Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption.

A simple method, sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with direct protein adsorption analysis (SDS-PAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentrations (in pg/µl). We used this method to characterize the effects of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures and its utility in developing preventive strategies of adsorption.

Can the positive buffer the negative? Testing the impact of protective childhood experiences on adjustment in adults following trauma exposure.

BACKGROUND: It is unclear if protective childhood experiences (PCEs), like emotional support and economic stability, exert influence on adulthood adjustment. Prior research suggests PCEs can promote childhood resilience through increased social connection. In contrast, research has demonstrated potential life-long negative impacts of adverse childhood experiences (ACEs) on psychological health. This study examined the role of PCEs and ACEs in psychological symptoms following potentially traumatic events (PTE) in adults. METHODS: Participants (N = 128) were adults admitted to two Level 1 Trauma Centers following violence, motor-vehicle crashes, or other accidents. Participants reported childhood experiences and completed assessments of depression, PTSD, and social support at one, four, and nine months post-PTE. RESULTS: Structural Equation Modeling was used to simultaneously model PCEs and ACEs as predictors of psychological symptoms over time, with potential mediation through social support. PCEs overall did not directly affect psychological symptoms nor indirectly through social support. However, the emotional support component of PCEs had an indirect effect on psychological symptoms at baseline through social support. ACEs predicted greater psychological symptoms at baseline and over time. CONCLUSION: PCEs consisting of childhood emotional support indirectly promote adjustment in adults after PTEs through initial social support, while ACEs exert direct effects on psychological symptoms.

Appendicular Lean Mass and Frailty among Geriatric Outpatients.

The objective of this observational study was to examine the association between appendicular lean mass and frailty in adults aged 60 years and older. This study was conducted in the Outpatient Department of the National Geriatric Hospital in Hanoi, Vietnam. Appendicular lean mass (kg) was assessed by using Dual energy X-ray absorptiometry scans. Frailty was defined according to Fried's frailty criteria. A total of 560 outpatients were included in the study, with a mean age of 70 years. The prevalence of frailty was 12.0%. Frail patients had significantly lower appendicular lean mass compared with non-frail outpatients (9.6 ± 2.0 kg vs. 11.7 ± 3.1 kg, p<0.001). On multivariable logistic regression models, higher appendicular lean mass was associated with significantly reduced odds for frailty (adjusted OR = 0.74, 95%CI 0.59 - 0.93). These findings suggest that the assessment of appendicular lean mass should be considered in older patients attending outpatient geriatric clinics.

Cellular pathways of recombinant adeno-associated virus production for gene therapy.

Recombinant adeno-associated viruses (rAAVs) are among the most important vectors for in vivo gene therapies. With the rapid development of gene therapy, current rAAV manufacturing capacity faces a challenge to meet the emerging demand for these therapies in the future. To examine the bottlenecks in rAAV production during cell culture, we focus here on an analysis of cellular pathways of rAAV production, based on an overview of assembly mechanisms first in the wild-type (wt) AAV replication and then in the common methods of rAAV production. The differences analyzed between the wild-type and recombinant systems provide insights into the mechanistic differences that may correlate with viral productivity. Based on these analyses, we identify potential barriers to high productivity of rAAV and discuss future directions for improvement to meet the emerging needs set by the growth of rAAV-based therapy and the needs of patients.

Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.

The optimization of upstream and downstream processes for production of recombinant adeno-associated virus (rAAV) with consistent quality depends on the ability to rapidly characterize critical quality attributes (CQAs). In the context of rAAV production, the virus titer, capsid content, and aggregation are identified as potential CQAs, affecting the potency, purity, and safety of rAAV-mediated gene therapy products. Analytical methods to measure these attributes commonly suffer from long turnaround times or low throughput for process development, although rapid, high-throughput methods are beginning to be developed and commercialized. These methods are not yet well established in academic or industrial practice, and supportive data are scarce. Here, we review both established and upcoming analytical methods for the quantification of rAAV quality attributes. In assessing each method, we highlight the progress toward rapid, at-line characterization of rAAV. Furthermore, we identify that a key challenge for transitioning from traditional to newer methods is the scarcity of academic and industrial experience with the latter. This literature review serves as a guide for the selection of analytical methods targeting quality attributes for rapid, high-throughput process characterization during process development of rAAV-mediated gene therapies.

Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells.

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Cephalopod-Derived Biopolymers for Ionic and Protonic Transistors.

Cephalopods (e.g., squid, octopuses, and cuttlefish) have long fascinated scientists and the general public alike due to their complex behavioral characteristics and remarkable camouflage abilities. As such, these animals are explored as model systems in neuroscience and represent a well-known commercial resource. Herein, selected literature examples related to the electrical properties of cephalopod-derived biopolymers (eumelanins, chitosans, and reflectins) and to the use of these materials in voltage-gated devices (i.e., transistors) are highlighted. Moreover, some potential future directions and challenges in this area are described, with the aim of inspiring additional research effort on ionic and protonic transistors from cephalopod-derived biopolymers.

Immediate postoperative extubation in patients undergoing free tissue transfer.

OBJECTIVES/HYPOTHESIS: Extubation (cessation of ventilatory support) is often delayed in free flap patients to protect the microvascular anastomosis, presumably by reducing emergence-related agitation. We sought to determine if immediate extubation in the operating room (OR) would improve the postoperative course compared to delayed extubation in the intensive care unit (ICU). STUDY DESIGN: Retrospective chart review. METHODS: Medical records of all patients undergoing free tissue transfer for head and neck reconstruction between January 2009 and July 2010 were reviewed (n = 52). Patients extubated immediately postoperatively in the OR (immediate group, n = 26) were compared to patients extubated in the ICU (delayed group, n = 26). RESULTS: Tobacco use, alcohol use, pulmonary history, case length, and free flap type were not significantly different between the two groups. Although the average ICU stay for the immediate group was significantly shorter than the delayed group (2.0 days vs. 3.4 days; P = .008), the reduction in overall hospital stay for the immediate group did not achieve statistical significance (8.2 days vs. 9.5 days; P = .21). Use of treatment for agitation (27% vs. 65%) and physical restraints (8% vs. 69%) were significantly lower in the immediate versus delayed group (P = .01 and P < .001, respectively). Although flap-related, surgical, and medical complication rates were not significantly different between the two groups, the delayed extubation group had a significantly higher incidence of pneumonia (15% vs. 0%; P = .05). CONCLUSIONS: Immediate postoperative extubation in the OR following head and neck microvascular free tissue transfer reduces ICU stay, anxiolytic use, restraint use, and incidence of pneumonia without an increase in flap- or wound-related complications.

Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities.

The hepatitis C virus (HCV)-specific CD8(+)-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8(+)-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8(+)-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8(+)-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

Human immunodeficiency virus type 1-hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses.

Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8(+)- and CD4(+)-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8(+)-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8(+)-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.