Suppression of telomere capping defects of Saccharomyces cerevisiae yku70 and yku80 mutants by telomerase.

The Ku complex performs multiple functions inside eukaryotic cells, including protection of chromosomal DNA ends from degradation and fusion events, recruitment of telomerase, and repair of double-strand breaks (DSBs). Inactivation of Ku complex genes YKU70 or YKU80 in cells of the yeast Saccharomyces cerevisiae gives rise to mutants that exhibit shortened telomeres and temperature-sensitive growth. In this study, we have investigated the mechanism by which overexpression of telomerase suppresses the temperature sensitivity of yku mutants. Viability of yku cells was restored by overexpression of the Est2 reverse transcriptase and TLC1 RNA template subunits of telomerase, but not the Est1 or Est3 proteins. Overexpression of other telomerase- and telomere-associated proteins (Cdc13, Stn1, Ten1, Rif1, Rif2, Sir3, and Sir4) did not suppress the growth defects of yku70 cells. Mechanistic features of suppression were assessed using several TLC1 RNA deletion derivatives and Est2 enzyme mutants. Supraphysiological levels of three catalytically inactive reverse transcriptase mutants (Est2-D530A, Est2-D670A, and Est2-D671A) suppressed the loss of viability as efficiently as the wild-type Est2 protein, without inducing cell senescence. Roles of proteins regulating telomere length were also determined. The results support a model in which chromosomes in yku mutants are stabilized via a replication-independent mechanism involving structural reinforcement of protective telomere cap structures.

Construction of a genetically modified T7Select phage system to express the antimicrobial peptide 1018.

Bacteriophage therapy was an ascendant technology for combating bacterial infections before the golden age of antibiotics, but the therapeutic potential of phages was largely ignored after the discovery of penicillin. Recently, with antibiotic-resistant infections on the rise, these phages are receiving renewed attention to combat problematic bacterial infections. Our approach is to enhance bacteriophages with antimicrobial peptides, short peptides with broad-spectrum antibiotic or antibiofilm effects. We inserted coding sequences for 1018, an antimicrobial peptide previously shown to be an effective broad-spectrum antimicrobial and antibiofilm agent, or the fluorescent marker mCherry, into the T7Select phage genome. Transcription and production of 1018 or mCherry began rapidly alter E. coli cultures were infected with genetically modified phages. mCherry fluorescence, which requires a 90 min initial maturation period, was observed in infected cultures after 2 h of infection. Finally, we tested phages expressing 1018 (1018 T7) against bacterial planktonic cultures and biofilms, and found the 1018 T7 phage was more effective than the unmodified T7Select phage at both killing planktonic cells and eradicating established biofilms, validating our phage-driven antimicrobial peptide expression system. The combination of narrow-spectrum phages delivering relatively high local doses of broad-spectrum antimicrobials could be a powerful method to combat resistant infections. The experiments we describe prove this combination is feasible in vitro, but further testing and optimization are required before genetically modified phages are ready for use in vivo.

Optimization of a peptide extraction and LC-MS protocol for quantitative analysis of antimicrobial peptides.

We optimized a peptide extraction and LC-MS protocol for identification and quantification of antimicrobial peptides (AMPs) in biological samples. Amphipathic AMPs were extracted with various concentrations of ethanol, methanol, acetonitrile, formic acid, acetic acid or trichloroacetic acid in water. Yields were significantly greater for extraction with 66.7% ethanol than other extraction methods. Liquid chromatography was accomplished on a C18 column with a linear gradient of acetonitrile-formic acid, and mass spectrometry detection was performed in the positive electrospray multiple reaction monitoring mode by monitoring the transitions at m/z 385.2/239.2 and m/z 385.2/112.0 (AMP 1018), m/z 418.1/104.1 and m/z 418.1/175.1 (Methionine-1018). This method was shown to be reliable and efficient for the identification and quantification of scorpion AMPs Kn2-7 and its D-isomer dKn2-7 in human serum samples by monitoring the transitions at m/z 558.7/120.2 and m/z 558.7/129.1 (Kn2-7/dKn2-7).

Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom.

Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A2 (PLA2) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA2 activity of Western cottonmouth venom in vitro, using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy.

Effect of Losartan on Mitral Valve Changes After Myocardial Infarction.

BACKGROUND: After myocardial infarction (MI), mitral valve (MV) tethering stimulates adaptive leaflet growth, but counterproductive leaflet thickening and fibrosis augment mitral regurgitation (MR), doubling heart failure and mortality. MV fibrosis post-MI is associated with excessive endothelial-to-mesenchymal transition (EMT), driven by transforming growth factor (TGF)-ß overexpression. In vitro, losartan-mediated TGF-ß inhibition reduces EMT of MV endothelial cells. OBJECTIVES: This study tested the hypothesis that profibrotic MV changes post-MI are therapeutically accessible, specifically by losartan-mediated TGF-ß inhibition. METHODS: The study assessed 17 sheep, including 6 sham-operated control animals and 11 with apical MI and papillary muscle retraction short of producing MR; 6 of the 11 were treated with daily losartan, and 5 were untreated, with flexible epicardial mesh comparably limiting left ventricular (LV) remodeling. LV volumes, tethering, and MV area were quantified by using three-dimensional echocardiography at baseline and at 60 ± 6 days, and excised leaflets were analyzed by histopathology and flow cytometry. RESULTS: Post-MI LV dilation and tethering were comparable in the losartan-treated and untreated LV constraint sheep. Telemetered sensors (n = 6) showed no significant losartan-induced changes in arterial pressure. Losartan strongly reduced leaflet thickness (0.9 ± 0.2 mm vs. 1.6 ± 0.2 mm; p < 0.05; 0.4 ± 0.1 mm sham animals), TGF-ß, and downstream phosphorylated extracellular-signal-regulated kinase and EMT (27.2 ± 12.0% vs. 51.6 ± 11.7% α-smooth muscle actin-positive endothelial cells, p < 0.05; 7.2 ± 3.5% sham animals), cellular proliferation, collagen deposition, endothelial cell activation (vascular cell adhesion molecule-1 expression), neovascularization, and cells positive for cluster of differentiation (CD) 45, a hematopoietic marker associated with post-MI valve fibrosis. Leaflet area increased comparably (17%) in constrained and losartan-treated sheep. CONCLUSIONS: Profibrotic changes of tethered MV leaflets post-MI can be modulated by losartan without eliminating adaptive growth. Understanding the cellular and molecular mechanisms could provide new opportunities to reduce ischemic MR.

Myocardial Infarction Alters Adaptation of the Tethered Mitral Valve.

BACKGROUND: In patients with myocardial infarction (MI), leaflet tethering by displaced papillary muscles induces mitral regurgitation (MR), which doubles mortality. Mitral valves (MVs) are larger in such patients but fibrosis sets in counterproductively. The investigators previously reported that experimental tethering alone increases mitral valve area in association with endothelial-to-mesenchymal transition. OBJECTIVES: The aim of this study was to explore the clinically relevant situation of tethering and MI, testing the hypothesis that ischemic milieu modifies mitral valve adaptation. METHODS: Twenty-three adult sheep were examined. Under cardiopulmonary bypass, the papillary muscle tips in 6 sheep were retracted apically to replicate tethering, short of producing MR (tethered alone). Papillary muscle retraction was combined with apical MI created by coronary ligation in another 6 sheep (tethered plus MI), and left ventricular remodeling was limited by external constraint in 5 additional sheep (left ventricular constraint). Six sham-operated sheep were control subjects. Diastolic mitral valve surface area was quantified by 3-dimensional echocardiography at baseline and after 58 ± 5 days, followed by histopathology and flow cytometry of excised leaflets. RESULTS: Tethered plus MI leaflets were markedly thicker than tethered-alone valves and sham control subjects. Leaflet area also increased significantly. Endothelial-to-mesenchymal transition, detected as α-smooth muscle actin-positive endothelial cells, significantly exceeded that in tethered-alone and control valves. Transforming growth factor-ß, matrix metalloproteinase expression, and cellular proliferation were markedly increased. Uniquely, tethering plus MI showed endothelial activation with vascular adhesion molecule expression, neovascularization, and cells positive for CD45, considered a hematopoietic cell marker. Tethered plus MI findings were comparable with external ventricular constraint. CONCLUSIONS: MI altered leaflet adaptation, including a profibrotic increase in valvular cell activation, CD45-positive cells, and matrix turnover. Understanding cellular and molecular mechanisms underlying leaflet adaptation and fibrosis could yield new therapeutic opportunities for reducing ischemic MR.

