CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.

Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and inverted Alu repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP that is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs) but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease-defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP, and their associated nuclease activities in protecting CFSs in mammalian cells.

Risk Factors for Bed Bugs Among Urban Emergency Department Patients.

Bed bugs are a significant and under-studied public health nuisance. We surveyed 706 emergency department patients and found bed bug infestations were more likely (p < 0.05) among subjects having persons 19-64 years of age in the house, living in a group home, a previous history with bed bugs, and knowing someone who currently has bed bugs. Sleeping in a hotel, recent homelessness, obtaining used clothing or furniture and using a laundromat were not associated with increased risk (p > 0.05) for infestation. Individuals with less education and income were more likely (p < 0.05) to have bed bugs and express concern about getting bed bugs. Younger persons were more likely to correctly identify a picture of a bed bug compared to those over the age of 60 years (p < 0.001).

Improving E. coli growth performance by manipulating small RNA expression.

Efficient growth of E. coli, especially for production of recombinant proteins, has been a challenge for the biotechnological industry since the early 1970s. By employing multiple approaches, such as different media composition, various growth strategies and specific genetic manipulations, it is now possible to grow bacteria to concentrations exceeding 100 g/L and to achieve high concentrations of recombinant proteins. Although the growth conditions are carefully monitored and maintained, it is likely that during the growth process cells are exposed to periodic stress conditions, created by fluctuations in pH, dissolved oxygen, temperature, glucose, and salt concentration. These stress circumstances which can occur especially in large volume bioreactors, may affect the growth and production process. In the last several years, it has been recognized that small non-coding RNAs can act as regulators of bacterial gene expression. These molecules are found to be specifically involved in E. coli response to different environmental stress conditions; but so far, have not been used for improving production strains. The review provides summary of small RNAs identified on petri dish or in shake flask culture that can potentially affect growth characteristics of E. coli grown in bioreactor. Among them MicC and MicF that are involved in response to temperature changes, RyhB that responds to iron concentration, Gady which is associated with lower pH, Sgrs that is coupled with glucose transport and OxyS that responds to oxygen concentration. The manipulation of some of these small RNAs for improving growth of E. coli in Bioreactor is described in the last part of the review. Overexpression of SgrS was associated with improved growth and reduced acetate expression, over expression of GadY improved cell growth at acidic conditions and over expression of OxyS reduced the effect of oxidative stress. One of the possible advantages of manipulating sRNAs for improving cell growth is that the modifications occur at a post-translational level. Therefore, the use of sRNAs may exert minimal effect on the overall bacterial metabolism. The elucidation of the physiological role of newly discovered sRNAs will open new possibilities for creating strains with improved growth and production capabilities.

Home blood pressure monitor use in patients with chronic kidney disease.

Home blood pressure monitoring (HBPM) is recommended in patients with chronic kidney disease (CKD) and hypertension (HTN). However, little is known about the use and pattern of HBPM in CKD patients. A cross-sectional study was conducted in an out-patient nephrology clinic. A total of 285 patients participated in the study. Of all patients, 66% reported using HBPM. Self-reported compliance with BP medications (93.9% vs. 85.1%, p 0.03), exercise >3 days/week (45.9 vs. 26.3%, p <0.001) and dietary sodium restriction (85.6% vs. 71.6%, p < 0.001) were more common in HBPM users vs. non-HBPM users. Most patients with HBPM used upper arm cuff (82.3%), reported receiving education on correct use of HBPM (82.5%), had perception that home BP was controlled (75.4%) and believed that HBPM is helpful in managing hypertension (85.4%). Most common reason for not using HBPM was lack of advice by a physician (43.4%). HBPM use is common in patients with CKD and HTN. HBPM users are more likely to follow life-style and dietary modifications for blood pressure control.

A survey of 0 + 5 vascular surgery residency candidates: Perceptions of an applicant pool.

BACKGROUND: Applications to integrated (0 + 5) vascular surgery residencies have increased, while total applications have stayed relatively constant. This survey sought to document the perceptions of 0 + 5 vascular surgery applicants. METHODS: Academic faculty conducted interviews for 0 + 5 residency match at an academic medical center in preparation for the National Resident Matching Program (NRMP) Main Residency Match. Applicant pool (n = 20) perceptions were determined with surveys. Participation was anonymous and voluntary. RESULTS: Nineteen interviewees (26.3% female: 73.7% male), age (26.8 ± 2.6 years) responded (95% response rate). Of 19 respondents, 68% became interested in vascular surgery in their third year with 53% becoming aware of 0 + 5 programs in their third year. All respondents identified a vascular surgery attending at their institution as significant mentors. Forty-seven percent identified their mentor during their third year of medical school. All respondents felt that 0 + 5 training would prepare them adequately for the workforce and board certification exams. Almost all (89%) had plans to seek jobs immediately upon completion of residency. CONCLUSION: Applicants remained positive about their planned training and career paths. Attending vascular surgeons were identified as the strongest mentors, yet most students decided only in their third and fourth years to pursue 0 + 5 residencies. Educational debt remains a concern, and there may be consideration for a concerted effort to recruit potential candidates sooner.

