Myocardial Fibrosis Induced by Exposure to Subclinical Lipopolysaccharide Is Associated with Decreased miR-29c and Enhanced NOX2 Expression in Mice

Background

Exposure to subclinical levels of lipopolysaccharide (LPS) occurs commonly and is seemingly well tolerated. However, recurrent LPS exposure induces cardiac fibrosis over 2 to 3 months in a murine model, not mediated by the renin-angiotensin system. Subclinical LPS induces cardiac fibrosis by unique mechanisms.

Methods

In C57/Bl6 mice, LPS (10 mg/kg) or saline (control) were injected intraperitoneally once a week for 1–4 weeks. Mice showed no signs of distress, change in activity, appetite, or weight loss. Mice were euthanized after 3 days, 1, 2, or 4 weeks to measure cardiac expression of fibrosis-related genes and potential mediators (measured by QRT-PCR), including micro-RNA (miR) and NADPH oxidase (NOX). Collagen fraction area of the left ventricle was measured with picrosirius red staining. Cardiac fibroblasts isolated from adult mouse hearts were incubated with 0, 0.1, 1.0 or 10 ng/ml LPS for 48 hours.

Results

Cardiac miR expression profiling demonstrated decreased miR-29c after 3 and 7 days following LPS, which were confirmed by QRT-PCR. The earliest changes in fibrosis-related genes and mediators that occurred 3 days after LPS were increased cardiac expression of TIMP-1 and NOX-2 (but not of NOX-4). This persisted at 1 and 2 weeks, with additional increases in collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, TIMP2, and periostin. There was no change in TGF-β or connective tissue growth factor. Collagen fraction area of the left ventricle increased after 2 and 4 weeks of LPS. LPS decreased miR-29c and increased NOX-2 in isolated cardiac fibroblasts.

Conclusions

Recurrent exposure to subclinical LPS induces cardiac fibrosis after 2–4 weeks. Early changes 3 days after LPS were decreased miR-29c and increased NOX2 and TIMP1, which persisted at 1 and 2 weeks, along with widespread activation of fibrosis-related genes. Decreased miR-29c and increased NOX2, which induce cardiac fibrosis in other conditions, may uniquely mediate LPS-induced cardiac fibrosis.     

The overlapping of local iron overload and HFE mutation in venous leg ulcer pathogenesis

Chronic venous stasis determines red blood cell extravasation and either dermal hemosiderin deposits or iron-laden phagocytes. Several authors have suspected that iron could play a role in the pathogenesis of venous leg ulcers. They hypothesized that local iron overload could generate free radicals or activate a proteolytic hyperactivity on the part of metalloproteinases (MMPs) or else down-regulate tissue inhibitors of MMPs. However, they were unable to explain why iron deposits, visible in the legs of patients with chronic venous disease (CVD), cause lesions in only some individuals, whereas in others they do not. We hypothesized that such individual differences could be genetically determined and investigated the role of the C282Y and H63D mutations of the HFE gene. C282Y mutation significantly increases the risk of ulcer in primary CVD more than six times (OR = 6.69; 1.45-30.8; p = 0.01). Patients carrying the H63D variant have an earlier age of ulcer onset, by almost 10 years (p > 0.004). The increased risk of skin lesion and the early age of onset of the disease in HFE carriers confirm in a clinical setting that intracellular iron deposits of mutated macrophages have less stability than those of the wild type. We hypothesize that the physiologic iron protective mechanisms are affected by the HFE mutations and should be investigated in all diseases characterized by the combination of iron overload and inflammation.     

Genetic variability of the narrow endemic Rhamnus persicifolia Moris (Rhamnaceae) and its implications for conservation

Rhamnus persicifolia Moris is an endemic small tree belonging to the Rhamnus cathartica group, growing along mountainous streams of Central-Eastern Sardinia (Italy). ISSR markers were used to detect the genetic diversity within and among six populations representative of the species distribution range. In spite of the limited distribution of this endemic taxon, fairly high levels of genetic diversity were detected. Percentage of polymorphic bands (PPB), gene diversity (H_S and H_T) and Shannon information measure (Sh) were calculated both at population (PPB = 30.70%, H_S = 0.1105, Sh = 0.1646) and at species level (PPB = 68.42%, H_T = 0.2066, Sh = 0.3139).The existence of a spatial distribution of genetic diversity in R. persicifolia was revealed by a low gene flow, a fairly high level of genetic differentiation (G_ST = 0.4583) among populations and a positive correlation between genetic and geographic distances (Mantel test, r = 0.71, p = 0.016). The spatial genetic structure was also confirmed with BAPS analysis. Our results show that a certain level of isolation by distance and sex-ratio bias may explain the distribution of genetic diversity among populations.Conservation measures are suggested on the basis of the genetic diversity detected, by implementing an integrated in situ and ex situ conservation program for each population, in order to ensure effective protection for this endemic species.     

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly <Superscript>15</Superscript>N labeled integral membrane proteins in magnetically aligned lipid bilayers

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring ¹H-¹⁵N dipolar couplings (DC) and ¹⁵N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([¹H,¹⁵N]-SE-PISEMA-PDSD). The incorporation of 2D ¹⁵N/¹⁵N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the ¹⁵N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.     