Twelve-Hour Hypothermic Machine Perfusion for Donor Heart Preservation Leads to Improved Ultrastructural Characteristics Compared to Conventional Cold Storage.

BACKGROUND Hypothermic machine perfusion of donor hearts has the theoretical advantage of continuous aerobic metabolism and washes out toxic metabolic byproducts. Here, we studied the effect of hypothermic machine perfusion on cardiac myocyte integrity when hearts are preserved for longer ischemic times (12 hours). MATERIAL AND METHODS Pig hearts were harvested and stored in Celsior® solution for 12 hours using either conventional cold storage on ice (12 h CS, n=3) or pulsatile perfusion with the Paragonix Sherpa Perfusion™ Cardiac Transport System at different flow rates (12 h PP, n=3 or 12 h PP low flow, n=2). After cold preservation, hearts were reperfused using an LV isovolumic Langendorff system. Controls (n=3) were reperfused immediately after organ harvest. Biopsies were taken from the apex of the left ventricle before storage, after storage and after reperfusion to measure ATP and endothelin-1 content in the tissue. TUNEL staining for signs of apoptosis and electron microscopy of the donor hearts were performed. RESULTS 12 h PP hearts showed significantly more weight gain than 12 h CS and controls after preservation. Pulsatile perfused hearts showed less ATP depletion, lower endothelin-1 levels and less apoptosis after preservation compared to CS. Electron microscopy showed damaged muscle fibers, endothelial cell rupture, and injury of mitochondria in the 12 h CS group, while machine perfusion could preserve the cell structures. CONCLUSIONS Hypothermic machine perfusion of donor hearts can preserve the cell structures better than conventional cold storage in prolonged ischemic times. Hypothermic pulsatile perfusion may therefore enable longer preservation times of donor hearts. Whether this method is able to avoid primary graft failure after orthotopic heart transplantation remains to be evaluated in further studies.

Sex-specific molecular genetic response to UVB exposure in Xiphophorus maculatus skin.

In both Xiphophorus fishes and humans, males are reported to have a higher incidence of melanoma than females. To better understand sex-specific differences in the molecular genetic response to UVB, we performed RNA-Seq experiments in skin of female and male Xiphophorus maculatus Jp 163 B following UVB doses of 8 or 16kJ/m(2) exposure. Male X. maculatus differentially express a significantly larger number of transcripts following exposure to 16kJ/m(2) UVB (1293 genes) compared to 8kJ/m(2) UVB (324 genes). Female skin showed differential gene expression in a larger number of transcripts following 8kJ/m(2) UVB (765) than did males; however, both females and males showed similar numbers of differentially expressed genes at 16kJ/m(2) UVB (1167 and1293, respectively). Although most modulated transcripts after UVB exposure represented the same dominant pathways in both females and males (e.g., DNA repair, circadian rhythm, and fatty acid biosynthesis), we identified genes in several pathways that exhibited opposite modulation in female vs. male skin (e.g., synaptic development, cell differentiation, wound healing, and glucose metabolism). The oppositely modulated genes appear related through uncoupling protein 3 (UCP3) that is involved with the regulation of fatty acid oxidation and serves to balance glucose and lipid metabolism. Overall, these results identify gender-specific differences in UVB-induced genetic profiles in the skin of females and males and show female and male X. maculatus respond to UVB differently through pathways involved in reactive oxygen species, wound healing, and energy homeostasis.

Preservation of donor hearts using hypothermic oxygenated perfusion.