Constitutive expression of the sRNA GadY decreases acetate production and improves E. coli growth.

BACKGROUND: Escherichia coli responds to acid stress by applying various physiological, metabolic, and proton-consuming mechanisms depending on the growth media composition, cell density, growth phase, pH, and aerobic or anaerobic growth conditions. It was reported that at mild acidic conditions (pH 5.8), the Hfq-associated sRNA GadY is activated. It was also reported that the two decarboxylase systems-the lysine decarboxylase system (LDS) and the glutamate decarboxylase system (GDS)-are activated to maintain intracellular balance of protons. The purpose of this study was to establish the role of GadY in high density growth of E. coli and to evaluate the possibility of using this small RNA to create an acid-resistant strain suitable for industrial applications. RESULTS: Parental E. coli K-12 and constitutively expressing GadY strains were grown to high cell densities in a bioreactor at pH 7.0 and pH 6.0. At pH 7.0, both strains grew to similar cell densities of 43 OD, but the constitutively expressing GadY strain produced around 6 g/L acetate compared with 10 g/L by the parental strain. At pH 6.0, the parental strain grew to an OD of 20 and produced 10 g/L of acetate while the GadY strain grew to an average OD of 31 and produced 4 g/L acetate. After analyzing 17 genes associated with acid stress, it was found that at pH 7.0 LDS was expressed in the early exponential phase and GDS was expressed in the late exponential phase in both strains. However, at pH.6.0, GDS was expressed in the late exponential phase only in the parental strain and not in the constitutively expressing GadY strain, while there was no difference in the LDS expression pattern; it was expressed in the early exponential phase in both strains. This indicates that GadY affects GDS expression at low pH since the GDS was not detected in the GadY strain at pH 6.0. CONCLUSIONS: The constitutive expression of GadY improves E. coli growth at pH 6.0 by deactivating the expression of the GDS in the late exponential growth phase. The expression of GadY also decreases acetate production regardless of pH, which decreases the inhibitory effect of this acid on bacterial growth.

Rad50 zinc hook is important for the Mre11 complex to bind chromosomal DNA double-stranded breaks and initiate various DNA damage responses.

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.

Real-World Multicenter Coronary Lithotripsy Registry: Long-Term Clinical Follow-Up.

OBJECTIVES: Coronary lithotripsy (CL) works by fracturing the calcified plaque, allowing mean area gain, enhancing vessel compliance, and facilitating stent deployment. This study reports the safety, effectiveness, and durability of the clinical benefit of CL at long-term follow-up of a real-world multicenter registry. METHODS: This was a prospective, multicenter, single-arm study that included consecutive patients with calcified lesions undergoing CL from August, 2018 to October, 2020 with a clinical follow-up of 20 months (interquartile range, 14.5-25). Exclusion criteria were a target lesion located in a vessel <2.5 mm and/or the presence of dissection prior to CL. The primary endpoint was the rate of major adverse cardiovascular event (MACE, defined as death or target-lesion revascularization [TLR] or myocardial infarction [MI]) at follow-up. RESULTS: This registry included 109 patients (128 lesions). The population was elderly (mean age, 74 years old), with high rates of diabetic patients (58%), renal insufficiency (32%), and multivessel disease (76%). Most of the lesions were predilated with semicompliant/noncompliant balloons (25 with cutting balloon). Rotational atherectomy was used in 20 lesions. On average, CL required the use of 1 balloon delivering a mean of 60 pulses. Twelve patients presented with ST-segment-elevation MI and a culprit calcified coronary lesion undergoing CL. Successful CL was achieved in 99% of cases. There were few procedural complications, with 30-day freedom from MACE rate of 98%. The MACE rate at long-term follow-up was 5.6%. CONCLUSION: This is the first real-world, multicenter registry that confirms the safety and long-term efficacy of percutaneous coronary intervention for calcified lesions using CL in an unselected and high-risk population with a low long-term follow-up MACE rate.

