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PDF file, 293KB, Quantification of proliferating cells in 4T1 tumors.

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Deregulation of TGF-β superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-β superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-β type II receptor-based, single-chain traps were designed, termed (TβRII)2 and (TβRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TβRII-ED), and a similar or slightly better potency than pan–TGF-β–neutralizing antibody 1D11 or an Fc-fused receptor trap (TβRII-Fc). Despite its short in vivo half-life (3) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential. Mol Cancer Ther; 11(7); 1477–87. ©2012 AACR.

PDF file, 832KB, Schematics and molecular models of single-chain activin and BMP traps.

PDF file, 243KB, Improved neutralization of BMP-2 using single-chain bivalent trap (BMPR1a)2 compared to monovalent receptor BMPR1a-ED.

PDF file, 2243KB, Molecular dynamics analysis of the (TBRII)2 trap in complex with TGF-B3.

PDF file, 297KB, TGF-B1 neutralization using a (TBRII)2 trap having a natural or artificial linker sequence.

Monitoring antibody response to SARS-CoV-2 is critical for assessing the humoral response, especially important considering the emergence of multiple SARS-CoV-2 variants of concern (VOCs).

INTRODUCTION: The COVID-19 pandemic has created unique challenges for primary care practices while also highlighting their importance in the pandemic response. To understand primary care practice needs, a survey was conducted of practices in Western North Carolina. METHODS: Phase 2 of a primary care needs assessment was administered to 63 practices in Western North Carolina over the course of six weeks, from July 23 to August 31, 2021. RESULTS: Most practices were operating with normal hours, though some still operated with reduced hours. Many practices reported insufficient personal protective equipment (PPE) supplies. While most practices provided at least some care via telehealth, practices cited different barriers to providing telehealth, with patient technology challenges being the most frequently cited. DISCUSSION: Practices have adapted to the restrictions of the pandemic, but many are still vulnerable, and the patients they serve may face reduced access to care due to practice limitations or barriers to telehealth. Practices play a critical role in providing care to patients throughout the pandemic and continue to assist in pandemic response by providing COVID-19 testing and other services. CONCLUSION: Primary care practices in Western North Carolina continue to provide care to patients and support the overall pandemic response. The pandemic has highlighted the need to include primary care in emergency response efforts. Ongoing work will allow North Carolina to reach practices more effectively in future crises via the newly created NC Responds system, which allows primary care practices to be contacted in the event of a public health emergency.

BACKGROUND: It is critical to ensure that Primary Care Providers (PCPs) have adequate personal protective equipment (PPE), supplies, training, staffing, and contingency planning during pandemics, particularly in rural areas. In March 2020, during the onset of the COVID-19 pandemic, the Mountain Area Health Education Center (MAHEC), in collaboration with the Cecil G. Sheps Center for Health Services Research at UNC Chapel Hill, rapidly created and conducted a needs assessment of PCPs in western North Carolina (WNC). METHODS: A group of twenty volunteers conducted a telephone survey of PCPs in a 16 county region of WNC. Practices were asked about their COVID-19 testing and telehealth offerings, PPE adequacy, and capacity to continue serving patients. The survey’s emergency alert feature linked practices to immediate support. Descriptive data were generated to identify regional needs. RESULTS: Out of 110 practices, 48 (43.6%) offered COVID-19 testing, with testing more common in rural counties (56.3% vs 33.9%). Telehealth services, including phone-only visits, were offered by almost all practices (91.8%). PPE needs included N-95 respirators (49.1%), face shields (45.5%), and staff gowns (38.2%). Rural practices were more likely to report the need for PPE. Assistance was requested for staff member childcare (34.5%) and providing or billing for telehealth (31.8%). The most urgent practice requests were related to finances, PPE, and telehealth. MAHEC’s Practice Support team linked practices to virtual coaching, tip sheets, case-based video didactics and communication forums, and newsletters. CONCLUSION: During a pandemic, it is crucial to ensure that PCPs can continue to serve their patients. A rapid needs assessment of PCPs can allow for immediate and ongoing support that matches regional and practice-specific needs. Rural practices may require more assistance than their urban counterparts. Our rapid survey process jumpstarted a statewide system for enhanced communications with PCPs to better prepare for future emergencies.

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine. Estación Experimental Agropecuaria Bariloche Fil: Mignaqui, Ana Clara. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Ferella, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina Fil: Cass, Brian. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil Mukankurayija, Larissa. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: L’Abbé, Denis. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: Bisson, Louis. Human Health Therapeutics Research Center, National Research Council Canada; Canada Fil: Sánchez, Cintia. Biogénesis-Bagó; Argentina Fil: Scian, Romina. Biogénesis-Bagó; Argentina Fil: Cardillo, Sabrina Beatriz. Biogénesis-Bagó; Argentina Fil: Durocher, Yves. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina

Background Web-based electronic patient-reported outcomes (ePRO) measures are increasingly used to facilitate patient-centered health assessments. However, it is unknown if ePRO completion is feasible for recently ill intensive care unit (ICU) survivors and their families. Objective To develop and evaluate the usability of a novel ePRO system (ePRO to Support People and Enhance Recovery [ePROSPER]) among ICU survivors and their families within an ongoing clinical trial. Methods Paper-based PROs were iteratively adapted to electronic forms (ePROs). Then, the usability of ePROSPER was assessed among 60 patients, their family members, and PRO and programming experts via questionnaires (eg, Systems Usability Scale), "think aloud" open-ended feedback, task completion times, and error rates. Results Input from patients and their families was used to incorporate user-experience modifications into ePROSPER. This feedback also led to inclusion of automated reminders for questionnaire completion and real-time alerts for staff triggered by high symptom levels. Median usability scores increased over testing cycles from 40 to 73 to 95, nearing the maximum score and showing excellent usability. All users completed ePROSPER within 20 minutes; 87% preferred it to a written version. ePROSPER was then implemented in a clinical trial without data errors. Conclusions Automated ePRO systems can be successfully integrated in a post-ICU clinical trial setting. The value of integrating such systems in direct clinical care should be assessed in future studies.

