16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

Biomechanical Comparison of Glutaraldehyde-Crosslinked Gelatin Fibrinogen Electrospun Scaffolds to Porcine Coronary Arteries.

Cardiovascular disease (CVD) is the leading cause of death for Americans. As coronary artery bypass graft surgery (CABG) remains a mainstay of therapy for CVD and native vein grafts are limited by issues of supply and lifespan, an effective readily available tissue-engineered vascular graft (TEVG) for use in CABG would provide drastic improvements in patient care. Biomechanical mismatch between vascular grafts and native vasculature has been shown to be the major cause of graft failure, and therefore, there is need for compliance-matched biocompatible TEVGs for clinical implantation. The current study investigates the biaxial mechanical characterization of acellular electrospun glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen cylindrical constructs, using a custom-made microbiaxial optomechanical device (MOD). Constructs crosslinked for 2, 8, and 24 hrs are compared to mechanically characterized porcine left anterior descending coronary (LADC) artery. The mechanical response data were used for constitutive modeling using a modified Fung strain energy equation. The results showed that constructs crosslinked for 2 and 8 hrs exhibited circumferential and axial tangential moduli (ATM) similar to that of the LADC. Furthermore, the 8-hrs experimental group was the only one to compliance-match the LADC, with compliance values of 0.0006±0.00018 mm Hg-1 and 0.00071±0.00027 mm Hg-1, respectively. The results of this study show the feasibility of meeting mechanical specifications expected of native arteries through manipulating GLUT vapor crosslinking time. The comprehensive mechanical characterization of cylindrical biopolymer constructs in this study is an important first step to successfully develop a biopolymer compliance-matched TEVG.

A local neighborhood volunteer network improves response times for simulated cardiac arrest.

AIM: Each minute is crucial in the treatment of out-of-hospital cardiac arrest (CA). Immediate chest compressions and early defibrillation are keys to good outcomes. We hypothesized that a coordinated effort of alerting trained local neighborhood volunteers (vols) simultaneously with 911 activation of professional EMS providers would result in substantial decreases in call-to-arrival times, leading to earlier CPR and defibrillation. METHODS: We developed a program of simultaneously alerting CPR- and AED-trained neighborhood vols and the local EMS system for CA events in a retirement residential neighborhood in Southern Arizona, encompassing approximately 440 homes. The closest EMS station is 3.3 miles from this neighborhood. Within this neighborhood, 15 vols and the closest EMS station were involved in multiple days of mock CA notifications and responses. RESULTS: The two groups differed significantly in distance to the mock CA event and in response times. The volunteers averaged 0.3 ±â€¯0.2 miles from the mock CA incidences while the closest EMS station averaged 3.4 ±â€¯0.1 miles away (p < 0.0001). Response times (time from call to arrival) also differed. Two volunteers, one bringing an AED, averaged 1 min 38 s ±â€¯53 s in Phase 1, while it took the EMS service an average of 7 min 20 s ±â€¯1 min 13 s to arrive on scene; p < 0.0001. CONCLUSION: Local neighborhood volunteers were geographically closer and arrived significantly sooner at the mock CA scene than did the EMS service. The approximate time savings from call to arrival with the volunteers was 4-6 min.

Bifidobacterium animalis subsp. lactis 420 mitigates the pathological impact of myocardial infarction in the mouse.

There is a growing appreciation that our microbial environment in the gut plays a critical role in the maintenance of health and the pathogenesis of disease. Probiotic, beneficial gut microbes, administration can directly attenuate cardiac injury and post-myocardial infarction (MI) remodelling, yet the mechanisms of cardioprotection are unknown. We hypothesised that administration of Bifidobacterium animalis subsp. lactis 420 (B420), a probiotic with known anti-inflammatory properties, to mice will mitigate the pathological impact of MI, and that anti-inflammatory T regulatory (Treg) immune cells are necessary to impart protection against MI as a result of B420 administration. Wild-type male mice were administered B420, saline or Lactobacillus salivarius 33 (Ls-33) by gavage daily for 14 or 35 days, and underwent ischemia/reperfusion (I/R). Pretreatment with B420 for 10 or 28 days attenuated cardiac injury from I/R and reduced levels of inflammatory markers. Depletion of Treg cells by administration of anti-CD25 monoclonal antibodies eliminated B420-mediated cardio-protection. Further cytokine analysis revealed a shift from a pro-inflammatory to an anti-inflammatory environment in the probiotic treated post-MI hearts compared to controls. To summarise, B420 administration mitigates the pathological impact of MI. Next, we show that Treg immune cells are necessary to mediate B420-mediated protection against MI. Finally, we identify putative cellular, epigenetic and/or post-translational mechanisms of B420-mediated protection against MI.

