pinecone/core-2020-05-10-deduplication huggingface dataset:
Photo inactivation of virus particles in microfluidic capillary systems

This is the peer reviewed version of the following article: REN, Y. ... et al, 2016. Photo inactivation of virus particles in microfluidic capillary systems. Biotechnology and Bioengineering, 113 (7), pp. 1481-1492., which has been published in final form at http://dx.doi.org/10.1002/bit.25912. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."It has long been established that UVC light is a very effective method for inactivating pathogens in a fluid, yet the application of UVC irradiation to modern biotechnological processes is limited by the intrinsic short penetration distance of UVC light in optically dense protein solutions. This experimental and numerical study establishes that irradiating a fluid flowing continuously in a microfluidic capillary system, in which the diameter of the capillary is turned to the depth of penetration of UVC light, uniquely treats the whole volume of the fluid to UVC light resulting in fast and effective inactivation of pathogens, with particular focus to virus particles. This was demonstrated by inactivating human herpes simplex virus type-1 (HSV-1, a large enveloped virus) on a dense 10% fetal calf serum solution in a range of fluoropolymer capillary systems, including a 0.75 mm and 1.50 mm internal diameter capillaries and a high-throughput MicroCapillary Film with mean hydraulic diameter of 206 μm. Up to 99.96% of HSV-1 virus particles were effectively inactivated with a mean exposure time of up to 10s, with undetectable collateral damage to proteins. The kinetics of virus inactivation matched well the results from a new mathematical model that considers the parabolic flow profile in the capillaries, and showed the methodology is fully predictable and scalable and avoids both the side effect of UVC light to proteins and the dilution of the fluid in current tubular UVC inactivation systems. This is expected to speed up the industrial adoption of non-invasive UVC virus inactivation in clinical biotechnology and biomanufacturing of therapeutic molecules
Photo inactivation of virus particles in microfluidic capillary systems

It has long been established that UVC light is a very effective method for inactivating pathogens in a fluid, yet the application of UVC irradiation to modern biotechnological processes is limited by the intrinsic short penetration distance of UVC light in optically dense protein solutions. This experimental and numerical study establishes that irradiating a fluid flowing continuously in a microfluidic capillary system, in which the diameter of the capillary is turned to the depth of penetration of UVC light, uniquely treats the whole volume of the fluid to UVC light resulting in fast and effective inactivation of pathogens, with particular focus to virus particles. This was demonstrated by inactivating human herpes simplex virus type-1 (HSV-1, a large enveloped virus) on a dense 10% fetal calf serum solution in a range of fluoropolymer capillary systems, including a 0.75 mm and 1.50 mm internal diameter capillaries and a high-throughput MicroCapillary Film with mean hydraulic diameter of 206 µm. Up to 99.96% of HSV-1 virus particles were effectively inactivated with a mean exposure time of up to 10s, with undetectable collateral damage to proteins. The kinetics of virus inactivation matched well the results from a new mathematical model that considers the parabolic flow profile in the capillaries, and showed the methodology is fully predictable and scalable and avoids both the side effect of UVC light to proteins and the dilution of the fluid in current tubular UVC inactivation systems. This is expected to speed up the industrial adoption of non-invasive UVC virus inactivation in clinical biotechnology and biomanufacturing of therapeutic molecules.This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/wol1/doi/10.1002/bit.25912/full
Decellularisation and histological characterisation of porcine peripheral nerves

yesPeripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair.Engineering and Physical Sciences Research Council. Grant Number: EPSRC EP/F500513/1Gol
Non-chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

