Background: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. Methods:We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated.Results: There was~40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low~4.7 × 10 10 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of2 .0 × 10 12 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. Conclusions: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.
The incidence of pancreatic cancer, the fourth leading cause of cancer death in United States, is increasing worldwide. Even though the cure rate has doubled in 40 years, it is abysmally poor at 6-7%. As surgical resection remains the only curative treatment and less than 20% of the newly diagnosed cancers are resectable, the major burden of disease management lies in early diagnosis, good prognostication, and proper neo-adjuvant and/or adjuvant therapy. With advancing technologies and their ease of availability, researchers have better tools to understand pancreatic cancer. In the post-genetic era, proteomic, phosphoproteomic, metabolomic, and more have brought us to a multi-omics era. These newer avenues bring promises of better screening modalities, less invasive diagnostics and monitoring, subtyping of pancreatic cancer, and fine tuning the treatment modalities not only to the right stage of tumor but also to the right tumor biology. As the multitudes of technologies are generating extensive amounts of incongruous data, they are giving clinicians a lot of non-actionable information. In this paper, we wish to encompass the newer technologies, sub-classifications, and future treatment modalities in personalized care of patients with pancreatic cancer.
Cadaver labs are one of the staples of medical education in the United States, yet it is relatively uncommon for nursing students to have the opportunity to engage in the direct observation, hands-on learning, and the efficiency of the immersive environment in a cadaver-based anatomy lab. The primary aim of this project was to determine if medical students could create and deliver a cadaver lab workshop for nursing students that would provide educational benefits to both groups of students at a neutral cost. The purpose of this activity was to evaluate how a cadaver lab for nursing students could increase understanding of clinicallyrelevant anatomy, disease, and indwelling medical devices, while enhancing overall clinical problem-solving skills. The participants, nursing and medical students, completed a five-hour workshop followed by completion of a four-question survey of their overall learning experience and the value of the workshop from an interprofessional perspective. The surveys were analyzed individually for qualitative central themes; similar central themes were compiled by question, and overarching themes were identified and reported. Self-reflections completed by the students revealed that this shared encounter between trainees resulted in a better understanding of the visualization of the size, spatial relations, and physical interactions between organ systems; increased confidence in patient care regarding the physical exam and medical device management; and a better-perceived understanding of each profession's approach in providing patientcentered care. Medical students may also benefit by participation in this interprofessional activity through the development of clinical teaching skills that are needed while working with patients and clinical colleagues.
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