Using Telemedicine and Infographics for Physician-Guided Home Drain Removal.

OBJECTIVE: Measures to decrease hospital length of stay and outpatient visits are crucial during the coronavirus disease 2019 (COVID-19) pandemic. Physician-guided home drain removal presents a potential opportunity for mitigating viral spread and transmission. METHODS: A prospective case series on patients undergoing major head and neck surgery with Jackson-Pratt drain placement was conducted. Patients were shown an infographic detailing drain care and removal at preoperative assessment and prior to discharge. At a 1-week follow-up telemedicine visit, patients were instructed to remove the drain under physician guidance. Patients were assessed 7 days after to determine complication rate and satisfaction. RESULTS: Twenty-five patients were enrolled with 100% patients undergoing successful drain removal at home with caregiver support. There were no complications reported at the 7-day postdrain removal time point, and overall patient satisfaction was high. DISCUSSION: Infographics and telemedicine are 2 synergistic strategies to guide safe and effective home drain removal. IMPLICATIONS FOR PRACTICE: This study demonstrates how telemedicine and an infographic can be effectively used in physician-guided home drain removal. During a time like the COVID-19 pandemic, innovative measures are necessary to curb transmission and infection rates. We propose a unique and replicable yet safe solution to limit unnecessary exposure and encourage other surgical providers to adopt a similar strategy.

Rationale and design of the granulocyte-macrophage colony stimulating factor in peripheral arterial disease (GPAD-3) study.

BACKGROUND: Lower extremity peripheral arterial disease (PAD) is a public health problem and many patients with PAD experience claudication despite adequate medical and/or surgical management. Mobilization of endogenous progenitor cells using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a novel therapeutic option that has shown promising results in experimental models and phase I/IIA clinical trials. The GPAD-3 trial will study the effect of two successive administrations of GM-CSF at 3-month interval for improving claudication among patients with lower extremity PAD. METHODS: We plan to recruit 176 patients in this ongoing randomized, double-blind, placebo-controlled Phase IIB trial. After screening for inclusion and exclusion criteria, eligible subjects undergo a 4-week screening phase where they perform subcutaneous placebo injections thrice weekly and walk at least three times a day until they develop claudication. After the screening phase, eligible subjects undergo baseline testing and are randomized 2:1 to receive 500 µg/day of GM-CSF subcutaneously thrice weekly for three weeks or placebo injections. After 3 months, follow-up endpoint testing is performed and subjects in the GM-CSF group receive the second administration of the drug for three weeks while subjects in placebo group receive matching placebo injections. All participants undergo endpoint testing at six-month and nine-month follow-up. The primary endpoint is change in 6-min walk distance between baseline and 6-month follow-up. CONCLUSION: GPAD-3 explores a novel approach to address the need for alternative therapies that can alleviate symptoms among patients with lower extremity PAD. If successful, this study will pave the way for a pivotal Phase III trial.

The Impact of Different Diagnostic Imaging Modalities on the Evaluation of Root Canal Anatomy and Endodontic Residents' Stress Levels: A Clinical Study.

INTRODUCTION: The purpose of this study was firstly to compare the impact of radiographs, cone-beam computed tomographic (CBCT) imaging, and 3D Endo software (Dentsply Sirona, Ballaigues, Switzerland) on the assessment of root canal anatomy and radiographic quality of endodontic treatment and secondly to assess stress levels in the same cohort of residents performing endodontic treatment. METHODS: Sixty patients requiring primary molar endodontic treatment were allocated randomly into 3 groups: group 1 (n = 20), conventional radiographs (periapical radiography [PR]) only; group 2 (n = 20), PR and CBCT imaging; and group 3 (n = 20), PR, CBCT imaging, and 3D Endo software. All treatment was performed using a standardized protocol. Residents completed a questionnaire to assess their stress levels and usefulness of the imaging modality used. The radiographic quality of completed cases was assessed by 2 experienced endodontists who were not involved in the supervision of the cases being assessed. RESULTS: Groups 2 (CBCT imaging) and 3 (PR, CBCT imaging, and 3D Endo) proved significantly better than group 1 (PR) (P < .001) for assessing the number of root canals and anatomy and estimating the working lengths. Group 3 provided a significantly more accurate determination of the working level (P = .002). There were significantly more cases with obturation short of the apex (<2 mm) and voids in group 1 compared with group 3 (P < .05) and a significantly higher number of cases with voids in group 1 compared with group 3 (P < .01). Clinicians found treatment to be moderately or very stressful in 75%, 5%, and 0% in groups 1, 2, and 3, respectively. CONCLUSIONS: 3D Endo software followed by CBCT imaging were found to be more desirable for the evaluation of root canal anatomy and working lengths and reducing the residents' stress levels.