BACKGROUND: Hypothermic machine perfusion of donor hearts enables continuous aerobic metabolism and washout of toxic metabolic byproducts. We evaluated the effect of machine perfusion on cardiac myocyte integrity in hearts preserved for 4 h in a novel device that provides pulsatile oxygenated hypothermic perfusion (Paragonix Sherpa Perfusion™ Cardiac Transport System). MATERIAL AND METHODS: Pig hearts were harvested and stored in Celsior® solution for 4 h using either conventional cold storage on ice (4-h CS, n=6) or the Sherpa device (4-h pulsatile perfusion (PP), n=6). After cold preservation, hearts were evaluated using a non-working heart Langendorff system. Controls (n=3) were reperfused immediately after organ harvest. Biopsies were taken from the apex of the left ventricle before storage, after storage, and after reperfusion to measure ATP content and endothelin-1 in the tissue. Ultrastructural analysis using electron microscopy was performed. RESULTS: Four-hour CS, 4-h PP, and control group did not show any significant differences in systolic or diastolic function (+dP/dt, -dP/dt, EDP). Four-hour PP hearts showed significantly more weight gain than 4-h CS after preservation, which shows that machine perfusion led to myocardial edema. Four-hour CS led to higher endothelin-1 levels after preservation, suggesting more endothelial dysfunction compared to 4-h PP. Electron microscopy revealed endothelial cell rupture and damaged muscle fibers in the 4-h CS group after reperfusion, but the cell structures were preserved in the 4-h PP group. CONCLUSIONS: Hypothermic pulsatile perfusion of donor hearts leads to a better-preserved cell structure compared to the conventional cold storage method. This may lead to less risk of primary graft failure after orthotopic heart transplantation.

A novel model of accelerated intimal hyperplasia in the pig iliac artery.

There is no good animal model of large artery injury-induced intimal hyperplasia (IH). Those available are reproducible, providing only a few layers of proliferating cells or have the disadvantage of the presence of a metallic stent that complicates histology evaluation. This study was designed to develop a new, simple model of accelerated IH based on balloon injury in conjunction with disruption of the Internal Elastic Lamina (IEL) in pig external iliac arteries. Iliac artery injury (n = 24) was performed in 12 Yorkshire pigs divided in two groups: Group I (n = 10), overdistention injury induced by an oversized non-compliant balloon; Group II (n = 14), arterial wall disruption by pulling back an isometric cutting balloon (CB) followed by stretching with a compliant Fogarty Balloon (FB). At two weeks, arteries were processed for morphometric analysis and immunohistochemistry (IHC) for smooth muscle cells (SMC) and proliferating cell nuclear antigen (PCNA). When comparing the two groups, at 2 weeks, arteries of group II had a higher incidence of IH (100%vs. 50%, P = 0.0059), increased intimal areas (2.54 ± 0.33 mm(2) vs. 0.93 ± 0.36 mm(2) , P = 0.004), increased intimal area/Media area ratios (0.95 ± 0.1 vs. 0.28 ± 0.05; P < 0.0001) and decreased lumen areas (6.24 ± 0.44 vs. 9.48 ± 1.56, P = 0.026). No thrombosis was noticed in Group II. Neointima was composed by proliferating SMC located with the highest concentration in the area of IEL disruption (IHC). Arterial injury by pulling back CB and FB induces significant IH in pig iliac arteries by two weeks without thrombosis. This model is superior to the classical overdistention non-compliant model and should be useful and cost-effective for preclinical testing of procedures designed to inhibit IH in large peripheral arteries.

Active adaptation of the tethered mitral valve: insights into a compensatory mechanism for functional mitral regurgitation.