Two-dimensional solid-state NMR reveals two topologies of sarcolipin in oriented lipid bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.     

Comparing Alternative Methods for Holding Virgin Honey Bee Queens for One Week in Mailing Cages before Mating

In beekeeping, queen honey bees are often temporarily kept alive in cages. We determined the survival of newly-emerged virgin honey bee queens every day for seven days in an experiment that simultaneously investigated three factors: queen cage type (wooden three-hole or plastic), attendant workers (present or absent) and food type (sugar candy, honey, or both). Ten queens were tested in each of the 12 combinations. Queens were reared using standard beekeeping methods (Doolittle/grafting) and emerged from their cells into vials held in an incubator at 34C. All 12 combinations gave high survival (90 or 100%) for three days but only one method (wooden cage, with attendants, honey) gave 100% survival to day seven. Factors affecting queen survival were analysed. Across all combinations, attendant bees significantly increased survival (18% vs. 53%, p<0.001). In addition, there was an interaction between food type and cage type (p<0.001) with the honey and plastic cage combination giving reduced survival. An additional group of queens was reared and held for seven days using the best method, and then directly introduced using smoke into queenless nucleus colonies that had been dequeened five days previously. Acceptance was high (80%, 8/10) showing that this combination is also suitable for preparing queens for introduction into colonies. Having a simple method for keeping newly-emerged virgin queens alive in cages for one week and acceptable for introduction into queenless colonies will be useful in honey bee breeding. In particular, it facilitates the screening of many queens for genetic or phenotypic characteristics when only a small proportion meets the desired criteria. These can then be introduced into queenless hives for natural mating or insemination, both of which take place when queens are one week old.     

The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP^Sc). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP^C) into infectious PrP^Sc. By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP^Sc. In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP^C allotype to PrP^Sc in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP^Sc with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP^C containing an M or V at residue 129 having a similar propensity to misfold into PrP^Sc thus causing sCJD. By contrast, PrP^Sc with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP^Sc containing V at residue 129. In both types of CJD, the PrP^Sc allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP^Sc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.     

New findings on seed ecology of Ribes sardoum: can it provide a new opportunity to prevent the extinction of a threatened plant species?

Ribes sardoum, the most threatened endemic plant of Sardinia, is included in the Habitats Directive (92/43/EEC) and it was considered Critically Endangered in the global IUCN Red Lists. This species has been reported to have an extremely low fertility, scarce fruit production, low seed viability and a general inability to reproduce sexually. Fruits were collected for the first time from the remnant population, and the requirements for seed germination were investigated in the laboratory. Seeds were incubated at different temperatures (10, 15, and 20°C) and, in addition, they were exposed to a warm stratification (W) or a move-along treatment characterized by three cold temperature regimes (CCC). Seeds were also sown on the surface of 1% agar water with 250 mg·L^?1 of GA₃. At maturity, seeds have a linear underdeveloped embryo. Germination percentage between 35% and 65% were detected in the control and W groups. A low germination percentage occurred after CCC and during GA₃ treatment. W treatment speeds up seed germination. Our results demonstrate that fruits of R. sardoum produce viable seeds, that are able to germinate under controlled conditions, with the assumption that the seeds have morphophysiological dormancy (MPD), and that propagation from the seeds is possible. Although the ability of seed germination was demonstrated, the lack of seedlings in the natural population seems to be a consequence of unfavourable climatic conditions for recruitment. However, our results indicate that seedlings obtained under controlled conditions could be useful for future translocation reducing and/or mitigating the extinction likelihood of this highly threatened plant.     

Distribution and correlation of three oenological traits inSaccharomyces cerevisiae

The improvement of yeast starters for wine making, with classical genetic techniques, relays on the effective independence of the main oenological traits in order to combine them optimally. The analysis of three characters (ethanol production, volatile acidity and fermenting rate) in 787Saccharomyces cerevisiae strains, isolated from several parts of the world and substrates, shows moderate correlation between the volatile acidity and the other two traits, whereas ethanol production and fermenting rate appear more correlated. The minimum number of strains necessary to start selection or genetic improvement programs is discussed. The correlation between the oenological traits and the source of isolation showed that the best strains can be obtained from wineries and wine, whereas the isolates from grapes and must are less performing on the average.     

Effects of pre-treatments and temperature on seed viability and germination of Juniperus macrocarpa Sm.

The effects of collecting season, collection site, laboratory pre-treatments and temperatures on seed viability and germination of Juniperus macrocarpa were investigated. Ripe cones were collected in four Sardinian dune systems, in two seasons, from plant and soil. Warm (W) and cold (C) stratifications, two combinations of them (W + C, C + W), and no pre-treatment (0) were applied. Seeds were incubated in a range of constant (10–25 °C) and an alternating (25/10 °C) temperature regime. Seed viability was low (ca. 40%) and varied significantly according to the collecting season. Seed germination was also low (ca. 10%), the 0 and W were the most effective pre-treatments on stimulating germination. The best germination temperature, without any pre-treatment, was 15 °C (ca. 20%). J. macrocarpa seeds are dormant and the achieved results suggested that the presence of secondary dormancy is induced by cold stratification. Spring appeared to be the best season for seed collecting, whereas autumn was the best for sowing. These results give new findings for restoration activities on this species.