aging biology in HFpEF pathophysiology, and provide a molecular framework for how ExT potentially reverses cardiac aging phenotypes in HFpEF.     

Pentoxifylline with metformin treatment improves biochemical parameters in patients with nonalcoholic steatohepatitis

BackgroundThe progression of the nonalcoholic fatty liver disease to nonalcoholic steatohepatitis (NASH) is multifactorial, and there is still a lack of approved medications for its treatment. The study aimed to evaluate the impact of combined treatment with Pentoxifylline and Metformin on biochemical parameters in patients with Nash. Setting: Outpatient hepatology clinic.MethodsA prospective trial was conducted. The first cohort included patients with biopsy-proven Nash, while the second cohort consisted of patients with biopsy-confirmed NAFLD. Blood tests were checked at baseline and every three months. Pentoxifylline at a dosage of 400 mg t.i.d. and Metformin at the dosage of 500 mg t.i.d. were introduced for six months in Nash group. The impact of the treatment was assessed based on biochemical results after combined treatment with low-cost medications.ResultsAll 33 Nash patients completed 24 weeks of treatment. We observed significant improvement (p<0.05) of median values after treatment for the following parameters: serum uric acid levels decreased by 51.0 mmol/L, calcium decreased for 0.27 mmoL/L, magnesium showed an increase of 0.11 mmoL/L. Insulin resistance improved as a reduction of HOMA - IR by 1.3 was detected. A significant decrease of median in liver enzymes, alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase by 24.0 U/L, 9.1 U/L, 10.8 U/L respectively, was noted.ConclusionsPentoxifylline and Metformin may provide possible treatment option in Nash. Some new potential benefit of the therapy in improving liver function whilst decreasing cardiovascular risk was perceived.     

Riociguat Reduces Infarct Size and Post-Infarct Heart Failure in Mouse Hearts: Insights from MRI/PET Imaging

Aim

Stimulation of the nitric oxide (NO) – soluble guanylate (sGC) - protein kinase G (PKG) pathway confers protection against acute ischaemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI) heart failure are less defined. The aim of this study was to not only determine whether the sGC stimulator riociguat reduces infarct size but also whether it protects against the development of post-MI heart failure.

Methods and Results

Mice were subjected to 30 min ischaemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l) were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI) and ¹⁸F-FDG-positron emission tomography (PET) were performed to determine infarct size. In the riociguat-treated mice, the resulting infarct size was smaller (8.5±2.5% of total LV mass vs. 21.8%±1.7%. in controls, p = 0.005) and LV systolic function analysed by MRI was better preserved (60.1%±3.4% of preischaemic vs. 44.2%±3.1% in controls, p = 0.005). After 28 days, LV systolic function by echocardiography treated group was still better preserved (63.5%±3.2% vs. 48.2%±2.2% in control, p = 0.004).

Conclusion

Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size and better long-term preservation of LV systolic function. These findings suggest that sGC stimulation during reperfusion therapy may be a powerful therapeutic treatment strategy for preventing post-MI heart failure.     

Variability of the Needle Essential Oils of Juniperus deltoides R.P.Adams from Different Populations in Serbia and Croatia

The essential‐oil compositions of one Croatian and three Serbian populations of Juniperus deltoides R.P.Adams have been determined by GC/MS analysis. In total, 147 compounds were identified, representing 97.3–98.3% of the oil composition. The oils were dominated by monoterpenes, which are characteristic components for the species of the section Juniperus. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, with a summed‐up average content of 49.45%. Statistical methods were used to determine the diversity of the terpene classes and the common terpenes between the newly described J. deltoides populations from Serbia and Croatia. Only reports on several specimens from this species have been reported so far, and there are no studies that treat population diversity. Cluster analysis of the oil contents of 21 terpenes showed high correlation with the geographical distribution of the populations, separating the Croatian from the Serbian populations. The comparison of the essential‐oil compositions obtained in the present study with literature data, showed the separation of J. deltoides and J. oxycedrus ssp. oxycedrus from the western Mediterranean region.     