Papillary muscle repair surgery in ischemic mitral valve patients.

BACKGROUND: Ischemic mitral regurgitation (MR), when ischemia/infarction has resulted in fibrotic degeneration and elongation of papillary muscles, carries a high risk for the patient and a technical challenge for the surgeon. We have developed a papillary-shortening plasty for this specific pathology. METHODS: Papillary muscle repair was performed in 88 patients (7.2%) where degenerated and fibrotic elongated papillary muscles were found, which resulted in a prolapse of one or more parts of the mitral valve leaflets (MR III-IV). All patients had a papillary muscle-shortening plasty using a pericardium pledged-reinforced polytetrafluoroethylene suture and a ring annuloplasty. Because the cause of regurgitation in this specific group of patients was ischemic, concomitant coronary bypass grafting was required in all patients, with 2.2 grafts/patient. RESULTS: There were five hospital deaths (5.7%). Postoperative mitral valve function was satisfactory in all patients: no residual mitral regurgitation (MR 0) was found in 80 patients (90.9%), mild regurgitation (MR I) in 5 patients (5.7%), and moderate regurgitation (MR I-II) was observed in 3 patients (3.4%). Within a short mean follow-up period of 18.6 months (3 to 40 months), there was one late death (1.2%). The actuarial freedom from reoperation and thromboembolic complications was 100%, but there were two anticoagulation-induced gastric bleeding complications (2.3%). All patients were in New York Heart Association functional class I or II at the time of follow-up. CONCLUSIONS: Our data show that careful assessment of papillary muscle pathology is mandatory, and that a papillary muscle-shortening plasty is a simple but valuable surgical tool to repair the mitral valve in this specific group of high-risk patients with ischemic mitral regurgitation.

Polymeric endoluminal paving. A family of evolving methods for extending endoluminal therapeutics beyond stenting.

This article briefly reviews the background and rationale for the development of polymer paving. The process of endoluminal paving is described both in its generic form as well as in three experimental embodiments. Several experimental studies with two forms of paving, solid paving and gel paving, are reviewed. Finally, the envisioned future clinical role for both solid and gel paving is described.

Polymeric endoaortic paving: Mechanical, thermoforming, and degradation properties of polycaprolactone/polyurethane blends for cardiovascular applications.

Polymeric endoaortic paving (PEAP) is a process by which a polymer is endovascularly delivered and thermoformed to coat or "pave" the lumen of the aorta. This method may offer an improvement to conventional endoaortic therapy in allowing conformal graft application with reduced risk of endoleak and customization to complex patient geometries. Polycaprolactone (PCL)/polyurethane (PU) blends of various blend ratios were assessed as a potential material for PEAP by characterizing their mechanical, thermoforming and degradation properties. Biaxial tension testing revealed that the blends' stiffness is similar to that of aortic tissue, is higher for blends with more PCL content, and may be affected by thermoforming and degradation. Tubes of blends were able to maintain a higher diameter increase after thermoforming at higher PCL content and higher heating temperatures; 50/50 blend tubes heated to 55 °C were able to maintain 90% of the diameter increase applied. Delamination forces of the blends ranged from 41 to 235 N m⁻². In a Pseudomonas lipase solution, the 50/50 blend had a 94% lower degradation rate than pure PCL, and the 10/90 blend exhibited no degradation. These results indicate that PEAP, consisting of a PCL/PU blend, may be useful in developing the next generation of endoaortic therapy.

