Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Cardiac-Specific Inhibition of Kinase Activity in Calcium/Calmodulin-Dependent Protein Kinase Kinase-β Leads to Accelerated Left Ventricular Remodeling and Heart Failure after Transverse Aortic Constriction in Mice

Background

The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated.

Methods and Results

We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβ^kd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβ^kd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβ^kd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβ^kd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβ^kd TG mice compared with WT mice after TAC.

Conclusions

These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.     

Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

Background

We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm.

Methods and Results

We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F_2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice.

Conclusions

VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA.     

Role of EC-SOD Overexpression in Preserving Pulmonary Angiogenesis Inhibited by Oxidative Stress

Angiogenesis is one of the most important processes for normal lung development. Oxidative stress can impair the pulmonary angiogenesis, leading to chronic lung disease or Bronchopulmonary dysplasia (BPD).

Objective

To investigate the protective effects of EC-SOD overexpression on pulmonary angiogenesis on neonates following exposure to acute hyperoxia.

Design/Methods

Transgenic (TG) and wild-type (WT) neonatal mice (10 mice per group) were exposed either to air (control group) or 95% O₂ for 7 days starting at birth. After exposure, all animals were sacrificed. ROS concentration was measured in lung homogenates using OxiSelect ROS assay kit. Mean vascular density (MVD) was measured using anti CD34 staining. RNA was extracted and the angiogenesis markers, VEGF, VEGFR1 and VEGFR2 and PECAM-1 were analyzed by RT-q PCR. VGEF protein was measured using Western blots. Endothelial progenitor cells (EPCs) was assayed by flow cytometer.

Results

There was a significant reduction of ROS in TG hyperoxic neonate group (156±14.2) compared to WT hyperoxic animals (255±35.1). Evaluation of MVD, using anti-CD34, showed marked significant increase of MVD in the TG group following hyperoxic exposure (85±12) in comparison to the WT hyperoxic group (62±8.4), (P<0.05). Among the hyperoxic groups, both RNA and protein of VEGF expression were significantly reduced in the WT animals compared to the TG group (P<0.05). The same trend was found in VEGFR 1 and 2 which were significantly reduced in WT group compared to the TG group (P<0.05). There was no significant difference between hyperoxia TG and control group (P>0.05). PECAM expression was significantly reduced in both hyperoxic compared to normoxic groups (P<0.05). EPC’s showed significant reduction in WT hyperoxic group compared to others (P>0.05).

Conclusions

EC-SOD plays a key role in preserving angiogenesis by scavenging free radicals which has an inhibitory effect on angiogenesis process in neonatal mice lung following exposure to hyperoxia.     

Hepatic Serum Amyloid A1 Aggravates T Cell-mediated Hepatitis by Inducing Chemokines via Toll-like Receptor 2 in Mice

Serum amyloid A is a proinflammatory molecule that induces leukocyte infiltration and promotes neutrophil adhesion to endothelial cells under inflammatory conditions. The aim of this study was to examine whether Saa1 aggravates T cell-mediated hepatitis by inducing chemokines in a liver-specific, Saa1-overexpressing, transgenic (TG) mouse model. We generated TG mice in which Saa1 was overexpressed specifically in liver tissue. The chemokines monocyte chemotactic protein 1 (MCP1), MIP1α, MIP1β, interferon γ-induced protein 10 (IP-10), and eotaxin were induced in Saa1 TG mice. After concanavalin A treatment, Saa1 expression was higher in Saa1 TG mice than in WT mice. More severe liver injury, increased hepatocyte apoptosis, and higher levels of hepatic enzymes were observed in Saa1 TG mice than in WT mice. Liver infiltration of CD4⁺ T cells and macrophages increased after inducing hepatitis. Activation of T cells was higher in Saa1 TG mice than in WT mice, and the populations of Th17 cells and regulatory T cells were altered by overexpressing Saa1 in TG mice. Secretion of various cytokines, such as interferon γ, tumor necrosis factor α, and interleukin 6, increased in Saa1 TG mice. Injecting a Toll-like receptor 2 (TLR2) antagonist in vivo inhibited chemokine expression and IκBα phosphorylation and showed that the induction of chemokines by Saa1 was dependent on TLR2. Hepatic Saa1 accelerated T cell-mediated hepatitis by inducing chemokine production and activating T cells by TLR2. Therefore, Saa1 might be a novel inflammatory factor that acts as a chemokine modulator in hepatitis.     

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]_i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

Isolation of UV-sensitive variants of CHO-K1 by nylon cloth replica plating.