Interoperable Platform to Report Polymerase Chain Reaction SARS-CoV-2 Tests From Laboratories to the Chilean Government: Development and Implementation Study.

BACKGROUND: Testing, traceability, and isolation actions are a central strategy defined by the World Health Organization to contain the COVID-19 pandemic. In this sense, the countries have had difficulties in counting the number of people infected with SARS-CoV-2. Errors in reporting results are a common factor, as well as the lack of interoperability between laboratories and governments. Approaches aimed at sending spreadsheets via email expose patients' privacy and have increased the probability of errors due to retyping, which generates a delay in the notification of results. OBJECTIVE: This study aims to design and develop an interoperable platform to report polymerase chain reaction (PCR) SARS-CoV-2 tests from laboratories to the Chilean government. METHODS: The methodology to design and develop the interoperable platform was comprised of six well-structured stages: (1) creation of a minimum data set for PCR SARS-CoV-2 tests, (2) modeling processes and end points where institutions interchange information, (3) standards and interoperability design, (4) software development, (5) software testing, and (6) software implementation. RESULTS: The interoperable Fast Healthcare Interoperability Resources (FHIR) platform to report PCR SARS-CoV-2 tests from laboratories to the Chilean government was successfully implemented. The platform was designed, developed, tested, and implemented following a structured methodology. The platform's performance to 1000 requests resulted in a response time of 240 milliseconds, throughput of 28.3 requests per second, and process management time of 131 milliseconds. The security was assured through a private network exclusive to the Ministry of Health to ensure confidentiality and integrity. The authorization and authentication of laboratories were implemented with a JavaScript Object Notation Web Token. All the PCR SARS-CoV-2 tests were accessible through an application programming interface gateway with valid credentials and the right access control list. CONCLUSIONS: The platform was implemented and is currently being used by UC Christus Laboratory. The platform is secure. It was tested adequately for confidentiality, secure authorization, authentication, and message integrity. This platform simplifies the reporting of PCR SARS-CoV-2 tests and reduces the time and probability of mistakes in counting positive cases. The interoperable solution with FHIR is working successfully and is open for the community, laboratories, and any institution that needs to report PCR SARS-CoV-2 tests.

Primary Angioplasty of Calcified Coronary Lesions Using Coronary Lithotripsy in Acute ST-Segment Elevation Myocardial Infarction.

BACKGROUND: This study reports procedural and short-term clinical outcomes from a real-world series with the use of coronary lithotripsy in the context of primary angioplasty in ST-segment elevation myocardial infarction (STEMI). METHODS AND RESULTS: This was a prospective registry conducted at 2 hospitals, which included 10 patients who presented a culprit calcified lesion within acute STEMI and underwent coronary lithotripsy during primary angioplasty, between July 2019 and July 2020. Mean age was 69.2 ± 11.8 years, and there was a high proportion of hypertension (70%) and dyslipidemia (60%). All lesions (type B/C) were predilated with a semicompliant balloon. Coronary lithotripsy was performed in all cases once macroscopic thrombus was successfully retrieved by thrombus aspiration catheter. Before lithotripsy, rotational atherectomy was used in 1 case and cutting balloon was used in 2 cases. On average, coronary lithotripsy required the use of 1 lithotripsy balloon (range, 1-2) delivering a mean of 70 pulses. Two lithotripsy balloons were ruptured during lithotripsy therapy without any adverse event. Successful coronary lithotripsy was achieved in 90%. There were no periprocedural cardiac complications. CONCLUSIONS: Coronary lithotripsy seems to be a safe and effective technique in patients with STEMI and a culprit calcified lesion undergoing primary angioplasty for calcium modification in the absence of angiographic thrombus, and a suitable option to achieve adequate stent expansion and apposition.

Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21).

BACKGROUND: The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encoding the glucose transporter IICBGlc), was studied in both E. coli K-12 (MG1655 and JM109) and E. coli B (BL21). It is known that the transcription level of sgrS increases when E. coli K-12 (MG1655 and JM109) is exposed to the non-metabolized glucose alpha methyl glucoside (αMG) or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICBGlc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars. RESULTS: In the course of the described work, it was found that E. coli B (BL21) and E. coli K-12 (JM109 and MG1655) responded similarly to αMG: both strains increased SgrS transcription and reduced ptsG transcription. However, the two strains reacted differently to high glucose concentration (40 g/L). E. coli B (BL21) reacted by increasing sgrS transcription and reducing ptsG transcription while E. coli K-12 (JM109 and MG1655) did not respond to the high glucose concentration, and, therefore, transcription of sgrS was not detected and ptsG mRNA level was not affected. CONCLUSIONS: The results suggest that E. coli B (BL21) tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.