Autophagic response to cellular exposure to titanium dioxide nanoparticles.

Titanium dioxide is "generally regarded as safe" and titanium dioxide nanoparticles (TiO2 NPs) are used in a wide variety of consumer products. Cellular exposure to TiO2 NPs results in complex effects on cell physiology including induction of oxidative stress and impairment of lysosomal function, raising concerns about the impact of TiO2 NPs on biological systems. We investigated the effects of TiO2 NPs (15, 50, and 100 nm in diameter) on the lysosome-autophagy system, the main cellular catabolic pathway that mediates degradation of nanomaterials. Specifically, we monitored a comprehensive set of markers of the lysosome-autophagy system upon cell exposure to TiO2 NPs, ranging from transcriptional activation of genes required for the formation of autophagic vesicles to clearance of autophagic substrates. This study reveals that uptake of TiO2 NPs induces a response of the lysosome-autophagy system mediated by the transcription factor EB and consequent upregulation of the autophagic flux. Prolonged exposure to TiO2 NPs, however, was found to induce lysosomal dysfunction and membrane permeabilization, leading to a blockage in autophagic flux. Results from this study will inform the design of TiO2 NP based devices with specific autophagy-modulating properties.

Evaluation of resources for analyzing drug interactions.

OBJECTIVE: The research sought to evaluate seven drug information resources, specifically designed for analyzing drug interactions for scope, completeness, and ease of use, and determine the consistency of content among the seven resources. METHODS: A cross-sectional study was conducted where 100 drug-drug and drug-dietary supplement interactions were analyzed using 7 drug information resources: Lexicomp Interactions module, Micromedex Drug Interactions, Clinical Pharmacology Drug Interaction Report, Facts & Comparisons eAnswers, Stockley's Drug Interactions (10th edition), Drug Interactions Analysis and Management (2014), and Drug Interaction Facts (2015). The interaction sample was developed based on published resources and peer input. Two independent reviewers gathered data for each interaction from each of the 7 resources using a common form. RESULTS: Eighty-two drug-drug and 18 drug-dietary supplement interactions were analyzed. Scope scores were higher for Lexicomp Interactions (97.0%), Clinical Pharmacology Drug Interaction Report (97.0%), and Micromedex Drug Interactions (93.0%) compared to all other resources (p<0.05 for each comparison). Overall completeness scores were higher for Micromedex Drug Interactions (median 5, interquartile range [IQR] 4 to 5) compared to all other resources (p<0.01 for each comparison) and were higher for Lexicomp Interactions (median 4, IQR 4 to 5), Facts & Comparisons eAnswers (median 4, IQR 4 to 5), and Drug Interaction Facts (4, IQR 4 to 5) compared to all other resources, except Micromedex (p<0.05 for each comparison). Ease of use, in terms of time to locate information and time to gather information, was similar among resources. Consistency score was higher for Micromedex (69.9%) compared to all other resources (p<0.05 for each comparison). CONCLUSIONS: Clinical Pharmacology Drug Interaction Report, Lexicomp Interactions, and Micromedex Drug Interactions scored highest in scope. Micromedex Drug Interactions and Lexicomp Interactions scored highest in completeness. Consistency scores were overall low, but Micromedex Drug Interactions was the highest.

Hematopoietic stem cell-derived cancer-associated fibroblasts are novel contributors to the pro-tumorigenic microenvironment.