Techinques are described which permit the identification and isolation of UV-sensitive variants from mutagenized populations of Chinese hamster ovary (CHO) cells. Identification is based on the observation that within two days after receiving a dose of approximately 240 ergs/mm2 of UV irradiation most of the cells in a colony of CHO detach from the surface of a plastic tissue culture dish. At a lower dose of UV, which does not kill or detach a significant number of parental cells, UV-sensitive colonies are killed and become detached. Thus a clear plaque is produced in a lawn of unirradiated parental cells, marking the site occupied by a sensitive colony. Live cells from such sensitive colonies have been recovered from a nylon cloth replica prepared prior to irradiation and characterized. One UV-sensitive variant (CHO-UV-1) is indistinguishable from parental cells in X-ray resistance, chromosome number, generation time, and duration of the phases of the cell cycle. For UV irradiation the hit number (-n), shoulder width (Dq), and mean lethal dose (Do) for the variant are 2.8, 21 ergs/mm2, and 21 ergs/mm2, respectively, as compared to 2.6, 36 ergs/mm2, and 45 ergs/mm2 for CHO-K1 cells. These values have not changed for a period of eight months in culture.     

Culture at a Higher Temperature Mildly Inhibits Cancer Cell Growth but Enhances Chemotherapeutic Effects by Inhibiting Cell-Cell Collaboration

Acute febrile infections have historically been used to treat cancer. To explore the underlying mechanism, we studied chronic effects of fever on cancer cell growth and chemotherapeutic efficacy in cell culture. We found that culturing cancer cells at 39°C mildly inhibited cell growth by arresting the cells at the G1 phase of the cell cycle. When cells were seeded in culture dishes at a lower density, e.g. about 1000–2000 cells per 35-mm dish, the growth inhibition was much greater, manifested as many fewer cell colonies in the 39°C dishes, compared with the results at a higher density seeding, e.g. 20,000 cells per dish, suggesting that cell-cell collaboration as the Allee effect in cell culture is inhibited at 39°C. Withdrawal of cells from serum enhanced the G1 arrest at 39°C and, for some cell lines such as A549 lung cancer cells, serum replenishment failed to quickly drive the cells from the G1 into the S and G2-M phases. Therapeutic effects of several chemotherapeutic agents, including clove bud extracts, on several cancer cell lines were more potent at 39°C than at 37°C, especially when the cells were seeded at a low density. For some cell lines and some agents, this enhancement is long-lasting, i.e. continuing after the cessation of the treatment. Collectively these results suggest that hyperthermia may inhibit cancer cell growth by G1 arrest and by inhibition of cell-cell collaboration, and may enhance the efficacy of several chemotherapeutic agents, an effect which may persist beyond the termination of chemotherapy.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

A Large Cohort of Hemoglobin Variants in Thailand: Molecular Epidemiological Study and Diagnostic Consideration

Background

Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects.

Materials and Methods

Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques.

Results

Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α^{74GAC-CAC, Asp-His}) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β^{136GGT-GAT, Gly-Asp}) and Hb Tak (β^{146+AC, Ter-Thr}) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics.

Conclusions

Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.     

Reversal Learning and Associative Memory Impairments in a BACHD Rat Model for Huntington Disease

Chorea and psychiatric symptoms are hallmarks of Huntington disease (HD), a neurodegenerative disorder, genetically characterized by the presence of expanded CAG repeats (>35) in the HUNTINGTIN (HTT) gene. HD patients present psychiatric symptoms prior to the onset of motor symptoms and we recently found a similar emergence of non motor and motor deficits in BACHD rats carrying the human full length mutated HTT (97 CAG-CAA repeats). We evaluated cognitive performance in reversal learning and associative memory tests in different age cohorts of BACHD rats. Male wild type (WT) and transgenic (TG) rats between 2 and 12 months of age were tested. Learning and strategy shifting were assessed in a cross-maze test. Associative memory was evaluated in different fear conditioning paradigms (context, delay and trace). The possible confound of a fear conditioning phenotype by altered sensitivity to a ‘painful’ stimulus was assessed in a flinch-jump test. In the cross maze, 6 months old TG rats showed a mild impairment in reversal learning. In the fear conditioning tasks, 4, 6 and 12 months old TG rats showed a marked reduction in contextual fear conditioning. In addition, TG rats showed impaired delay conditioning (9 months) and trace fear conditioning (3 months). This phenotype was unlikely to be affected by a change in ‘pain’ sensitivity as WT and TG rats showed no difference in their threshold response in the flinch-jump test. Our results suggest that BACHD rats have a profound associative memory deficit and, possibly, a deficit in reversal learning as assessed in a cross maze task. The time course for the emergence of these symptoms (i.e., before the occurrence of motor symptoms) in this rat model for HD appears similar to the time course in patients. These data suggest that BACHD rats may be a useful model for preclinical drug discovery.     