Application of intraluminal ultrasound imaging to vascular stenting.

Over the past few years catheter-based intraluminal ultrasound (IVUS) has emerged as a promising and imaginative technique which can significantly extend our understanding of atherosclerotic lesions both before and following interventions. In relation to stent implantation intravascular ultrasound appears well suited as an imaging modality--providing information as to lumen shape lesion surface and topography, as well as wall composition. To this end arteriography is unable to provide such detailed information. The purpose of this review is to outline the characteristics of stents and endoluminal support devices, currently under investigation, and the role of ultravascular ultrasound in this respect.

Polymeric endoluminal gel paving: therapeutic hydrogel barriers and sustained drug delivery depots for local arterial wall biomanipulation.

Polymeric endoluminal paving is a process in which biodegradable polymers may be locally applied percutaneously to blood vessels as endoluminal liners, resurfacing or 'paving', the underlying vascular wall. Depending upon the type of polymer selected, endoluminal polymer layers may function as wall supports, barriers, therapeutic biomaterials or depots for local sustained drug delivery. In the original description of the paving process, that is solid paving, structural polymers were utilized. In this article a second form of paving--gel paving is described. In this process, hydrogel polymers are locally applied or polymerized on vascular endoluminal surfaces. Endoluminal hydrogel layers have been demonstrated to function as physical non-pharmacological barriers limiting cell and protein deposition and effectively reducing underlying arterial wall thrombogenicity. Hydrogel paving layers also provide a means for prolonged local arterial wall drug delivery. In this report an update on gel paving is provided. The overall process of polymeric endoluminal paving is initially reviewed. Gel paving and the rationale for this approach is described. Both thermoreversible as well as photopolymerizable PEG-lactide hydrogel paving systems are outlined. Recent experimental studies with gel paving examining polymer application, haemocompatability and endoluminal surface thromboprotection, effects on post-injury neointimal thickening and local drug delivery, are then reviewed. Finally, the role of gel paving in future approaches to vascular therapy is discussed.

Pre-conditioning of smooth muscle cells via induction of the heat shock response limits proliferation following mechanical injury.

Arterial smooth muscle cell (SMC) proliferation is a significant component of post-angioplasty restenosis. We evaluated whether pre-conditioning of SMCs, via induction of the heat shock response prior to actual physical injury, would result in an alteration in cell proliferation following injury. Rat aortic SMCs were pretreated with either chemical or thermal heat shock inducers and then subjected to scrape-wound injury in vitro. Cell proliferation at 24 hrs was measured via 3H-thymidine (Tdr) incorporation and compared with scrape wounded unstressed controls. A significant decline in cell proliferation post scrape-wound injury was observed for both chemical and thermal heat shock pre-conditioned cultures, compared to untreated controls. Increased expression of heat shock protein 72 was confirmed serially throughout the 24 hr study period for both chemical and thermal inducers. Despite reduced proliferation heat shocked cells remained viable as evidenced by fluorescent cell viability assay and preserved migration. Pre-conditioning of SMCs through induction of the heat shock response prior to physical injury may be a useful approach to limit aggressive proliferation observed with mechanical revascularization injury.

Inhibition of thrombosis and intimal thickening by in situ photopolymerization of thin hydrogel barriers.

Thin hydrogel barriers formed on the inner surface of injured arteries by interfacial photopolymerization dramatically reduced thrombosis and intimal thickening in rat and rabbit models of vascular injury. This polymerization technique allowed the synthesis of a thin hydrogel barrier that conformed to the vessel wall, directly blocking contact between blood and the damaged vessel. The illumination conditions could be varied to control the thickness of the barrier from 10 microns to > 50 microns. The hydrogel was designed to degrade by nonenzymatic hydrolysis. In rats in which the carotid artery had been severely injured by crushing, treatment with the hydrogel barrier completely eliminated thrombosis (P < 0.01) and preserved long-term patency (P < 0.01). Treatment in a rabbit model of balloon injury inhibited thrombosis (P < 0.02) and reduced long-term intimal thickening by approximately 80% (P < 0.003). These results suggest that blood-borne signals acting in the early phases of healing play an important role in stimulating thickening of the intima.