Targeting the tumor microenvironment is critical toward improving the effectiveness of cancer therapeutics. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types of the tumor microenvironment, playing an important role in tumor progression. Multiple origins for CAFs have been proposed including resident fibroblasts, adipocytes, and bone marrow. Our laboratory previously identified a novel hematopoietic stem cell (HSC) origin for CAFs; however, the functional roles of HSC-derived CAFs (HSC-CAFs) in tumor progression have not yet been examined. To test the hypothesis that HSC-CAFs promote tumor progression through contribution to extracellular matrix (ECM) and paracrine production of pro-angiogenic factors, we developed a method to isolate HSC-CAFs. HSC-CAFs were profiled on the basis of their expression of hematopoietic and fibroblastic markers in two murine tumor models. Profiling revealed production of factors associated with ECM deposition and remodeling. Functional in vivo studies showed that co-injection of HSC-CAFs with tumor cells resulted in increased tumor growth rate and significantly larger tumors than tumor cells alone. Immunohistochemical studies revealed increased blood vessel density with co-injection, demonstrating a role for HSC-CAFs in tumor vascularization. Mechanistic in vitro studies indicated that HSC-CAFs play a role in producing vascular endothelial growth factor A and transforming growth factor-ß1 in endothelial tube formation and patterning. In vitro and in vivo findings suggest that HSC-CAFs are a critical component of the tumor microenvironment and suggest that targeting the novel HSC-CAF may be a promising therapeutic strategy.

Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction.

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

Targeted overexpression of tissue inhibitor of matrix metalloproteinase-4 modifies post-myocardial infarction remodeling in mice.

RATIONALE: Myocardial infarction (MI) causes an imbalance between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMPs) and is associated with adverse left ventricular (LV) remodeling. A uniform reduction in TIMP-4 post-MI has been observed. OBJECTIVE: To examine post-MI remodeling with cardiac-restricted overexpression of TIMP-4, either through a transgenic or viral delivery approach. METHODS AND RESULTS: MI was induced in mice and then randomized to targeted injection of an adenoviral construct (10 µL; 8×10(9) plaque forming units/mL) encoding green fluorescent protein (GFP) and the full-length human TIMP-4 (Ad-GFP-TIMP4) or GFP. A transgenic construct with cardiac-restricted overexpression TIMP-4 (hTIMP-4exp) was used in a parallel set of studies. LV end-diastolic volume, an index of LV remodeling, increased by >60% from baseline at 5 days post-MI and by >100% at 21 days post-MI in the Ad-GFP only group. However, LV dilation was reduced by ≈50% in both the Ad-GFP-TIMP4 and hTIMP-4exp groups at these post-MI time points. LV ejection fraction was improved with either Ad-GFP-TIMP-4 or hTIMP-4exp. Fibrillar collagen expression and content were increased within the MI region with both TIMP-4 interventions, suggestive of matrix stabilization. CONCLUSIONS: This study is the first to demonstrate that selective myocardial targeting for TIMP-4 induction through either a viral or transgenic approach favorably altered the course of adverse LV remodeling post-MI. Thus, localized induction of endogenous matrix metalloproteinase inhibitors, such as TIMP-4, holds promise as a means to interrupt the progression of post-MI remodeling.

Mechanistic relationship between membrane type-1 matrix metalloproteinase and the myocardial response to pressure overload.

BACKGROUND: Although matrix metalloproteinases (MMPs) were initially thought to result primarily in extracellular matrix degradation, certain MMP types, such as membrane type-1 (MT1) MMP, may also be involved in profibrotic cascades through hydrolysis of latency-associated transforming growth factor-binding protein (LTBP-1) and activation of transforming growth factor-dependent profibrotic signaling. The present study tested the hypothesis that MT1-MMP plays a direct role in the matrix remodeling response to a left ventricular (LV) pressure overload (PO) stimulus. METHODS AND RESULTS: Wild-type (WT) and transgenic mice with cardiac-restricted MT1-MMP overexpression or MT1-MMP reduced expression underwent PO for 4 weeks. PO resulted in a 57% increase in LV mass (no change in LV end diastolic volume, resulting in an increase in the LV mass/volume ratio consistent with concentric remodeling), a 60% increase in MT1-MMP-mediated LTBP-1 hydrolysis and a 190% increase in collagen content in WT mice. Although LV mass was similar among WT, MT1-MMP overexpression, and MT1-MMP reduced expression after PO, significant differences in LV function, MT1-MMP-mediated LTBP-1 hydrolysis, and collagen content occurred. PO in MT1-MMP overexpression increased LTBP-1 hydrolysis (18%), collagen content (60%), and left atrial dimension (19%; indicative of LV diastolic dysfunction) when compared with WT. PO in MT1-MMP reduced expression reduced left atrial dimension (19%), LTBP-1 hydrolysis (40%), and collagen content (32%) when compared with both WT. CONCLUSIONS: Despite an equivalent PO stimulus and magnitude of LV myocardial growth, altering MT1-MMP levels caused specific matrix-dependent changes in remodeling, thereby demonstrating a mechanistic role in the development of the maladaptive remodeling and myocardial fibrotic response to PO.