Aberrant Forms of Streptococcus cremoris HP

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37°C or in the presence of penicillin. Serial plating and incubation at 37°C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25°C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30°C were transitional phase variants but at 37°C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.     

An interaction effect between glucokinase gene variation and carbohydrate intakes modulates the plasma triglyceride response to a fish oil supplementation

A large inter-individual variability in the plasma triglyceride (TG) response to fish oil consumption has been observed. The objective was to investigate the gene–diet interaction effects between single-nucleotide polymorphisms (SNPs) within glucokinase (GCK) gene and dietary carbohydrate intakes (CHO) on the plasma TG response to a fish oil supplementation. Two hundred and eight participants were recruited in the greater Quebec City area. The participants completed a 6-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Thirteen SNPs within GCK gene were genotyped using TAQMAN methodology. A gene–diet interaction effect on the plasma TG response was observed with rs741038 and CHO adjusted for age, sex and BMI (p = 0.008). In order to compare the plasma TG response between genotypes according to CHO, participants were divided according to median CHO. Homozygotes of the minor C allele of rs741038 with high CHO >48.59 % had a greater decrease in their plasma TG concentrations following the intake of fish oil (p < 0.05) than C/C homozygotes with low CHO and also than the other genotypes either with high or low CHO. The plasma TG response to a fish oil supplementation may be modulated by gene–diet interaction effects involving GCK gene and CHO.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0395-5) contains supplementary material, which is available to authorized users.     

The Yellow Gorgonian Eunicella cavolini: Demography and Disturbance Levels across the Mediterranean Sea

The yellow octocoral Eunicella cavolini is one of the most common gorgonians thriving in Mediterranean hard-bottom communities. However, information regarding its distribution and ecology in several parts of the Mediterranean is lacking, while population trends and conservation status remain largely unknown. We investigated 19 populations of E. cavolini over three representative geographic regions: the NW Mediterranean, CE Adriatic, and N Aegean. Focusing on the upper bathymetric range of the species (<40 m), data were collected on the populations’ upper depth limit, density, colony height, and extent of injury. A three-level hierarchical sampling design was applied to assess the existence of spatial patterns, using: a) regions (located thousands of km apart), b) localities within regions (tens to hundreds of km apart), and c) sites within localities (hundreds of m to a few km apart). In the NW Mediterranean and CE Adriatic, the upper distribution limit was at depths ≤15 m, whereas in the N Aegean most populations were found deeper than 30 m. Population density ranged between 4.46-62 colonies per m², while mean colony height was 15.6±8.9 SD cm with a maximum of 62 cm. The NW Mediterranean sites were characterized by dense populations dominated by small colonies (<20 cm), periodic recruitment, and low proportion of large gorgonians (>30 cm). The CE Adriatic displayed intermediate densities, with well-structured populations, and continuous recruitment. In the N Aegean, most populations presented low densities, high proportion of large colonies, but low number of small colonies, signifying limited recruitment. Disturbance levels, as a function of extent and type of injury, are discussed in relation to past or present human-induced threats. This work represents geographically the most wide ranging demographic study of a Mediterranean octocoral to date. The quantitative information obtained provides a basis for future monitoring at a Mediterranean scale.     

Azo Dye Colony Staining Technique To Detect Fungal Mutants Lacking β-Glucosidase Activity

A simple, rapid, convenient, easily interpreted azo dye colony-staining technique was developed to detect fungal mutants lacking β-glucosidase activity on a 6-bromo-2-naphthyl substrate. The procedure includes net replication of colonies and chemical restriction of colony growth to allow testing of many colonies per culture dish.     