BACKGROUND: In patients with left ventricular infarction or dilatation, leaflet tethering by displaced papillary muscles frequently induces mitral regurgitation, which doubles mortality. Little is known about the biological potential of the mitral valve (MV) to compensate for ventricular remodeling. We tested the hypothesis that MV leaflet surface area increases over time with mechanical stretch created by papillary muscle displacement through cell activation, not passive stretching. METHODS AND RESULTS: Under cardiopulmonary bypass, the papillary muscle tips in 6 adult sheep were retracted apically short of producing mitral regurgitation to replicate tethering without confounding myocardial infarction or turbulence. Diastolic leaflet area was quantified by 3-dimensional echocardiography over 61+/-6 days compared with 6 unstretched sheep MVs. Total diastolic leaflet area increased by 2.4+/-1.3 cm(2) (17+/-10%) from 14.3+/-1.9 to 16.7+/-1.9 cm(2) (P=0.006) with stretch with no change in the unstretched valves despite sham open heart surgery. Stretched MVs were 2.8 times thicker than normal (1.18+/-0.14 versus 0.42+/-0.14 mm; P<0.0001) at 60 days with an increased spongiosa layer. Endothelial cells (CD31(+)) coexpressing alpha-smooth muscle actin were significantly more common by fluorescent cell sorting in tethered versus normal leaflets (41+/-19% versus 9+/-5%; P=0.02), indicating endothelial-mesenchymal transdifferentiation. alpha-Smooth muscle actin-positive cells appeared in the atrial endothelium, penetrating into the interstitium, with increased collagen deposition. Thickened chordae showed endothelial and subendothelial alpha-smooth muscle actin. Endothelial-mesenchymal transdifferentiation capacity also was demonstrated in cultured MV endothelial cells. CONCLUSIONS: Mechanical stresses imposed by papillary muscle tethering increase MV leaflet area and thickness, with cellular changes suggesting reactivated embryonic developmental pathways. Understanding such actively adaptive mechanisms can potentially provide therapeutic opportunities to augment MV area and reduce ischemic mitral regurgitation.

A novel model to test accuracy and reproducibility of MDCT scan protocols for coronary calcium in vivo.

OBJECTIVES: We compared the accuracy and reliability of prospectively triggered, retrospectively ECG gated, and non-gated CT image reconstruction for measurements of coronary artery calcification (CAC) in vivo using a novel animal model. MATERIALS AND METHODS: In six Yorkshire farm pigs, prefabricated chains of cortical bone fragments were sutured over the epicardial bed of the major coronary arteries. Using a 4-slice MDCT scanner, each animal was imaged with two different protocols: sequential acquisition with prospective ECG triggering, and spiral acquisition with retrospectively ECG gated image reconstruction- non-gated reconstructions were also generated from these latter scans. Two independent observers measured the 'Agatston score' (AS), the calcified volume (CV), and mineral mass (MM). To calculate accuracy of MM measurements the ash weight of the burned bone fragments was compared to MDCT derived MM. RESULTS: Six pigs successfully underwent surgery and CT imaging (mean heart rate: 86+/-12 bpm). MM measurements from prospectively ECG triggered CT sequential scans were more accurate (p<0.02) and reproducible (p=0.05) than sequential CT scans without ECG triggering or spiral acquisition using retrospective ECG gating. CONCLUSIONS: At high heart rates prospective ECG triggered image reconstruction is more accurate and reproducible for CAC scoring than retrospective ECG gated reconstruction and non-gated reconstruction.

A stentless trileaflet valve from a sheet of decellularized porcine small intestinal submucosa.

PURPOSE: The purpose of this study was to investigate the function of a trileaflet pulmonary valve constructed from a sheet of porcine small intestinal submucosa. DESCRIPTION: In four sheep, the native pulmonary valve and a segment of the pulmonary trunk was excised and replaced with a trileaflet valve constructed from decellularized porcine small intestinal submucosa. The valve construct was created from a sheet of the xenograft material by a method of involuting flaps of tissue inside a cylinder of itself. The function of the valve was assessed by echocardiography, catheter pullback across the valve, and observation of an excised valve in a flow simulator. EVALUATION: The valve constructs exhibited low gradients and symmetrical leaflet movement with good mobility when tested under physiologic conditions in an acute sheep model. CONCLUSIONS: This method offers a means to create a functional trileaflet valve replacement from a sheet of tissue.

Optimization of vessel orientation for robotic coronary artery bypass grafting.

