Design, Assembly, Production, and Transfection of Synthetic Modified mRNA.

Proteins are drivers of cell functions and are targets of many therapies. Exogenous protein expression techniques, therefore, have been essential for research and medicine. The most common method for exogenous protein expression relies on DNA-based viral or non-viral vectors. However, DNA-based vectors have the potential to integrate into the host genome and cause permanent mutations. RNA-based vectors solve this shortcoming. In particular, synthetic modified mRNA provides non-viral, integration-free, zero-footprint method for expressing proteins. Modified mRNA can direct cell fate specification and cellular reprogramming faster and more efficiently than other methods. Furthermore, when simultaneously express multiple different proteins, mRNA vectors allow for greater flexibility and control over stoichiometric ratios, dose titrations, and complete silencing of expressions. Additionally, modified mRNAs have been shown to be viable and safe as therapeutic agents for gene therapy and vaccine, providing an alternative approach to address diseases. Despite these advantages, technical challenge, mRNA instability, and host immunogenicity have caused significant barriers to widespread use of this technology. The comprehensive method presented here addresses all of these shortcomings. This stepwise protocol describes every step necessary for the synthesis of modified mRNA from any coding DNA sequence of interest. The meticulously detailed protocol enables the users to make alterations to each component of modified mRNA for even more significant customization, allowing the researchers to apply this technology to a wide range of uses. This non-cytotoxic synthetic modified mRNA can be used for protein expression, regulation of cell reprogramming or differentiation, and drug delivery.

Evidence-based management of central cord syndrome.

OBJECT: Evidence-based medicine is used to examine the current treatment options, timing of surgical intervention, and prognostic factors in the management of patients with traumatic central cord syndrome (TCCS). METHODS: A computerized literature search of the National Library of Medicine database, Cochrane database, and Google Scholar was performed for published material between January 1966 and February 2013 using key words and Medical Subject Headings. Abstracts were reviewed and selected, with the articles segregated into 3 main categories: surgical versus conservative management, timing of surgery, and prognostic factors. Evidentiary tables were then assembled, summarizing data and quality of evidence (Classes I-III) for papers included in this review. RESULTS: The authors compiled 3 evidentiary tables summarizing 16 studies, all of which were retrospective in design. Regarding surgical intervention versus conservative management, there was Class III evidence to support the superiority of surgery for patients presenting with TCCS. In regards to timing of surgery, most Class III evidence demonstrated no difference in early versus late surgical management. Most Class III studies agreed that older age, especially age greater than 60-70 years, correlated with worse outcomes. CONCLUSIONS: No Class I or Class II evidence was available to determine the efficacy of surgery, timing of surgical intervention, or prognostic factors in patients managed for TCCS. Hence, there is a need to perform well-controlled prospective studies and randomized controlled clinical trials to further investigate the optimal management (surgical vs conservative) and timing of surgical intervention in patients suffering from TCCS.

Interleukin-8 as a modulator of response to bevacizumab in preclinical models of head and neck squamous cell carcinoma.

OBJECTIVES: Bevacizumab, a monoclonal antibody to VEGF-A, is under active clinical evaluation in head and neck squamous cell carcinoma (HNSCC) and appears to be a promising therapy in at least a subset of patients. However, there are no reliable predictive biomarkers to identify those patients most likely to benefit. In this study, we assessed the efficacy of bevacizumab in HNSCC xenograft models to characterize escape mechanisms underlying intrinsic resistance and identify potential biomarkers of drug response. MATERIALS AND METHODS: We evaluated the angiogenic profile of HNSCC cells from sensitive and resistant cell lines using antibody array. We further examined the role of interleukin-8 (IL-8) in contributing to resistance both in vitro and in vivo, using a loss- and gain-of-function approach. RESULTS: Angiogenic profiling indicated that resistant cells expressed higher levels of proangiogenic factors including IL-8, interleukin-1α (IL-1α), vascular endothelial growth factor (VEGF), fibroblast growth factor-a (FGF-a), and tumor necrosis factor-α (TNF-α). IL-8 was the most differentially expressed protein. IL-8 signaling compensated for VEGF inhibition in endothelial cells. Downregulation of IL-8 resulted in sensitization of resistant tumors to bevacizumab by disrupting angiogenesis and enhancing endothelial cell apoptosis. Overexpression of IL-8 in sensitive tumors conferred resistance to bevacizumab. Serum analysis of HNSCC patients treated with a bevacizumab-containing regime revealed high baseline IL-8 levels in a subset of patients refractory to treatment but not in responders. CONCLUSIONS: These results implicate IL-8 in mediating intrinsic resistance to bevacizumab in HNSCC. Hence, co-targeting of VEGF and IL-8 may help overcome resistance and enhance therapeutic efficacy.

Nerve block of the infrapatellar branch of the saphenous nerve in knee arthroscopy: a prospective, double-blinded, randomized, placebo-controlled trial.

BACKGROUND: With the rising use of outpatient knee arthroscopy over the past decade, interest in peripheral nerve blocks during arthroscopy has increased. Femoral nerve blocks are effective but are associated with an inherent risk of the patient falling postoperatively because of quadriceps weakness. We studied blocks of the infrapatellar branch of the saphenous nerve, which produce analgesia in the knee that is similar to that resulting from a femoral nerve block but without associated quadriceps weakness. METHODS: Thirty-four patients were enrolled into each arm of this prospective, randomized, double-blinded trial comparing 10 mL of 0.25% bupivacaine used as a block of the infrapatellar branch of the saphenous nerve with a placebo during simple knee arthroscopy. Immediate outcome measures included Numeric Rating Scale (NRS) pain scores (0 to 10 points), mobility and discharge times, opioid usage, subjective adverse side effects, and forty-eight-hour anesthesia recovery surveys. Short-term measures included one-week and twelve-week Lysholm knee scores. RESULTS: No adverse effects or increased quadriceps weakness were observed following use of the nerve block. Improvement in early NRS scores and subjective nausea (p = 0.03) were detected. Patients for whom the block was successful also had improved twelve-week Lysholm knee scores (p = 0.04). No differences in opioid usage, mobility time, forty-eight-hour anesthesia recovery scores, or one-week Lysholm knee scores were found. CONCLUSIONS: No significant adverse effect or disadvantage was identified for blocks of the infrapatellar branch of the saphenous nerve used in simple knee arthroscopy. In addition to decreased early NRS scores and nausea, blocks of the infrapatellar branch of the saphenous nerve demonstrated potential benefit at twelve weeks after simple knee arthroscopy.