Reproducible porcine model of thoracic aortic aneurysm.

BACKGROUND: Thoracic aortic aneurysms (TAAs) develop secondary to abnormal aortic extracellular matrix remodeling, resulting in a weakened and dilated aortic wall that progressed to rupture if left unattended. Currently, no diagnostic/prognostic tests are available for the detection of TAA disease. This is largely driven by the lack of a large animal model, which would permit longitudinal/mechanistic studies. Accordingly, the objective of the present study was to establish a reproducible porcine model of aortic dilatation, which recapitulates the structural and biochemical changes observed during human TAA development. METHODS AND RESULTS: Descending TAAs were induced in Yorkshire pigs (20-25 kg; n=7) through intra-adventitial injections of collagenase (5 mL, 0.35 mg/mL) and periadventitial application of crystalline CaCl2 (0.5 g). Three weeks after TAA induction, aortas were harvested and tissue was collected for biochemical and histological measurements. A subset of animals underwent MRI preoperatively and at terminal surgery. Results were compared with sham-operated controls (n=6). Three weeks after TAA induction, aortic luminal area increased by 38 ± 13% (P=0.018 versus control). Aortic structural changes included elastic lamellar degradation and decreased collagen content. The protein abundance of matrix metalloproteinases 3, 8, 9, and 12 increased in TAA tissue homogenates, whereas tissue inhibitors of metalloproteinases 1 and 4 decreased. CONCLUSIONS: These data demonstrate aortic dilatation, aortic medial degeneration, and alterations in matrix metalloproteinase/tissue inhibitors of metalloproteinase abundance, consistent with TAA formation. This study establishes for the first time a large animal model of TAA that recapitulates the hallmarks of human disease and provides a reproducible test bed for examining diagnostic, prognostic, and therapeutic strategies.

Pulmonary artery endothelial cell phenotypic alterations in a large animal model of pulmonary arteriovenous malformations after the Glenn shunt.

BACKGROUND: Longevity of the superior cavopulmonary connection (SCPC) is limited by the development of pulmonary arteriovenous malformations (PAVM). The goal of this study was to determine whether phenotypic changes in pulmonary artery endothelial cells (PAEC) that favor angiogenesis occur with PAVM formation. METHODS: A superior vena cava to right pulmonary artery connection was constructed in 5 pigs. Pulmonary arteries were harvested at 6 to 8 weeks after surgery to establish cultures of PAEC and smooth muscle cells, to determine cell proliferation, gene expression, and tubule formation. Abundance of proteins related to angiogenesis was measured in lung tissue. RESULTS: Contrast echocardiography revealed right-to-left shunting, consistent with PAVM formation. While the proliferation of smooth muscle cells from the right pulmonary artery (shunted side) and left pulmonary artery (nonshunted side) were similar, right PAEC proliferation was significantly higher. Expression profiles of genes encoding cellular signaling proteins were higher in PAECs from the right pulmonary artery versus left pulmonary artery. Protein abundance of angiopoietin-1, and Tie-2 (angiopoietin receptor) were increased in the right lung (both p < 0.05). Tubule formation was increased in endothelial cells from the right pulmonary artery compared with the left pulmonary artery (404 ± 16 versus 199 ± 71 tubules/mm(2), respectively; p < 0.05). CONCLUSIONS: These findings demonstrate that PAVMs developed in a clinically relevant animal model of SCPC concomitantly with differential changes in PAEC proliferative ability and phenotype. Moreover, there was a significant increase in the angiopoietin/Tie-2 complex in the right lung, which may provide novel therapeutic targets to attenuate PAVM formation after a SCPC.

Plasma biomarkers for distinguishing etiologic subtypes of thoracic aortic aneurysm disease.

