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Christopher M Bloom,1 Ryan J Post,1 Joshua Mazick,1 Brittany Blumenthal,1 Caroline Doyle,1 Brenna Peters,1 Jeff Dyche,2 D Gene Davenport3 1Providence College, Providence, RI, USA; 2James Madison University, Harrisonburg, VA, USA; 3Saint Louis University, St Louis, MO, USA Abstract: Traditionally, the signaled avoidance (SA) paradigm has been used in an attempt to better understand human phobia. Animal models of this type have been criticized for ineffectively representing phobia. The SA model characterizes phobia as an avoidance behavior by presenting environmental cues, which act as warning signals to an aversive stimulus (ie, shock). Discriminated conditioned punishment (DCP) is an alternative paradigm that characterizes phobia as a choice behavior in which fear serves to punish an otherwise adaptive behavior. The present study quantifies the differences between the paradigms and suggests that DCP offers an alternative paradigm for phobia. Rats trained on either SA or DCP were compared on a number of behavioral variables relevant to human phobia. Results indicate that rats in the DCP paradigm responded significantly earlier to warning signals and were more effective at preventing shocks than rats in the SA paradigm. Implications of this alternative paradigm are discussed. Keywords: animal models, avoidance, fear conditioning, anxiety disorders

User interface designers are challenged to design for diverse users, including those of different genders, cultures and abilities; however, little research has been directed at this problem. One factor which may inhibit such research is its cost. This paper presents an approach which offers a way to seek out important characteristics of designs in a cost-effective way and reports on the results. In a study reported here, subjects from different nationalities and of both genders evaluated three dialog boxes specifically designed for 'white American women'. 'European adult male intellectuals', and 'English-speaking-internationals'. The dialog boxes were evaluated with conjoint techniques of preference rankings, and factor-analysed adjective ratings. These results showed that female subjects had stronger and more consistent patterns of preferences than the male subjects. All subjects preferred interfaces rated high on an accessibility factor and disliked complex layouts; this effect was even stronger for women. Nationality did not effect ratings. Gender had a stronger effect on the outcome than nationality. [ABSTRACT FROM AUTHOR]

Generally injuries that remove or disrupt the endothelial cells lining blood vessels stimulate formation of vascular lesions composed of smooth muscle cells. One of the first events after such endothelial cell disruption is the generation of thrombin at the site of injury. This leads to platelet activation and thrombus formation. Evidence suggests that thrombus formation may stimulate smooth muscle-cell proliferation through the action of any number of factors emanating from the thrombus including platelet- or macrophage-derived factors, platelet-derived growth factor, basic fibroblast growth factor or thrombin. Atherosclerotic plaques continue to grow for many years. The slow indolent process of nondenuding chemical injury of the endothelium and lesion formation may be accelerated periodically by thrombi forming on the lumenal surface at sites of small denuding injuries leading to progressive atherosclerotic disease. Genistein, an isoflavonoid derived from soy products, has been shown to inhibit thrombin formation and platelet activation in vitro in addition to its antigrowth factor activity. Should it have similar actions in vivo, this compound has the potential to affect the progression of atherosclerotic disease by modifying coagulation responses. To assess the potential of genistein as a therapeutic for vascular disease, additional studies will be required to establish its effect on experimental vascular lesion formation. [ABSTRACT FROM AUTHOR]

El Niño events owe their name to their tendency to be locked to the seasonal cycle. A simple explanation is proposed here for the locking of the peak of ENSO's basin-scale warming to the end of the calendar year. The explanation is based on incorporating a seasonally varying coupled ocean--atmosphere instability strength into the delayed oscillator mechanism for the ENSO cycle. It is shown that the seasonally varying amplification of the Rossby and Kelvin ocean waves by the coupled instability forces the events to peak when this amplification is at its minimum strength, at the end of the calendar year. The mechanism is demonstrated using a simple delayed oscillator model and is further analyzed using the Cane--Zebiak model. Being based on the oversimplified delayed oscillator paradigm of ENSO, the proposed mechanism cannot be expected to fully explain the locking of observed events to the end of the year. However, the wave dynamics perspective it offers to approaching the ENSO phase-locking problem may serve as a first step toward a fuller explanation based on more realistic models and additional data analysis. [ABSTRACT FROM AUTHOR]

Vertebral body aplasia or hypoplasia leads to rapidly progressive spinal deformity and neurologic defects when unrecognized in infancy. Four patients with this unusual defect are described. [ABSTRACT FROM AUTHOR]