Acetate accumulation through alternative metabolic pathways in ackA (-) pta (-) poxB (-) triple mutant in E. coli B (BL21).

Individual deletions of acs and aceA genes in E. coli B (BL21) showed little difference in the metabolite accumulation patterns but deletion of the ackA gene alone or together with pta showed acetic acid gradually accumulated to 3.1 and 1.7 g/l, respectively, with a minimal extended lag in bacterial growth and a higher pyruvate formation. Single poxB deletion in E. coli B (BL21) or additional poxB deletion in the ackA-pta mutants did not change the acetate accumulation pattern. When the acetate production genes (ackA-pta-poxB) were deleted in E. coli B (BL21) acetate still accumulated. This may be an indication that perhaps acetate is not only a by-product of carbon metabolism; it is possible that acetate plays also a role in other cellular metabolite pathways. It is likely that there are alternative acetate production pathways.

Prolonged QT Interval in SARS-CoV-2 Infection: Prevalence and Prognosis.

BACKGROUND: The prognostic value of a prolonged QT interval in SARS-Cov2 infection is not well known. OBJECTIVE: To determine whether the presence of a prolonged QT on admission is an independent factor for mortality in SARS-Cov2 hospitalized patients. METHODS: Single-center cohort of 623 consecutive patients with positive polymerase-chain-reaction test (PCR) to SARS Cov2, recruited from 27 February to 7 April 2020. An electrocardiogram was taken on these patients within the first 48 h after diagnosis and before the administration of any medication with a known effect on QT interval. A prolonged QT interval was defined as a corrected QT (QTc) interval >480 milliseconds. Patients were followed up with until 10 May 2020. RESULTS: Sixty-one patients (9.8%) had prolonged QTc and only 3.2% had a baseline QTc > 500 milliseconds. Patients with prolonged QTc were older, had more comorbidities, and higher levels of immune-inflammatory markers. There were no episodes of ventricular tachycardia or ventricular fibrillation during hospitalization. All-cause death was higher in patients with prolonged QTc (41.0% vs. 8.7%, p < 0.001, multivariable HR 2.68 (1.58-4.55), p < 0.001). CONCLUSIONS: Almost 10% of patients with COVID-19 infection have a prolonged QTc interval on admission. A prolonged QTc was independently associated with a higher mortality even after adjustment for age, comorbidities, and treatment with hydroxychloroquine and azithromycin. An electrocardiogram should be included on admission to identify high-risk SARS-CoV-2 patients.

Coronary lithoplasty for calcified lesions: real-world multicenter registry.

INTRODUCTION AND OBJECTIVES: Coronary lithoplasty (CL) is a balloon-based technique used to treat calcified lesions. This study reports the initial experience of treatment of calcified lesions with CL in an unselected and high-risk population. METHODS: This was a prospective, multicenter registry, which included all consecutive cases with calcified coronary lesions that underwent CL between August, 2018 and August, 2019. Exclusion criteria consisted of a target lesion located in a small vessel (< 2.5mm) and the presence of dissection prior to CL. Quantitative coronary angiography and intravascular ultrasound/optical coherence tomography analysis were completed by an independent central core laboratory. RESULTS: This registry included 57 patients (66 lesions). The population was elderly (72.6±9.4 years) with high proportions of patients with diabetes (56%), chronic kidney disease (35%), and multivessel disease (84%). All lesions were classified as type B/C. More than 75% of lesions were predilated with noncompliant/semicompliant balloons or cutting-balloon. Rotablator was used in 5 lesions (7.6%) prelithoplasty. On average, CL required 1.17 balloons delivering a mean of 60 pulses. Successful CL was achieved in 98%. In 13% of cases, lithoplasty balloon was broken during therapy. There were few procedural complications: 2 cases of significant dissections (none related to lithoplasty balloon rupture) were successfully treated with drug-eluting stent implantation. One patient experienced stent thrombosis 2 days after successfully undergoing target lesion revascularization. CONCLUSIONS: This is a real-world multicenter registry, which supports the feasibility, safety, and short-term efficacy of PCI for calcified coronary lesions using CL in an unselected and high-risk population with promising results.

Cimicosis in Persons Previously Fed Upon by Bed Bugs.