BACKGROUND: Thoracic aortic aneurysms (TAAs) develop through an asymptomatic process resulting in gross dilation that progresses to rupture if left undetected and untreated. If detected, patients with TAA are followed over time until the risk of rupture outweighs the risk of surgical repair. Current methodologies for tracking TAA size are limited to expensive computed tomography or magnetic resonance imaging because no acceptable population screening tools are currently available. Previous studies from this laboratory and others have identified differential protein profiles for the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), in ascending TAA tissue from patients with bicuspid aortic valves (BAVs), versus patients with idiopathic degenerative disease and a tricuspid aortic valve (TAV). In addition, altered microRNA (miR) expression levels have also been reported in TAAs compared with normal aortic tissue. The objective of our study was to identify circulating factors within plasma that could serve as potential biomarkers for distinguishing etiologic subtypes of aneurysm disease. METHODS: Ascending TAA tissue and plasma specimens were obtained from patients with BAV (n = 21) and TAV (n = 21) at the time of surgical resection. The protein abundance of key MMPs (1, 2, 3, 8, and 9), TIMPs (1, 2, 3, and 4), and miRs (1, 21, 29a, 133a, 143, and 145) was examined using a multianalyte protein profiling system or by quantitative polymerase chain reaction, respectively. Results were compared with normal aortic tissue and plasma obtained from patients without aortic disease (n = 10). RESULTS: Significant (P < .05) differences in standardized miR-1 and miR-21 abundance between BAV and TAV aortic tissue samples and different tissue and plasma profiles of analyte differences from normal aorta where observed between the BAV and TAV groups. Linear regression analysis revealed significant linear relationships in plasma and tissue measurements only for MMP-8 and TIMP-1, TIMP-3, and TIMP-4 (P < .05). Receiver operator curve analysis revealed specific cassettes of analytes predictive of TAA disease. Relative to normal aorta, BAV proteolytic balance was significantly increased for MMP-1, MMP-2, and MMP-7, and for decreased MMP-8 and MMP-9. In contrast, TAV proteolytic balance relative to normal aorta was significantly increased only for MMP-1 and decreased for MMP-8 and MMP-9. CONCLUSIONS: Taken together, these unique data demonstrate differential plasma profiles of MMPs, TIMPs, and miRs in ascending TAA specimens from patients with BAV and TAV. These results suggest that circulating biomarkers may form the foundation for a broader platform of biomarkers capable of detecting the presence of TAA using a simple blood test and may also be useful in personalized strategies to distinguish between etiologic subtypes of TAAs in patients with aneurysm disease.

Direct regulation of membrane type 1 matrix metalloproteinase following myocardial infarction causes changes in survival, cardiac function, and remodeling.

The membrane type 1 matrix metalloproteinase (MT1-MMP) is increased in left ventricular (LV) failure. However, the direct effects of altered MT1-MMP levels on survival, LV function, and geometry following myocardial infarction (MI) and the proteolytic substrates involved in this process remain unclear. MI was induced in mice with cardiac-restricted overexpression of MT1-MMP (MT1-MMPexp; full length human), reduced MT1-MMP expression (heterozygous; MT1-MMP(+/-)), and wild type. Post-MI survival was reduced with MT1-MMPexp and increased with MT1-MMP(+/-) compared with WT. LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT post-MI and was higher in the MT1-MMP(+/-) mice. In vivo localization of MT1-MMP using antibody-conjugated microbubbles revealed higher MT1-MMP levels post-MI, which were the highest in the MT1-MMPexp group and the lowest in the MT1-MMP(+/-) group. LV collagen content within the MI region was higher in the MT1-MMPexp vs. WT post-MI and reduced in the MT1-MMP(+/-) group. Furthermore, it was demonstrated that MT1-MMP proteolytically processed the profibrotic molecule, latency-associated transforming growth factor-1-binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by over fourfold in the post-MI MT1-MMPexp group and reduced in the MT1-MMP(+/-) group, which was directionally paralleled by phospho-Smad-3 levels, a critical signaling component of the profibrotic transforming growth factor pathway. We conclude that modulating myocardial MT1-MMP levels affected LV function and matrix structure, and a contributory mechanism for these effects is through processing of profibrotic signaling molecules. These findings underscore the diversity of biological effects of certain MMP types on the LV remodeling process.

Continuous localized monitoring of plasmin activity identifies differential and regional effects of the serine protease inhibitor aprotinin: relevance to antifibrinolytic therapy.

