Experimental Myocardial Infarction Induces Altered Regulatory T Cell Hemostasis,and Adoptive Transfer Attenuates Subsequent Remodeling

Background

Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling.

Methods and Results

The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling.

Conclusion

Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.     

Selector Screening for Enantioseparation of dl‐α‐Methyl Phenylglycine Amide by Liquid–Liquid Extraction

Enantioseparation through liquid extraction technology is an emerging field, e.g., enantioseparations of amino acids (and derivatives thereof), amino alcohols, amines, and carboxylic acids have been reported. Often, when a new selector is developed, the versatility of substrate scope is investigated. From an industrial point of view, the problem is typically approached the other way around, and for a target racemate, a selector needs to be found in order to accomplish the desired enantioseparation. This study presents such a screening approach for the separation of the enantiomers of dl ‐α‐methyl phenylglycine amide (dl ‐α‐MPGA), a model amide racemate with high industrial relevance. Chiral selectors that were reported for other classes of racemates were investigated, i.e., several macrocyclic selectors and Pd‐BINAP complexes. It appeared very challenging to obtain both high extraction yields and good enantioselectivity for most selectors, but Pd‐BINAP‐based selectors performed well, with enantioselectivities up to 7.4 with an extraction yield of the desired enantiomer of 95.8%. These high enantioselectivities were obtained using dichloromethane as solvent. Using less volatile chlorobenzene or 1‐chloropentane, reasonable selectivities of up to 1.7 were measured, making these the best alternative solvents for dichloromethane. Chirality 27:123–130, 2015. © 2014 Wiley Periodicals, Inc.     

Analyses of volatiles produced by the African fruit fly species complex (Diptera,Tephritidae)

Ceratitis fasciventris, Ceratitis anonae and Ceratitis rosa are polyphagous agricultural pests originating from the African continent. The taxonomy of this group (the so-called Ceratitis FAR complex) is unclear. To clarify the taxonomic relationships, male and female-produced volatiles presumably involved in pre-mating communication were studied using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) followed by multivariate analysis, and gas chromatography combined with electroantennographic detection (GC-EAD). GC×GC-TOFMS analyses revealed sex specific differences in produced volatiles. Male volatiles are complex mixtures that differ both qualitatively and quantitatively but share some common compounds. GC-EAD analyses of male volatiles revealed that the antennal sensitivities of females significantly differ in the studied species. No female volatiles elicited antennal responses in males. The results show clear species-specific differences in volatile production and provide complementary information for the distinct delimitation of the putative species by chemotaxonomic markers.     

On the relationship between the non-photochemical quenching of the chlorophyll fluorescence and the Photosystem II light harvesting efficiency. A repetitive flash fluorescence induction study

Plants respond to excess light by a photoprotective reduction of the light harvesting efficiency. The notion that the non-photochemical quenching of chlorophyll fluorescence can be reliably used as an indicator of the photoprotection is put to a test here. The technique of the repetitive flash fluorescence induction is employed to measure in parallel the non-photochemical quenching of the maximum fluorescence and the functional cross-section (sigma(PS II)) which is a product of the photosystem II optical cross-section a(PS II) and of its photochemical yield Phi(PS II) (sigma (PS II) = a(PS II) Phi(PS II)). The quenching is measured for both, the maximum fluorescence found in a single-turnover flash (F(M) (ST)) and in a multiple turnover light pulse (F(M) (MT)). The experiment with the diatom Phaeodactylum tricornutum confirmed that, in line with the prevalent model, the PS II functional cross-section sigma (PS II) is reduced in high light and restored in the dark with kinetics and amplitude that are closely matching the changes of the F(M) (ST) and F(M) (MT) quenching. In contrast, a poor correlation between the light-induced changes in the PS II functional cross-section sigma (PS II) and the quenching of the multiple-turnover F(M) (MT) fluorescence was found in the green alga Scenedesmus quadricauda. The non-photochemical quenching in Scenedesmus quadricauda was further investigated using series of single-turnover flashes given with different frequencies. Several mechanisms that modulate the fluorescence emission in parallel to the Q(A) redox state and to the membrane energization were resolved and classified in relation to the light harvesting capacity of Photosystem II.     