Beta3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration.

BACKGROUND: Smooth muscle cell (SMC) migration is a vital component in the response of the arterial wall to revascularization injury. Cell surface integrin-extracellular matrix interactions are essential for cell migration. SMCs express both beta1- and beta3-integrins. In this study, we examined the relative functional roles of beta1- and beta3-integrin-matrix interactions in postinjury SMC migration. METHODS AND RESULTS: Flow cytometry and fluorescence microscopy of migrating SMCs immunostained with anti-beta1 and anti-alpha(v)beta3/5 antibodies (Abs) revealed expression of both beta1- and beta3-integrins, with beta1 observed as linear streaks and beta3 found in focal contacts. In a scrape-wound migration assay, anti-beta1 Abs (92.0+/-10.7% of control, P=.1) and 0.5 mmol/L linear RGD (105+/-5% of control, P=.2) did not alter SMC migration at 48 hours after injury. Beta3-blockade, however, via Abs (anti-beta3/5 35.7+/-4.5% of control, anti-beta3 61+/-12% of control, both P<.001) and cyclic RGD (0.5 mmol/L) (12+/-10% of control, P<.001) decreased migration. Neither beta1- nor beta3-inhibition altered postinjury [3H]thymidine incorporation. In the rat carotid injury model, local adventitial polymer-based delivery of radiolabeled linear or cyclic RGD led to uptake and retention of label, for both peptides, over a 72-hour period after injury. Local arterial wall beta1-blockade via polymer-based delivery of linear RGD had no effect on SMC migration at 4.5 days (11.5+/-3.2 versus 12.8 SMCs per x600 field [control], P=.6) or on neointimal thickening at 14 days (I/M area ratio, 0.664+/-0.328 versus 1.179+/-0.324 [control], P=.6) after injury. In contrast, local beta3-blockade via cRGD limited migration (0.8+/-0.8 versus 12.8+/-4.4 SMCs per x600 field [control], P<.01) and thickening (I/M area ratio, 0.004+/-0.008 versus 1.179+/-0.324 [control], P<.01). CONCLUSIONS: In postinjury migrating SMCs, beta3- rather than beta1-integrin-matrix interactions are of greater functional significance in adhesive processes essential for SMC migration in vitro and in vivo. Blockade of dominant SMC integrin (beta3)-matrix interactions may be a valuable approach for limiting injury-induced SMC migration and late arterial renarrowing.

Paradoxical effects of exercise on the QT interval in patients with polymorphic ventricular tachycardia receiving type Ia antiarrhythmic agents.

We analyzed the results of exercise testing performed in the absence of all antiarrhythmic drugs in 11 case patients with newly documented polymorphic ventricular tachycardia in response to type Ia antiarrhythmic agents. These results were compared with those found in 11 control patients matched for age, sex, and heart disease to determine whether the response of the QT interval to exercise testing was abnormal in patients who developed worsening of arrhythmia while taking antiarrhythmic drugs. QT, RR, and QTc intervals (by Bazett's method) were evaluated at rest and at 3 minutes of exercise in both groups. At rest, there was no significant difference in the QT interval (410 +/- 13 vs. 386 +/- 11 msec), RR interval (890 +/- 56 vs. 781 +/- 43 msec), or corrected QT interval (438 +/- 10 vs. 438 +/- 4 msec) in the case patients and the control patients. Both groups demonstrated a similar chronotropic response to exercise. The QT interval shortened in both groups with exercise (p less than 0.001), but the degree of shortening tended to be greater in the control patients (to 310 +/- 9 msec) than in the case patients (to 357 +/- 11 msec) (p = 0.06). Thus, there was a paradoxical increase in the QTc interval in the patients who experienced a proarrhythmic effect of type Ia drugs but not in the control patients (to 482 +/- 8 vs. 431 +/- 5 msec; p less than 0.001). Ten of 11 case patients but only one of 11 control patients had an increase in QTc interval of more than 10 msec with exercise (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)