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP₂) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP₂. However, the interaction with PIP₂ is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.Pollen tube growth provides a unique model system for studying the role of exocytosis in cell morphogenesis. Pollen tubes are characterized by a highly rapid polarized unidirectional tip growth. Given the relative simplicity of their structure, fast growth rates, haploid genome content, and ability to grow under in vitro culture conditions, pollen tubes provide an extremely attractive system for studying cell morphogenesis. Furthermore, the growth characteristics of pollen tubes resemble those of root hairs, moss protonema, and fungal hyphae and to some extent can be paralleled to neurite growth (Chebli and Geitmann, 2007; Cheung and Wu, 2008; Guan et al., 2013; Hepler and Winship, 2015).It is well established that oscillating polarized exocytosis is fundamental for pollen tube development and determines growth rate (Bove et al., 2008; McKenna et al., 2009; Chebli et al., 2013). Exocytosis is required for the delivery of membrane and cell wall components to the growing tip. Yet, the exact location where exocytosis takes place is under debate. Ultrastructural studies showing the accumulation of vesicles at the tip suggested that exocytosis takes place at the tip (Lancelle et al., 1987; Lancelle and Hepler, 1992; Derksen et al., 1995), which was further supported by studies on the dynamics of cell wall thickness (Rojas et al., 2011), secretion of pectin methyl esterase (PME) and PME inhibitor, and staining of pectin by propidium iodide (PI; Röckel et al., 2008; Rounds et al., 2014). Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al., 2008; Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 μm away from the tip (Silva et al., 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al., 2008; Moscatelli et al., 2012; Idilli et al., 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al., 2014). The tip-based growth is in agreement with exocytosis taking place at the tip. Presumably, part of the disagreement regarding the site of exocytosis resulted from the lack of intracellular markers for exocytosis (Cheung and Wu, 2008; Hepler and Winship, 2015), and as a result, the relationship between the FM dye-labeled inverted cone and exocytotic events during pollen tube growth is not fully understood.In many cell types, the process of secretory vesicles tethering and docking prior to fusion with the plasma membrane is initially mediated by an evolutionarily conserved tethering complex known as the exocyst. The exocyst is a heterooligomeric protein complex composed of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Studies originally based on budding yeast (Saccharomyces cerevisiae) have shown that the exocyst functions as an effector of Rab and Rho small GTPases that specifies the sites of vesicle docking and fusion at the plasma membrane in both space and time (Guo et al., 2001; Zhang et al., 2001). Support for the function of the exocyst in vesicle tethering was demonstrated recently by ectopic Sec3p-dependent vesicle recruitment to the mitochondria (Luo et al., 2014).Land plants contain all subunits of the exocyst complex, which were shown to form the functional complex (Elias et al., 2003; Cole et al., 2005; Synek et al., 2006; Hála et al., 2008). Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al., 2005; Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010; Kulich et al., 2010; Pecenková et al., 2011; Safavian and Goring, 2013; Wu et al., 2013; Safavian et al., 2015; Zhang et al., 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al., 2005; Hála et al., 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al., 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al., 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al., 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013; Safavian et al., 2015) have implicated the exocyst in polarized exocytosis in plants. Given their function, it was likely that exocyst subunits could be used as markers for polarized exocytosis. Furthermore, it could also be hypothesized that, by studying the mechanisms that underlie the association of the exocyst complex with the plasma membrane, it should be possible to identify mechanisms underlying the regulation of polarized exocytosis (Guan et al., 2013). Moreover, since the interaction of exocytotic vesicles with the exocyst is transient and marks the site(s) of active exocytosis in the membrane, fluorescently labeled exocyst subunits could be used as markers for exocytosis while avoiding potential imaging artifacts stemming from pollen tube tips densely populated with vesicles.We have shown previously that the ROP effector ICR1 can interact with SEC3a and that ROPs can recruit SEC3a-ICR1 complexes to the plasma membrane (Lavy et al., 2007). However, ICR1 is not expressed in pollen tubes, suggesting that SEC3a membrane binding in these cells is likely dependent on other factors. In yeast, the interaction of Sec3p and Exo70p subunits with the plasma membrane is critical for exocyst function (He and Guo, 2009). It has been shown that the membrane binding of both Sec3p and Exo70p is facilitated by their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂; He et al., 2007; Zhang et al., 2008). The yeast Exo70p interacts with PIP₂ via a number of positively charged residues distributed along the protein, with the highest number located at the C-terminal end (Pleskot et al., 2015). It has been suggested that yeast Sec3p interacts with PIP₂ through N-terminal basic residues (Zhang et al., 2008). These data were further corroborated by x-ray crystallography studies, which showed that the yeast Sec3p N-terminal region forms a Pleckstrin homology (PH) domain fold (Baek et al., 2010; Yamashita et al., 2010), a PIP₂ interaction motif (Lemmon, 2008).The localization of the exocyst subunits has been addressed in several studies. In Arabidopsis root hairs and root epidermis cells, SEC3a-GFP was observed in puncta distributed throughout the cell (Zhang et al., 2013). Studies on the Arabidopsis EXO70 subunits EXO70E2, EXO70A1, and EXO70B1 revealed them to be localized in distinct compartments that were termed exocyst-positive organelles (Wang et al., 2010). The exocyst-positive organelles, visualized mostly by ectopic expression, were shown to be cytoplasmic double membrane organelles that can fuse with the plasma membrane and secrete their contents to the apoplast in an exosome-like manner. It is not yet known whether other exocyst subunits also are localized to the same organelles and what might be the biological function of this putative compartment (Wang et al., 2010; Lin et al., 2015). In differentiating xylem cells, two coiled-coil proteins termed VESICLE TETHERING1 and VESICLE TETHERING2 recruit EXO70A1-positive puncta to microtubules via the GOLGI COMPLEX2 protein (Oda et al., 2015). Importantly, the functionality of the XFP fusion proteins used for the localization studies described above was not tested, and in most cases, the fusion proteins were overexpressed. Therefore, the functional localization of the exocyst is still unclear.Here, we studied the function and subcellular localization of the Arabidopsis exocyst SEC3a subunit using a combination of genetics, cell biology, biochemistry, and structural modeling approaches. Our results show that SEC3a is essential for the determination of pollen tube tip germination site and growth. Partial complementation of sec3a resulted in the formation of pollen with multiple pollen tube tips. In Arabidopsis growing pollen tubes, SEC3a localization is dynamic, and it accumulates in domains of polarized secretion, at or close to the tip plasma membrane (PM). Labeling of GFP-SEC3-expressing pollen with FM4-64 revealed the spatial correlation between polarized exocytosis and endocytic recycling. Furthermore, the association of SEC3a with PM at the tip marks the direction of tube elongation and positively correlates with the deposition of PI-labeled pectins and specific anti-esterified pectin antibodies in the cell wall. In tobacco (Nicotiana tabacum), the mechanisms underlying SEC3a interaction with the PM and its subcellular distribution depend on pollen tube growth mode and involve the interaction with PIP2 through the N-terminal PH domain. Collectively, our results highlight the function of SEC3a as a polarity determinant that links between polarized exocytosis and cell morphogenesis. The correlation between exocyst function and distribution in pollen tubes provides an explanation for some of the current discrepancies regarding the localization of exocytosis.     