The availability of telemanipulation robots has not yet resulted in the emergence of a reliable endoscopic coronary bypass procedure. A major challenge in performing a closed-chest coronary operation is creating a high-quality anastomosis in a reasonable period of time. In this experimental study, the impact of distal vessel orientation on the speed and accuracy of anastomosis was quantifed. We found that vessel orientation and the relative angle of the surgical plane influence anastomosis speed, the trauma to the vessel, the accuracy of stitch placement, and the eventual achievement of hemostasis. Our results suggest that the speed and accuracy of a robotically performed anastomosis of a vessel graft to a coronary artery can be improved by making small changes in vessel orientation. Vessels should be positioned between the horizontal and diagonal orientation and inclined between the horizontal and +45 degrees . Because the 6-o'clock stitch is particularly challenging, surgeons may benefit from an orientation that moves the heel or the toe of the anastomosis away from this critical position.

Continuous addition of adenosine with a micropump system improves warm whole blood cardioplegia.

BACKGROUND: Micropump additive systems allow for continuous modification of cardioplegia composition during heart surgery. Although the use of such systems in warm heart surgery is theoretically desirable, the role of the systems has been clinically limited by coronary vasoreactivity with higher potassium concentration and unreliable mechanical arrest at lower potassium concentration. Adenosine, a potent coronary vasodilator and arresting agent, has the potential to reduce the potassium concentration required for arrest and to improve distribution of cardioplegia. However, clinical use of adenosine has been limited by a short half-life in blood and difficulty in titrating the dose. This study tested the hypothesis that continuous addition of adenosine with an in-line linear micropump system would facilitate whole blood hyperkalemic perfusion for cardiac surgery. METHODS: Canine hearts (n = 9) were randomized to 20 minutes of arrest with whole blood cardioplegia or cardioplegia with adenosine at either low (0.5 M) or high (8 M) concentration. Potassium was supplemented at an arresting dose (24 mEq/L) for 5 minutes and then at a maintenance dose (6 mEq/L) for an additional 15 minutes. Coronary flow was held constant (4 mL/kg per minute), and aortic root pressure was measured. Myocardial performance was assessed by measurement of the end-diastolic pressure to stroke volume relationship at constant afterload. Myocardial tissue perfusion was evaluated with colored microspheres. RESULTS: During the initial period of high-concentration potassium arrest, coronary resistance rose progressively regardless of adenosine addition. Coronary resistance remained elevated during the period of low potassium perfusion, except when high-concentration adenosine was added. With addition of 8 M adenosine, coronary resistance returned to baseline, and left ventricular endocardial perfusion was augmented. Electromechanical quiescence improved with adenosine perfusion and was complete with high-dose adenosine addition. Function was preserved in all hearts. CONCLUSION: Use of a modern micropump system allowed for continuous addition of adenosine and potassium to whole blood cardioplegia. Adenosine minimized potassium-induced coronary vasoconstriction and improved endocardial perfusion and mechanical quiescence. These findings supported addition of adenosine to the perfusate during warm whole blood cardioplegia.

Endoscopic multivessel coronary artery bypass grafting using automated device for proximal anastomosis.

BACKGROUND: The use of computer-enhanced telemanipulation robots in cardiothoracic surgery can reduce the need for open surgical access and enable closed-chest, endoscopic procedures, but these procedures hav e been limited to anterior target vessels. The feasibility of fully endoscopic multivessel, coronary artery bypass grafting (CABG) was examined. METHODS: Fully endoscopic, multivessel CABG solely through surgical ports was performed on 23 dogs weighing 75 to 85 pounds. A proximal anastomosis was made with the Symmetry bypass system aortic connector. The aorta was cross clamped, and cardioplegia solution was administered through the vein graft into the ascending aorta. RESULTS: Eighteen of 23 procedures yielded successful proximal anastomoses and 1 to 3 distal anastomoses. CONCLUSION: Endoscopic anastomosis to the ascending aorta is feasible with the Symmetry bypass connector. Antegrade cardioplegia and aortic root venting can then be easily accomplished. This approach simplifies closed chest cardioplegic arrest for mulitivessel CABG.