Abstract Periodic epidemics of malaria are a major public health problem for many sub-Saharan African countries. Populations in epidemic prone areas have a poorly developed immunity to malaria and the disease remains life threatening to all age groups. The impact of epidemics could be minimized by prediction and improved prevention through timely vector control and deployment of appropriate drugs. Malaria Early Warning Systems are advocated as a means of improving the opportunity for preparedness and timely response. Rainfall is one of the major factors triggering epidemics in warm semi-arid and desert-fringe areas. Explosive epidemics often occur in these regions after excessive rains and, where these follow periods of drought and poor food security, can be especially severe. Consequently, rainfall monitoring forms one of the essential elements for the development of integrated Malaria Early Warning Systems for sub-Saharan Africa, as outlined by the World Health Organization. The Roll Back Malaria Technical Resource Network on Prevention and Control of Epidemics recommended that a simple indicator of changes in epidemic risk in regions of marginal transmission, consisting primarily of rainfall anomaly maps, could provide immediate benefit to early warning efforts. In response to these recommendations, the Famine Early Warning Systems Network produced maps that combine information about dekadal rainfall anomalies, and epidemic malaria risk, available via their Africa Data Dissemination Service. These maps were later made available in a format that is directly compatible with HealthMapper, the mapping and surveillance software developed by the WHO's Communicable Disease Surveillance and Response Department. A new monitoring interface has recently been developed at the International Research Institute for Climate Prediction (IRI) that enables the user to gain a more contextual perspective of the current rainfall estimates by comparing them to previous seasons and climatological averages. These resources are available at no cost to the user and are updated on a routine basis.