α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current

Drug-induced long-QT syndrome (diLQTS) is often due to drug block of I_Kr, especially in genetically susceptible patients with subclinical mutations in the I_Kr-encoding KCHN2. Few variants in the cardiac Na_V1.5 Na⁺ channel complex have been associated with diLQTS. We tested whether a novel SNTA1 (α1-syntrophin) variant (p.E409Q) found in a patient with diLQTS increases late sodium current (I_Na-L), thereby providing a disease mechanism. Electrophysiological studies were performed in HEK293T cells co-expressing human Na_V1.5/nNOS/PMCA4b with either wild type (WT) or SNTA1 variants (A390V-previously reported in congenital LQTS; and E409Q); and in adult rat ventricular cardiomyocytes infected with SNTA1 expressing adenoviruses (WT or one of the two SNTA1 variants). In HEK293T cells and in cardiomyocytes, there was no significant difference in the peak I_Na densities among the SNTA1 WT and variants. However, both variants increased I_Na-L (% of peak current) in HEK293T cells (0.58±0.10 in WT vs. 0.90±0.11 in A390V, p = 0.048; vs. 0.88±0.07 in E409Q, p = 0.023). In cardiomyocytes, I_Na-L was significantly increased by E409Q, but not by A390V compared to WT (0.49±0.14 in WT vs.0.94±0.23 in A390V, p = 0.099; vs. 1.12±0.24 in E409Q, p = 0.019). We demonstrated that a novel SNTA1 variant is likely causative for diLQTS by augmenting I_Na-L. These data suggest that variants within the Na_V1.5-interacting α1-syntrophin are a potential mechanism for diLQTS, thereby expanding the concept that variants within congenital LQTS loci can cause diLQTS.     

Acute Administration of n-3 Rich Triglyceride Emulsions Provides Cardioprotection in Murine Models after Ischemia-Reperfusion

Dietary n-3 fatty acids (FAs) may reduce cardiovascular disease risk. We questioned whether acute administration of n-3 rich triglyceride (TG) emulsions could preserve cardiac function and decrease injury after ischemia/reperfusion (I/R) insult. We used two different experimental models: in vivo, C57BL/6 mice were exposed to acute occlusion of the left anterior descending coronary artery (LAD), and ex-vivo, C57BL/6 murine hearts were perfused using Langendorff technique (LT). In the LAD model, mice treated with n-3 TG emulsion (1.5g/kg body weight), immediately after ischemia and 1h later during reperfusion, significantly reduced infarct size and maintained cardiac function (p<0.05). In the LT model, administration of n-3 TG emulsion (300mgTG/100ml) during reperfusion significantly improved functional recovery (p<0.05). In both models, lactate dehydrogenase (LDH) levels, as a marker of injury, were significantly reduced by n-3 TG emulsion. To investigate the mechanisms by which n-3 FAs protects hearts from I/R injury, we investigated changes in key pathways linked to cardioprotection. In the ex-vivo model, we showed that n-3 FAs increased phosphorylation of AKT and GSK3β proteins (p<0.05). Acute n-3 TG emulsion treatment also increased Bcl-2 protein level and reduced an autophagy marker, Beclin-1 (p<0.05). Additionally, cardioprotection by n-3 TG emulsion was linked to changes in PPARγ protein expression (p<0.05). Rosiglitazone and p-AKT inhibitor counteracted the positive effect of n-3 TG; GSK3β inhibitor plus n-3 TG significantly inhibited LDH release. We conclude that acute n-3 TG injection during reperfusion provides cardioprotection. This may prove to be a novel acute adjunctive reperfusion therapy after treating patients with myocardial infarction.     

Accuracy of an experimental whole-blood test for detecting reactivation of echinococcal cysts

BackgroundCystic echinococcosis (CE) is a complex disease for which clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. We evaluated the accuracy of a whole-blood stimulation test based on Interleukin (IL)-4 detection in response to Antigen B (AgB) of Echinococcus granulosus sensu lato to discriminate cyst viability and detect cyst reactivation in patients with hepatic CE.Methodology/Principal findingsThirty patients with CE3b cysts and 37 patients with spontaneously-inactivated CE4-CE5 cysts were recruited (T0). After enrollment, 5 patients with CE3b cysts received albendazole, resulting in cyst solidification (CE4) in 4/5. Within a two-year follow-up, the whole-blood test was repeated at two time-points, in ≥14 (T1) and in ≥4 (T2) patients per group. IL-4 and a panel of other soluble factors were measured in the stimulated plasma.Baseline IL-4 levels were significantly higher in patients with CE3b compared to those with CE4 cysts (p = 0.006). Test accuracy for CE3b diagnosis had a sensitivity of 33–60% and a specificity of 76–95%, depending on the cut-off applied. Overall, IL-4 levels did not change significantly over time in either group; however, patients within the CE3b group showed a significant decrease of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF at T1 compared to T0 (p≤0.042).Conclusions/SignificanceWhole-blood IL-4-response to AgB is significantly higher in patients with active compared to inactive CE but apparently not modulated over time after treatment. On the contrary, the levels of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients’ follow-up.