BACKGROUND: Antifibrinolytic therapy, such as the use of the serine protease inhibitor aprotinin, was a mainstay for hemostasis after cardiac surgery. However, aprotinin was empirically dosed, and although the pharmacological target was the inhibition of plasmin activity (PLact), this was never monitored, off-target effects occurred, and led to withdrawn from clinical use. The present study developed a validated fluorogenic microdialysis method to continuously measure PLact and tested the hypothesis that standardized clinical empirical aprotinin dosing would impart differential and regional effects on PLact. METHODS/RESULTS: Pigs (30 kg) were instrumented with microdialysis probes to continuously measure PLact in myocardial, kidney, and skeletal muscle compartments (deltoid) and then randomized to high-dose aprotinin administration (2 mKIU load/0.5 mKIU/hr infusion; n = 7), low-dose aprotinin administration (1 mKIU load/0.250 mKIU/hr infusion; n = 6). PLact was compared with time-matched vehicle (n = 4), and PLact was also measured in plasma by an in vitro fluorogenic method. Aprotinin suppressed PLact in the myocardium and kidney at both high and low doses, indicative that both doses exceeded a minimal concentration necessary for PLact inhibition. However, differential effects of aprotinin on PLact were observed in the skeletal muscle, indicative of different compartmentalization of aprotinin. CONCLUSIONS: Using a large animal model and a continuous method to monitor regional PLact, these unique results demonstrated that an empirical aprotinin dosing protocol causes maximal and rapid suppression in the myocardium and kidney and in turn would likely increase the probability of off-target effects and adverse events. Furthermore, this proof of principle study demonstrated that continuous monitoring of determinants of fibrinolysis might provide a novel approach for managing fibrinolytic therapy.

Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system.

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the beta-tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446-bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of L-arginine, inadvertent gene silencing of bcass1 may result in partial L-arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild-type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or beta-glucuronidase using the pLOB1-based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.

Long-term localized high-frequency electric stimulation within the myocardial infarct: effects on matrix metalloproteinases and regional remodeling.

BACKGROUND: Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contributes to left ventricular wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high-frequency stimulation (LHFS) using low-amplitude electric pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning. METHODS AND RESULTS: At 3 weeks after MI, pigs were randomized for LHFS (n=7; 240 bpm, 0.8 V, 0.05-ms pulses) or were left unstimulated (UNSTIM; n=10). At 4 weeks after MI, left ventricular wall thickness (echocardiography; 0.89+/-0.07 versus 0.67+/-0.08 cm; P<0.05) and regional stiffness (piezoelectric crystals; 14.70+/-2.08 versus 9.11+/-1.24; P<0.05) were higher with LHFS than in UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage; 943+/-59 versus 1210+/-72 U; P<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower and TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all P<0.05). Transforming growth factor-beta receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electric stimulation (4 Hz) of isolated fibroblasts resulted in reduced MMP-2 and MT1-MMP levels but increased TIMP-1 levels compared with unstimulated fibroblasts. CONCLUSIONS: These unique findings demonstrate that LHFS of the MI region altered left ventricular wall thickness and material properties, likely as a result of reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify left ventricular remodeling after MI.

The pOT and pLOB vector systems: improving ease of transgene expression in Botrytis cinerea.

This paper outlines the construction of a novel vector system comprising interchangeable terminators, as well as a multiple cloning site (MCS), to facilitate the transformation of the fungal plant pathogen Botrytis cinerea. Previous molecular studies on B. cinerea have relied upon the pLOB1 based vector system (controlled by the Aspergillus nidulans oliC promoter and a region reported to be the B. cinerea tubA terminator). Investigations, however, have revealed that, rather than the genuine B. cinerea tubA terminator, the pLOB1 terminator fragment is from another gene locus within the genome. Because previous studies have found that terminators aide in transcript stability, the main aims of this study were to develop and evaluate both vector systems, pOT (controlled by the A. nidulans oliC promoter and A. nidulans trpC terminator) and pLOB, with a range of exogenous genes, including enhanced green fluorescent protein (eGFP), monomeric red fluorescent protein (mRFP), luciferase (LUC) and beta-glucuronidase (GUS). Our investigations demonstrate that pLOB and pOT based vectors are capable of expressing all four reporter genes and may be applied to future molecular studies on B. cinerea and other related ascomycetes. Additionally, this is the first reported expression of mRFP and LUC in B. cinerea.