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.     

Dependence of the Q10 values on the depth of the soil temperature measuring point

The parameter Q₁₀ is commonly used to express the relationship between soil CO₂ efflux and soil temperature. One advantage of this parameter is its application in a model expression of respiration losses of different ecosystems. Correct specification of Q₁₀ in these models is indispensable. Soil surface CO₂ efflux and soil temperature at different depths were measured in a 21-year-old Norway spruce stand and a mountain grassland site located at the Experimental Ecological Study Site Bily Kriz, Beskydy Mts. (NE Czech Republic), using automated gasometric systems. A time-delay and goodness-of-fit between soil CO₂ efflux and soil temperature at different measuring depths were determined. Wide ranges of values for the time-delay of CO₂ efflux in response to temperature, Q₁₀ and the determination coefficient (R²) between CO₂ efflux and temperature were obtained at the both sites. The values of Q₁₀ and the CO₂ time-delay increased with depth, while the R² of the CO₂-temperature relationship significantly decreased. Soil temperature records obtained close to the soil surface showed the highest values of R² and the lowest value of the time-delay at both sites. Measurement of soil temperature at very shallow soil layer, preferably at the soil surface, is highly recommended to determine useable values of Q₁₀. We present a new procedure to normalize Q₁₀ values for soil temperatures measured at different depths that would facilitate comparison of different sites.     

Single point mutations in the zinc finger motifs of the human immunodeficiency virus type 1 nucleocapsid alter RNA binding specificities of the gag protein and enhance packaging and infectivity

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Psi) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.     

Epidermal UV-shielding and photosystem II adjustment in wild type and chlorina f2 mutant of barley during exposure to increased PAR and UV radiation

The wild-type barley (WT; Hordeum vulgare L.) and its chlorophyll (Chl) b-less mutant chlorina f2 (clo f2) grown under shaded conditions in a greenhouse were transferred to outdoor conditions in early June with predominantly bright sunny days. During 6 days following transfer of plants we monitored the content of photosynthetic pigments, functional state of photosystem II (PSII) by means of Chl fluorescence induction kinetics and epidermal UV-shielding efficiency using Chl fluorescence imaging technique. Clo f2 mutant was more sensitive to exposure to an enhanced natural solar irradiance than WT barley. Nevertheless, clo f2 as well as WT were able to cope with stressful outdoor conditions, as was documented by the recovery of Chl a content and the maximal photochemical efficiency of PSII (F_V/F_M) after an initial decline. This was due to the immediate carotenoid-mediated photoprotection, reflected by strongly increased total carotenoids content and thermal energy dissipation localized within light-harvesting complexes of PSII (assessed by non-photochemical quenching of minimal fluorescence level). The positive acclimation response was further documented by an enhanced light-saturated electron transport rate through PSII (ETR). Based on the ratios of blue- to UV-excited Chl fluorescence we found that for both WT and clo f2 epidermal UV-shielding increased clearly after transfer to outdoor conditions and reached a saturation level after 3 days. In comparison with WT, clo f2 exhibited lower ability to induce UV-shielding. The kinetics of UV-shielding development during the outdoor treatment was different for the particular leaf regions. We suggest that this is related to the different age and developmental stage of the tissue along the leaf blade. The complementarity of carotenoid-mediated photoprotection and UV-shielding in acclimation of the assimilatory apparatus to increased visible and UV radiation is discussed.     

Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory‐reared and field‐collected harlequin ladybirds (Harmonia axyridis). For laboratory‐reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field‐collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect’s ability to respond to an immune challenge is significantly reduced in the course of overwintering.