Rational design of AAV capsids is a simple method for enhancing AAV transduction efficiency. AAV-DJ is a highly recombinogenic hybrid vector created from DNA shuffling of eight AAV serotypes, which mediates efficient gene expression both in vitro and in vivo. AAV2 and AAV8 are the closest parental vectors of AAV-DJ and it has been reported that mutations on the 137/251/503 ubiquitination or phosphorylation sites of the AAV2 or AAV8 capsid lead to dramatic enhancement of gene delivery. Here, we aimed to find out whether the same point mutations on the AAV-DJ capsid could lead to significant improvement for gene delivery both in vitro and in vivo. We constructed three single point mutants (K137R/T251A/S503A) of AAV-DJ and the transduction efficiency of these mutants and AAV-DJ were investigated using two reporter gene systems including green fluorescent protein (GFP) and dual-luciferase (Gaussia luciferase and Firefly luciferase). Data indicated that single point mutations T251A/S503A lead to significant improvement of dual-luciferase expression in vivo after tail vein (TV) injection in mice respectively, despite limited enhancement of GFP expression in 293 T, Hela and HepG2 cells in vitro. Moreover, in vivo bioluminescence image and viral genome DNA copy number in tissue analysis showed that these mutants reserved the liver tropism characteristics, consistent with AAV-DJ. Single point mutations on the 251/503 sites of AAV-DJ capsid can lead to a significant improvement for in vivo gene expression. These enhanced AAV vectors have great potential in gene therapy applications.
INTRODUCTION: Parry-Romberg syndrome (PRS) is a rare degenerative disease of the face marked by unilateral atrophy of the skin, soft tissues, muscles, and facial bones. Although multiple series of patients and their treatments have been described in the literature, there is little consensus regarding the optimal management of PRS. We describe our surgical experience with PRS and explore the optimal approach to surgical reconstruction and describe a new classification scheme with both prognostic and therapeutic implications. METHODS: The cases of 18 patients with Parry-Romberg Syndrome treated over a 27-year period at UCLA were examined. In total, 68 procedures were performed during the study period. Major procedures included autologous fat grafting, bone grafting, and corrective orthognathic surgery. Ancillary procedures such as de-fatting of soft tissue grafts, , canthopexy, scar revision, and blepharoplasty were utilized as necessary to optimize aesthetic outcomes. Based on this collective clinical experience, a new classification system was developed. Type A: atrophy of the skin and subcutaneous tissues, without evidence of bone involvement. Type B: atrophy of the skin and subcutaneous tissue with skeletal hypoplasia or other bony malformation but without clinically significant malocclusion. Type C: Atrophy of the skin and subcutaneous tissue with skeletal hypoplasia or other bony malformation causing severe malocclusion. RESULTS: Patients with Type A disease (n=7) averaged 1.8 procedures each and achieved excellent results with either serial fat grafting or free flap reconstruction. Patients with Type B disease (n=4) required an average of 7 procedures each and achieved moderate to excellent results by a combination of fat grafting, free flaps, and bone grafting. Patients with Type C disease (n=7) averaged 4.4 procedures and achieved moderate to excellent results with a combination of orthognathic surgery, bone grafting, and soft tissue transfer. CONCLUSION: In this study we present a series of 18 cases of Parry-Romberg Syndrome. Based on this experience, we have developed a new classification scheme with both prognostic and therapeutic value. This will assist with patient counseling, categorization and communication between colleagues, and standardization of management plans to help improve overall patient care and maximize functional and aesthetic outcomes.
INTRODUCTION: TPeripheral nerve injuries can result in lifelong disability. Primary nerve repair is used for short nerve defects. Though autologous nerve can bridge longer defects, the harvesting procedure creates donor site morbidity. Nerve conduits lack an aligned internal scaffold to support and guide axonal regeneration. E2 peptide amphiphiles (PA) can self-assemble into aligned nanofibers, and can potentially mimic the native internal architecture of peripheral nerve. Bioactive epitopes IKVAV (Ile-Lys-Val-Ala-Val) and RGDS (Arg-Gly-Asp-Ser) can be incorporated into E2PA nanofibers and have been shown to promote neuronal cell adhesion, growth, and migration. There are no studies to date that examine the ability of E2PA nanofibers to support the proliferation of Schwann cells, key components in peripheral nerve healing. In this preliminary study, we investigate the proliferation of rat Schwann cells after incorporation into aligned E2PA gels combined with bioactive epitopes. METHODS: E2PA was synthesized by Stupp et al, aqueously dissolved, and combined with Schwann cells (cell line RT4-D6P2T). Gels were then formed by pipetting E2PA/cell suspensions into CaCl2 solution. WST-1 proliferation assay was then performed on these gels and compared to collagen-cell gels and 2-dimensional cell culture. WST-1 cell proliferation assays were then performed at days 3,7,14, and 21. Following determination of cell viability in E2PA gels, E2PA and bioactive epitopes (also synthesized by Stupp et al) were then aqueously dissolved, divided into 5 groups (E2 only, E2+IKVAV, E2+RGDS, E2+VVIAK control, and E2+RSDG control) , incorporated with rat Schwann cells at the same cell density, and made into aligned gels. WST-1 assays were performed at days 1 and 7. Immunocytochemistry (ICC) was performed on Schwann cells cultured in aligned E2PA and E2PA combined with RGDS compared to collagen controls, staining for actin and cell-adhesion-molecules. RESULTS: Schwann cells demonstrated increasing proliferation in E2PA gels for at least 21 days. Schwann cells also demonstrated proliferation in aligned gels, which was increased by IKVAV and RGDS compared to E2 alone. ICC revealed attachment and alignment of Schwann cells to aligned E2 gels. CONCLUSION: Schwann cells embedded in aligned E2PA gels combined with epitopes RGDS and RSDG exhibit increasing proliferation compared with E2PA alone. Schwann cells demonstrated successful attachment and alignment to aligned E2. Currently we are investigating the effect that E2PA and epitope composition have on rat dorsal root ganglia neuronal migration, and will use these results for future in-vivo studies.