Objective: To compare the safety of sleeve gastrectomy and gastric bypass in a large cohort of commercially insured bariatric surgery patients from the IBM MarketScan claims database, while accounting for measurable and unmeasurable sources of selection bias in who is chosen for each operation., Summary of Background Data: Sleeve gastrectomy has rapidly become the most common bariatric operation performed in the United States, but its longer-term safety is poorly described, and the risk of worsening gastroesophageal reflux requiring revision may be higher than previously thought. Prior studies comparing sleeve gastrectomy to gastric bypass are limited by low sample size (in randomized trials) and selection bias (in observational studies)., Methods: Instrumental variables analysis of commercially insured patients in the IBM MarketScan claims database from 2011 to 2018. We studied patients undergoing bariatric surgery from 2012 to 2016. We identified re-interventions and complications at 30 days and 2 years from surgery using Comprehensive Procedural Terminology and International Classification of Disease (ICD)-9/10 codes. To overcome unmeasured confounding, we use the prior year's sleeve gastrectomy utilization within each state as an instrumental variable-exploiting variation in the timing of payers' decisions to cover sleeve gastrectomy as a natural experiment., Results: Among 38,153 patients who underwent bariatric surgery between 2012 and 2016, the share of sleeve gastrectomy rose from 52.6% (2012) to 75% (2016). At 2 years from surgery, patients undergoing sleeve gastrectomy had fewer re-interventions (sleeve 9.9%, bypass 15.6%, P < 0.001) and complications (sleeve 6.6%, bypass 9.6%, P = 0.001), and lower overall healthcare spending ($47,891 vs $55,213, P = 0.003), than patients undergoing gastric bypass. However, at the 2-year mark, revisions were slightly more common in sleeve gastrectomy than in gastric bypass (sleeve 0.6%, bypass 0.4%, P = 0.009)., Conclusions and Relevance: In a large cohort of commercially insured patients, sleeve gastrectomy had a superior safety profile to gastric bypass up to 2 years from surgery, even when accounting for selection bias. However, the higher risk of revisions in sleeve gastrectomy merits further exploration., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To investigate the relationship between surgeon gender and stress during the Covid-19 pandemic., Background: Although female surgeons face difficulties integrating work and home in the best of times, the Covid-19 pandemic has presented new challenges. The implications for the female surgical workforce are unknown., Methods: This cross-sectional, multi-center telephone survey study of surgeons was conducted across 5 academic institutions (May 15-June 5, 2020). The primary outcome was maximum stress level, measured using the validated Stress Numerical Rating Scale-11. Mixed-effects generalized linear models were used to estimate the relationship between surgeon stress level and gender., Results: Of 529 surgeons contacted, 337 surgeons responded and 335 surveys were complete (response rate 63.7%). The majority of female respondents were housestaff (58.1%), and the majority of male respondents were faculty (56.8%) (P = 0.008). A greater proportion of male surgeons (50.3%) than female surgeons (36.8%) had children ≤18 years (P = 0.015). The mean maximum stress level for female surgeons was 7.51 (SD 1.49) and for male surgeons was 6.71 (SD 2.15) (P < 0.001). After adjusting for the presence of children and training status, female gender was associated with a significantly higher maximum stress level (P < 0.001)., Conclusions: Our findings that women experienced more stress than men during the Covid-19 pandemic, regardless of parental status, suggest that there is more to the gendered differences in the stress experience of the pandemic than the added demands of childcare. Deliberate interventions are needed to promote and support the female surgical workforce during the pandemic., Competing Interests: The authors declare no conflict of interests., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To explore the impact of the Covid-19 pandemic on the stress levels and experience of academic surgeons by training status (eg, housestaff or faculty)., Background: Covid-19 has uniquely challenged and changed the United States healthcare system. A better understanding of the surgeon experience is necessary to inform proactive workforce management and support., Methods: A multi-institutional, cross-sectional telephone survey of surgeons was conducted across 5 academic medical centers from May 15 to June 5, 2020. The exposure of interest was training status. The primary outcome was maximum stress level, measured using the validated Stress Numerical Rating Scale-11 (range 0-10)., Results: A total of 335 surveys were completed (49.3% housestaff, 50.7% faculty; response rate 63.7%). The mean maximum stress level of faculty was 7.21 (SD 1.81) and of housestaff was 6.86 (SD 2.06) (P = 0.102). Mean stress levels at the time of the survey trended lower amongst housestaff (4.17, SD 1.89) than faculty (4.56, SD 2.15) (P = 0.076). More housestaff (63.6%) than faculty (40.0%) reported exposure to individuals with Covid-19 (P < 0.001). Subjects reported inadequate personal protective equipment in approximately a third of professional exposures, with no difference by training status (P = 0.557)., Conclusions: During the early months of the Covid-19 pandemic, the personal and professional experiences of housestaff and faculty differed, in part due to a difference in exposure as well as non-work-related stressors. Workforce safety, including adequate personal protective equipment, expanded benefits (eg, emergency childcare), and deliberate staffing models may help to alleviate the stress associated with disease resurgence or future disasters., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Despite the clear benefits of palliative care, surgical patients are less likely to receive palliative care consultations when compared to their medical counterparts. In this context, we conducted semi-structured interviews with a diverse range of practicing surgeons to better understand surgeon attitudes and experiences with both palliative care approaches and specialty services., Methods: Forty-six surgeons from community, tertiary-care, and academic institutions across the state of Michigan agreed to participate in this study. Each participant's interview lasted between 30 and 60 minutes and was digitally recorded. Audiotapes were transcribed verbatim and de-identified for names and places. The data were analyzed through iterative steps informed by thematic analysis., Results: Six major themes emerged describing surgeon-reported barriers to palliative care approaches and use of palliative care services, which were observed at the surgeon-level, patient and family-level, and system-level. At the surgeon-level, the following three major themes emerged: surgeon knowledge and attitudes, prognostication challenges, and surgeon identity. At the patient and family-level, two major themes were identified: expectations and discordance. At the system-level, two major themes emerged: culture and resources., Conclusions: Among our cohort of surgeons, several key factors influenced their use of palliative approaches and specialty palliative care services. A better understanding of surgeon-perceived barriers may lead to future work aimed at creating meaningful, surgeon-specific interventions that address the underuse of this important care for surgical patients and patients being considered for surgery.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Drug hypersensitivity reactions are immune mediated, with T lymphocytes being stimulated by the drugs via their T-cell antigen receptor (TCR). In the nonpathogenic state, the TCR is activated by foreign peptides presented by major histocompatibility complex molecules (pMHC). Foreign pMHC binds with sufficient affinity to TCRαβ and thereby elicits phosphorylation of the cytoplasmic tails of the TCRαβ-associated CD3 subunits. The process is called TCR triggering. In this review, we discuss the current models of TCR triggering and which drug properties are crucial for TCR stimulation. The underlying molecular mechanisms mostly include pMHC-induced exposure of the CD3 cytoplasmic tails or alterations of the kinase-phosphatase equilibrium in the vicinity of CD3. In this review, we also discuss triggering of the TCR by small chemical compounds in context of these general mechanisms.

The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.