Introduction Bed bug infestations have risen dramatically in many industrialized nations in recent decades. Most people fed upon by bed bugs will develop a pruritic rash although the frequency with which this occurs is not definitively known and may depend on host factors including the duration of the infestation. Methods Surveys were completed from 706 emergency department (ED) patients in Cleveland, OH about their current and past exposure with bed bugs. Subjects were asked about any post-bed bug feeding rashes that developed. Results There were 24% (169/698) of subjects reporting either a current or past home bed bug infestation, with 37% (253/698) reporting they had previously been fed upon by a bed bug. Of those reporting a previous bed bug feeding, 68% (172/253) reported a pruritic post-bed bug feeding rash and 24% (57/237) reported developing a blister. Overall, 5% (37/705) of ED patients reported currently having a rash, but only 2% (14/698) of ED patients reported currently have bed bugs at home and of those, only 14% (2/14) said they currently had a rash. Conclusion While 68% of ED patients reported a pruritic post-bed bug feeding pruritic rash, almost a third of persons did not report developing the rash. Post-bed bug feeding blister reactions are less common. Asking ED patients about a rash had a low sensitivity of 14% (2-43%) and a specificity 95% (93-96%) to identify persons reporting home bed bugs.

Characteristics of Bed Bug Infested Patients in the Emergency Department.

Cimex lectularius L., the common bed bug, is a hematophagous human ectoparasite that has undergone a global resurgence in the past two decades. We surveyed 706 active emergency department (ED) patients about their experiences with bed bugs. We found that 2% of ED patients reported having a current bed bug infestation, significantly more than the historical number of ED patients upon which we find bed bug; 37% of ED patients report previously having been fed on by a bed bug; 15% currently know someone with an active infestation; and 59% know someone that has had an infestation within ≤ 5 years. Only 18% of bed bug infested patients reported their infestation to emergency medicine providers and only 21% were put in isolation precautions. We found that 25% of patients with bed bugs worried about receiving worse healthcare because of their infestation. Persons with bed bugs were more likely compared to those without bed bugs to be older (52 vs. 41 years) and arrive by ambulance (57% vs. 14%) (p < 0.05), but not reporting insomnia (50% vs. 49%) (p = 1.0). Bed bug infested patients can be common in the ED. Most bed bug infested patients are older, arrive to the ED by ambulance, do not report their infestation to healthcare providers, and are not adequately placed into isolation precautions, potentially putting other patients and providers at risk for acquiring the infestation.

Strategies for manufacturing recombinant adeno-associated virus vectors for gene therapy applications exploiting baculovirus technology.

The development of recombinant adeno-associated virus (rAAV) gene therapy applications is hampered by the inability to produce rAAV in sufficient quantities to support pre-clinical and clinical trials. Contrasting with adherent cell cultures, suspension cultures provide a straightforward means for expansion, however, transiently expressing the necessary, but cytotoxic virus proteins remains the challenge for rAAV production. Both the expansion and expression issues are resolved by using the baculovirus expression vector (bev) and insect cell culture system. This review addresses strategies for the production of rAAV exploiting baculovirus technology at different scales using different configurations of bioreactors as well as processing and product characterization issues. The yields obtained with these optimized processes exceed approximately 1 x 10(14) vector particles per liter of cell culture suitable for pre-clinical and clinical trials and possible commercialization.

Large-scale production of recombinant adeno-associated viral vectors.

Current and future demands of viral vectors for the development of successful pre-clinical and clinical studies in human gene therapy and possible commercialization of gene therapy products require well-established large-scale production processes. One of the most promising vectors for human gene therapy is recombinant adeno-associated virus vectors (rAAVs). Some of the attractive features of rAAV are broad tissue tropism, low immunogenicity, ability to transduce both mitotic and post-mitotic cells, and long-term gene expression in non-dividing cells. Recently, we developed a novel technology for the production of these vectors exploiting baculovirus expression vectors (BEV: ) in insect cell cultures. Initially developed in small, shake flask format, this process has been successfully scaled to larger volumes. In an effort to standardize rAAV production in stirred tank bioreactors, we characterized the culture conditions to derive a set of parameters correlated with high rAAV yields. Measuring capacitance and dielectric spectroscopy with a permittivity probe enabled us to determine optimal times of infection and harvest. Consistent yields of rAAV, 2 x 10(13) DNase-resistant vector genomes (vg) [1 x 10(12) transducing units (tu)] per liter of cell culture were obtained in bioreactors with working volumes ranging from 10 to 40 l. This represents significant progress toward establishing a robust large-scale process at industry level.

Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales.

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g. > or =10(16) particles per production run, utilizes baculovirus expression vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred tank bioreactors are single-use, disposable bioreactors, e.g. Wave. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave bioreactors. Comparable yields of rAAV, approximately 2E+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration.

Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4.

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.