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies.

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. Crown copyright © 2004 Published by Elsevier Inc. All rights reserved.

Deregulation of TGF-β superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-β superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-β type II receptor-based, single-chain traps were designed, termed (TβRII)2 and (TβRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TβRII-ED), and a similar or slightly better potency than pan–TGF-β–neutralizing antibody 1D11 or an Fc-fused receptor trap (TβRII-Fc). Despite its short in vivo half-life (

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His) 8 to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. Crown Copyright © 2004.

Monoclonal antibodies have been successfully engineered as approved therapeutics. However, their large size is considered a major factor preventing them from having a more efficient tissue penetration. As the first step to establish a possibly more efficient antibody platform, we present here transient expression, purification and characterization of six chimeric heavy chain antibodies (cHCAbs), or fusion of camelid single domain antibodies (sdAbs) to human fragment crystallizable (Fc). All six HCAbs have a MW of not, vert, similar80 kDa, expressed well in a HEK293 expression system and have G0, G1 and G2 types of glycosylation. The transient expression also provided a very fast way to generate high milligram to low gram amount of proteins for in vitro assays and preliminary animal studies.

Background: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. Results: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. Conclusion: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b., available, unlimited, public

The vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors play essential and complementary roles in angiogenesis, and combined inhibition of these receptors has been shown to result in potent antitumor activity in vivo. In this study, we report that ellagic acid, a natural polyphenol found in fruits and nuts, inhibits VEGF-induced phosphorylation of VEGFR-2 in EC as well as PDGF-induced phosphorylation of PDGFR in smooth muscle cells, leading to the inhibition of downstream signaling triggered by these receptors. Ellagic acid also specifically inhibited VEGF-induced migration of endothelial cells as well as their differentiation into capillary-like tubular structures, and abolished PDGF-dependent smooth muscle cell migration. Interestingly, ellagic acid presents a greater selectivity for normal cells than for tumor cells since the migration of the U87 and HT1080 cell lines were much less affected by this molecule. The identification of ellagic acid as a naturally occurring dual inhibitor of VEGF and PDGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer.

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.

Top of pageAbstract Recombinant adeno-associated viruses (rAAV) represent a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broaden their usage is the availability of a production process able to provide sufficient quantities for preclinical and human trials. We present here a successful process for rAAV-2 production in low-serum and serum-free medium performed in a 3L stirred-tank bioreactor. The process is based on the triple transfection of suspension-growing HEK293 cells employing polyethylenimine (PEI) as the DNA carrier. Production of AAV-GFP in a 3L serum-free medium bioreactor yielded titers of 5.1 |[times]| 1011 infectious viral particles (IVP), corresponding to 2 |[times]| 1012 viral genome (VG) or 6.8 |[times]| 1012 viral particles (VP). The cell-specific and total viral titers obtained in suspension culture were about three-fold higher to those obtained with adherent cells. The process is very simple and robust as it does not require a medium exchange, either before or after transfection, making it particularly attractive for the generation of large and homogeneous stocks of rAAV vectors.

Background Web-based decision aids are increasingly important in medical research and clinical care. However, few have been studied in an intensive care unit setting. The objectives of this study were to develop a Web-based decision aid for family members of patients receiving prolonged mechanical ventilation and to evaluate its usability and acceptability. Methods Using an iterative process involving 48 critical illness survivors, family surrogate decision makers, and intensivists, we developed a Web-based decision aid addressing goals of care preferences for surrogate decision makers of patients with prolonged mechanical ventilation that could be either administered by study staff or completed independently by family members (Development Phase). After piloting the decision aid among 13 surrogate decision makers and seven intensivists, we assessed the decision aid’s usability in the Evaluation Phase among a cohort of 30 surrogate decision makers using the Systems Usability Scale (SUS). Acceptability was assessed using measures of satisfaction and preference for electronic Collaborative Decision Support (eCODES) versus the original printed decision aid. Results The final decision aid, termed ‘electronic Collaborative Decision Support’, provides a framework for shared decision making, elicits relevant values and preferences, incorporates clinical data to personalize prognostic estimates generated from the ProVent prediction model, generates a printable document summarizing the user’s interaction with the decision aid, and can digitally archive each user session. Usability was excellent (mean SUS, 80 ± 10) overall, but lower among those 56 years and older (73 ± 7) versus those who were younger (84 ± 9); p = 0.03. A total of 93% of users reported a preference for electronic versus printed versions. Conclusions The Web-based decision aid for ICU surrogate decision makers can facilitate highly individualized information sharing with excellent usability and acceptability. Decision aids that employ an electronic format such as eCODES represent a strategy that could enhance patient-clinician collaboration and decision making quality in intensive care. Electronic supplementary material The online version of this article (doi:10.1186/s13613-015-0045-0) contains supplementary material, which is available to authorized users.