Quantitative biology requires high precision measurement of cellular parameters such as surface areas or volumes. Here, we have developed an integrated approach in which the data from 3D confocal microscopy and 2D high-resolution transmission electron microscopy were combined. The volumes and diameters of the cells within one population were automatically measured from the confocal data sets. The perimeter of the cell slices was measured in the TEM images using a semi-automated segmentation into background, cytoplasm and nucleus. These data in conjunction with approaches from stereology allowed for an unbiased estimate of surface areas with high accuracy. We have determined the volumes and surface areas of the cells and nuclei of six different immune cell types. In mast cells for example, the resulting cell surface was 3.5 times larger than the theoretical surface assuming the cell was a sphere with the same volume. Thus, our accurate data can now serve as inputs in modeling approaches in systems immunology.

The transplantation of skeletal myoblasts (SkM) might improve cardiac function after myocardial infarction via paracrine action. We used scaffold-based cell transfer by using vascular endothelial growth factor (VEGF)-overexpressing myoblasts. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were transfected with pCINeo-VEGF(121) and seeded on polyurethane (PU) scaffolds. The seeded scaffolds were epicardially implanted in rats 2 weeks after myocardial infarction (group: PU-VEGF-SkM). Before this intervention and 6 weeks later, pressure/volume loops were analyzed followed by histology. Additional study groups (n = 10 per group) were injected with VEGF-overexpressing myoblasts (Inj-VEGF-SkM) or unmodified myoblasts (Inj-SkM) or underwent a sham operation. Overexpression of VEGF was verified in vitro. The transplantation of growth factor producing myoblast-seeded scaffolds resulted in enhanced angiogenesis of ischemically damaged myocardium in vivo. However, the infarction size was not reduced. In group Inj-SkM, hemodynamics remained unchanged. Systolic function as measured by dP/dt(max) was not significantly altered in PU-VEGF-SkM (preinterventionally 2,156 ± 1,222 mmHg vs. postinterventionally 2,134 ± 850 mmHg). Other systolic function and diastolic function parameters as measured by dP/dt(min), tau, and pressure half-time were not restored in groups PU-VEGF-SkM and Inj-VEGF-SkM either. Transplantation of VEGF-overexpressing skeletal myoblasts leads to neovascularization in infarcted hearts. No functional myocardial recovery was observed. Scaffold-based transfer of genetically-modified cells remains a useful tool to study paracrine stem cell action.

Stem cells transplanted to an injured heart affect the host myocardium indirectly. The cytokine hepatocyte growth factor (HGF) may play a key role in this paracrine activity. We hypothesized that HGF-overexpressing stem cells would restore cardiac function after myocardial infarction (MI). Because there is a high rate of cell death when injecting the cells intramyocardially, we used scaffold-based cell transfer. Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huHGF and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. The seeded scaffolds were transplanted in rats two weeks after MI (group: PU-HGF-SkM) or the infection solution was intramyocardially injected (group: Inj-HGF-SkM). Two groups (Inj-SkM and PU-SkM) have been prepared with untransfected cells and sham group without any cell therapy served as control (n = 10 each group). At the beginning of treatment (baseline) and six weeks later, hemodynamic parameters were assessed. At the end of the study, histological analysis was employed. In sham animals we detected a decrease in systolic and diastolic function during the observation time. Treatment with untransfected myoblasts did not lead to any significant changes in hemodynamic parameters between the intervention and six weeks later. In group PU-HGF-SkM, systolic parameters like dP/dt(max), dP/dt(min) and isovolumic contraction improved significantly from baseline to study end. Some diastolic parameters were inferior as compared to baseline (SB-Ked, pressure half time [PHT], Tau). In group Inj-HGF-SkM, only PHT was impaired as compared to preinterventional values. Histological analysis showed significantly more capillaries in the infarction border zone in groups PU-HGF-SkM than in sham and Inj-SkM group. The infarction size was not affected by the therapy. Transplanting HGF-transfected myoblasts after MI can limit the development of ventricular dysfunction. Scaffold-based therapy in combination with gene therapy accelerates this capacity. This hemodynamic amelioration is accompanied by neovascularization, but not by smaller infarction sizes., (© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Objective: Stromal cell-derived factor-1 (SDF-1) is a potent chemotaxin. Increased SDF-1 levels can be found in ischemic myocardium and might protect against ischemia-reperfusion injury. We hypothesized that transplantation of stem cells overexpressing SDF-1 might improve cardiac function after myocardial infarction (MI). We compared intramyocardial injection with a scaffold-based application of SDF-1-transfected cells., Methods: Skeletal myoblasts (SkMs) were isolated and expanded from newborn Lewis rats. Cells were transfected with pcDNA3-huSDF-1 and seeded on polyurethane (PU) scaffolds or diluted in medium for cell injection. Two weeks after myocardial infarction, seeded scaffolds were implanted epicardially into rats (group: PU-SDF-1-SkM) or the injection solution was applied intramyocardially (Inj-SDF-1-SkM). Additional groups were treated with non-transfected myoblasts either by injection (Inj-SkM) or by scaffold-based application (PU-SkM) or received a sham operation (Sham). Before this intervention and 6 weeks later, hemodynamic parameters were measured. Infarction size and neovascularization were assessed by histology at study end., Results: In sham animals, we detected a clear decrease in systolic function from intervention to study end. In group Inj-SkM and PU-SkM, all hemodynamic parameters that were assessed remained unchanged during observation time. Systolic function as measured by dP/dt(max) and SB-Emax was significantly improved in groups Inj-SDF-1-SkM and PU-SDF-1-SkM at study end without a difference between the two SDF-1 groups. Diastolic function measured by post-interventional dP/dt(min) was also increased in group Inj-SDF-1-SkM but not in PU-SDF-1-SkM. Histological analysis revealed a reduced infarction size in all treatment groups at study end but enhanced neovascularization was not observable., Conclusions: Transplantation of myoblasts overexpressing SDF-1 improves cardiac function after MI. The restoration of hemodynamic parameters is accompanied by a reduction in infarction size. This reverse remodeling capacity is independent of a scaffold-based application of the SDF-1-transfected cells., (Copyright © 2011 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.)