Essential Elements as a Distinguishing Factor between Mycorrhizal Potentials of Two Cohabiting Truffle Species in Riparian Forest Habitat in Serbia

True truffles (Tuber sp.) that establish ectomycorrhizal symbiosis (ECM) with trees in the Mediterranean and temporal regions have species specific abilities to assimilate soil born elements. Suitable habitats are usually inhabited by few truffle species, while distinguishing their symbiotic potentials appeared very difficult. Two species that commonly inhabit riparian forests in Serbia are the most prized one, Tuber magnatum Pico (Piedmont white truffle) and not so highly valued Tuber brumale Vitt . In order to assess potential differences between their assimilation and accumulation abilities, the differences between contents of elements that may be the subjects of the symbiotic trade between the host plant and fungi were evaluated in accumulation target (ascocarps) and their source (the soil). Essential (K, Na, Ca, Mg, Fe, P, S, and Zn) and essential trace elements (Co, Cr, Cu, Mn, and Se) in truffles and soil samples were determined by means of inductively coupled plasma with optical emission spectrometry (ICP‐OES). Their concentrations (mg/kg) in ascocarps were in the range from 1.364±0.591 (Cr) to 10760.862±16.058 (K), while in soil ranged from 23.035±0.010 (Cr) to 20809.300±122.934 (Fe). Element accumulation potential (bioaccumulation factor) was calculated in the system truffle/soil. The statistical approaches were used for establishing the differences, while the possible differentiation between symbiotic potentials of two mycelia in the defined soil conditions was discussed.     

The encounter of distinct Morimus asper (Coleoptera: Cerambycidae) phylogeographic lineages on the Balkan Peninsula: conservation implications

Journal of Insect Conservation - The first step in monitoring and management of any taxa includes resolving taxonomic ambiguities which complicate the identification of its management units. One of...     

Arsenic in Agricultural Soils of a Historically Mined and Industrial Region of Southern Serbia and Northern Kosovo: Bioavailability and Uptake by Plants Species Zea mays L. and Solanum tuberosum L.

This article reports the results of a study focused on the presence and bioavailability of arsenic in agricultural soil in the mining and industrial regions of northern Kosovo and southern Serbia, as well as uptake and bioaccumulation of arsenic in two commonly cultivated plant species (Zea mays L. and Solanum tuberosum L.). This area was one of the most important mining districts in Europe. The collected soil samples were subjected to a modified BCR three-step sequential extraction procedure in order to investigate the chemical partitioning of arsenic in the soils. The general distribution of arsenic in various fractions was: exchangeable < reducible < oxidizable fractions. Highest concentrations of total arsenic in soil were found close to industrial facilities and tailing ponds. In addition, fluvisols were significantly more enriched with arsenic than soils at a distance from the river flows. The edible parts of the plant specimen showed different As contents, suggesting that these plant species have different propensities for the uptake and bioaccumulation of arsenic from soil.     

Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.