Infusion pumps have been widely used in clinical settings for the administration of medications and fluids. We present the digital droplet infusion (DDI) device, a low-cost, high-precision digital infusion system, utilizing a microfluidic discretization unit to convert continuous flow into precisely delivered droplet aliquots and a valving unit to control the duration and frequency of flow discretization. The DDI device relies on a distinct capillarity-dominated process of coalescence and pinch-off of droplets for flow digitization, which is monitored by a pair of conductive electrodes located before and after the junction. The digital feedback-controlled flow rate can be employed to adjust a solenoid valve for refined infusion management. With this unique digital microfluidic approach, the DDI technology enables a simple yet powerful infusion system with an ultrahigh resolution of digital droplet transfer volume, as small as 57 nL, which is three orders of magnitude lower than that of clinical standard infusion pumps, as well as a wide range of digitally adjustable infusion rates ranging from 0.1 mL h-1 to 10 mL h-1, in addition to an array of programmable infusion profiles and safety features. Its modular design enables fast assembly using only off-the-shelf and 3D-printed components. Overall, benefiting from its simple device architecture and excellent infusion performance, the DDI technology has great potential to become the next-generation clinical standard for drug delivery with its high precision and ultimate portability at a low cost.
Though tremendous progress has been made in advancing the rights of the LGBTQ+ community, sexual minorities continue to face overt discrimination in American prison housing.The American prison system routinely neglects and abuses LGBTQ+ prisoners.The aim of this paper is to examine the abuse of LGBTQ+ prisoners in American correctional facilities and propose possible solutions.This paper recommends the government to build upon pre-existing safety standards by instituting self-identification policies; the state ought to provide flexibility and limited autonomy for LGBTQ+ prisoners to protect vulnerable sexual minorities.
Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy–chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.
Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.
INTRODUCTION: Traumatic peripheral nerve injury often leads to disability. Primary nerve coaptation is effective in the repair of short nerve defects, but treatment is limited for more complex nerve injuries. Autologous nerve grafts are limited by finite sources, deficit at the donor site, and donor site morbidity. Nerve conduits lack an aligned internal scaffold to support and guide axonal regeneration. Nanoscale peptide amphiphiles can self-assemble into aligned fibers and shown to promote nerve regeneration in a central nervous system model. There are no studies to date that examine the ability of PA-nanofibers support peripheral nerve regeneration. We investigate the application of this biomaterial to a peripheral nerve repair model. METHODS: PA-nanofibers were synthesized incorporating cultured rat Schwann cells into aligned fiber construct; adherence, viability, and proliferation of this cells within the nanofiber aggregates was confirmed. Evaluating peripheral nerve regeneration, PLGA conduits filled with PA-nanofibers were used to reconstruct a rat critical-sized sciatic nerve defect. Motor and sensory function tests evaluate regeneration. Results were compared to defects reconstructed with empty PLGA conduits and autologous nerve grafts. Unrepaired defects served as controls. RESULTS: Schwann cells were able to adhere to PA-nanofibers In Vitro, remaining viable and demonstrating proliferation throughout the culture period. In Vivo, sciatic nerve defects repaired with PA-nanofibersyielded return of motor and sensory function that was improved relative to controls. Restoration of function in these experimental animals was similar to that seen in animals repaired with autologous nerve graft and improved relative to animals repaired with empty conduit. CONCLUSIONS: PA-nanofiber scaffolds support attachment, survival, and proliferation of Schwann cells In Vitro. PA-nanofiber constructs support peripheral nerve regeneration In Vivo. Functional recovery using PA-nanofibersis equivalent to autologous nerve grafting. These findings warrant further investigation of PA-nanofibers to serve as a viable nerve graft substitutes in the treatment of complex peripheral nerve injury.
Pycnodysostosis is a rare autosomal recessive skeletal disorder involving a constellation of craniofacial manifestations including midface retrusion. We report the case of a 13-year-old girl with pycnodysostosis who presented with exorbitism, midface retrusion, malocclusion, and obstructive sleep apnea. Here, we describe the successful use of subcranial Le Fort III advancement using distraction osteogenesis with internal Kawamoto distracters. After a latency of 5 days, distraction for 10 days, and consolidation for 12 weeks, her midface was advanced by 10 mm with slight overcorrection at the occlusion level. At 2 years postoperatively, the patient had complete remission of her sleep apnea, resolution of her exorbitism, and amelioration of her class III malocclusion to class I. To the best of our knowledge, this is the first report of a successful subcranial Le Fort III midface advancement with distraction osteogenesis for craniofacial reconstruction of a pycnodysostosis. Our report highlights the surgical options that have been described for this craniofacial deformity and presents a novel and expedient approach for patients with pycnodysostosis presenting with exorbitism, midface retrusion, and/or sleep apnea.
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