P2X₇R deficiency is associated with a less severe outcome in acute and chronic inflammatory disorders. Recently, we demonstrated that extracellular adenosine triphosphate is involved in the pathogenesis of asthma by modulating the function of dendritic cells (DCs). However, the role of the purinergic receptor subtype P2X₇ is unknown. To elucidate the role of P2X₇R in allergic airway inflammation (AAI) in vitro and in vivo, P2X₇R expression was measured in lung tissue and immune cells of mice or in humans with allergic asthma. By using a specific P2X₇R-antagonist and P2X₇R-deficient animals, the role of this receptor in acute and chronic experimental asthma was explored. P2X₇R was found to be up-regulated during acute and chronic asthmatic airway inflammation in mice and humans. In vivo experiments revealed the functional relevance of this finding because selective P2X₇R inhibition or P2X₇R deficiency was associated with reduced features of acute and chronic asthma in the ovalbumin-alum or HDM model of AAI. Experiments with bone marrow chimeras emphasized that P2X₇R expression on hematopoietic cells is responsible for the proasthmatic effects of P2X₇R signaling. In the DC-driven model of AAI, P2X₇R-deficient DCs showed a reduced capacity to induce Th2 immunity in vivo. Up-regulation of P2X₇R on BAL macrophages and blood eosinophils could be observed in patients with chronic asthma. Our data suggest that targeting P2X₇R on hematopoietic cells (e.g., DCs or eosinophils) might be a new therapeutic option for the treatment of asthma.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schägger and von Jagow 1991); (Schägger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Myoblast-based therapy can improve cardiac function after infarction and is conventionally performed by direct injection. A scaffold-based transfer could overcome injection-associated problems. In upgrading this approach we transplanted skeletal myoblasts (SkM) overexpressing the prosurvival gene Akt1. SkM were transfected with pcDNA3-huda-Akt1 and seeded on polyurethane scaffolds. These scaffolds were transplanted in rats 2 weeks after myocardial infarction. Hemodynamics were analyzed before therapy and 6 weeks later. Infarction size and capillary density were performed thereafter. Additional groups received injections of Akt1-transfected or untransfected myoblasts, scaffolds seeded with untransfected myoblasts, or sham operation. Deterioration of global systolic left ventricular function could be inhibited by all therapeutic approaches. In addition, transplantation of Akt1-transfected cells, either scaffold-based or injected, was superior with regard to systolic properties of the left ventricular wall. This effect was accompanied by smaller infarction sizes and angiogenesis. Scaffolds with untransfected myoblasts yielded also smaller infarctions than injections of untransfected myoblasts. Both Akt groups profited with regard to dP/dt(min). In contrast, other diastolic parameters pointed at impaired relaxation and stiffer myocardium especially in the Akt1-scaffold group. In conclusion, SkM overexpressing Akt1 can maintain myocardial function after infarction, reduce infarction size, and induce neovascularization. Scaffold-based cell transfer does not augment this reverse remodeling capacity.