Inspired by nature, synthetic mineralizing proteins have been developed to synthesize various structures of silica-based nanomaterials under environmentally friendly conditions. However, the development of bioprocesses able to assist in the translation of these new materials has lagged the development of the materials themselves. The development of cost-effective and scalable bioprocesses which minimize reliance on chromatography to recover biomolecules from microbial cell factories remains a significant challenge. This paper reports a simplified purification process for a recently reported recombinant catalytic modular (D4S2) protein (M(DPSMKQLADS-LHQLARQ-VSRLEHA)4EPSRKKRKKRKKRKKGGGY; M 13.3 kDa; pI 10.9), which combines a variant of the established designer biosurfactant protein DAMP4 with a new biomimetic sequence (RKKRKKRKKRKKGGGY), providing for a bi-modular functionality (emulsification and biosilicification). The four-helix bundle structure of the protein has been demonstrated to remain stable and soluble under high temperature and high salt conditions, which confers simplified bioprocessing character. However, the high positive charge on the biosilification sequence necessitates removal of DNA contaminants from crude cell-extract at an early stage in the process by adding poly(ethyleneimine) (PEI). In this process, cellular protein contaminants were selectively precipitated by adding Na2SO4 to the protein mixture up to a high concentration (1 M) and mixed at high temperature (90°C, 5 min) where D4S2 remained stable and soluble due to its four-helix bundle structure. Further increase of the Na2SO4 concentration to 1.8 M precipitated, thus separated, D4S2 from residual PEI. The overall yield of the protein D4S2 was 28.8 mg per 800 mL cells (final cultivation OD600 ∼2) which gives an approximate 79% D4S2-protein yield. In comparison with the previously reported chromatographic purification of D4S2 protein (Wibowo et al., 2015), the final yield of D4S2 protein is increased fourfold in this study. The bio-produced protein D4S2 was proved to retain it emulsification and biosilicification functionalities enabling the formation of oil-core silica-shell nanocapsules at near-neutral pH and room temperature without the use of any toxic organic solvents, confirming no adverse effects due to bioprocess simplification. This work demonstrates that, through proper bioprocess engineering including the removal of critical contaminants such as DNA, a more efficient, simple, and scalable purification process can be used for the high-yield bio-production of a recombinant templating protein useful in the synthesis of bio-inspired nanomaterials. This simplified process is expected to be easily adapted to recover other mineralizing helix bundle-based functional proteins from microbial cell factories. Biotechnol. Bioeng.Full Tex
MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc
Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimise the productivity of these cultures it is important to monitor cellular metabolism, for example the utilisation of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalise on the wealth of genetic operons for metabolite sensing available in Nature for the development of other whole-cell biosensors
Development of an optical system for the non-invasive tracking of stem cell growth on microcarriers

This work is made available according to the conditions of the Creative Commons Attribution  4.0 International (CC BY 4.0) licence. Full details of this licence are available at: http://creativecommons.org/licenses/by/4.0/The emergence of medicinal indications for stem cell\ud
therapies has seen a need to develop the manufacturing capacity for\ud
adherent cells such as mesenchymal stem cells (MSCs). One such\ud
development is in the use of microcarriers, which facilitate enhanced\ud
cell densities for adherent stem cell cultures when compared with 2D\ud
culture platforms. Given the variety of stem cell expansion systems\ud
commercially available, novel methods of non-invasive and automated\ud
monitoring of cell number, confluence, and aggregation, within\ud
disparate environments, will become imperative to process control,\ud
ensuring reliable and consistent performance. The in situ epiillumination\ud
of mouse embryonic fibroblasts and human mesenchymal\ud
stem cells attached to Cytodex 1 and 3 microcarriers was achieved using\ud
a bespoke microscope. Robust image processing techniques were\ud
developed to provide quantitative measurements of confluence,\ud
aggregate recognition, and cell number, without the need for fluorescent\ud
labeling or cell detachment. Large datasets of cells counted on individual\ud
microcarriers were statistically analyzed and compared with NucleoCounter\ud
measurements, with an average difference of less than 7%\ud
observed from days 0 to 6 of a 12-day culture noted, prior to the onset of\ud
aggregation. The developed image acquisition system and postprocessing\ud
methodologies were successfully applied to dynamically\ud
moving colonized microcarriers. The proposed system offers a novel\ud
method of cell identification at the individual level, to consistently and\ud
accurately assess viable cell number, confluence, and cell distribution,\ud
while also minimizing the variability inherent in the current invasive\ud
means by which cells adhered to microcarriers are analyzed
Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor

This is an Open Access Article. It is published by Wiley under the Creative Commons Attribution 4.0 International Licence (CC BY). Full details of this licence are available at: http://creativecommons.org/licenses/by/4.0/Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS . The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency
Mortality from laparoscopic antireflux surgery in a nationwide cohort of the working-age population

Background: Both medication and surgery are effective treatments for severe gastro-oesophageal reflux disease (GORD). Postoperative risks have contributed to decreased use of antireflux surgery. The aim of this study was to assess short-term mortality following primary laparoscopic fundoplication. 
Methods: This was a population-based nationwide cohort study including all Swedish hospitals that performed laparoscopic fundoplication between 1997 and 2013. All patients aged 18-65 years with GORD who underwent primary laparoscopic fundoplication during the study interval were included. The primary outcome was absolute all-cause and surgery-related 90- and 30-day mortality. Secondary outcomes were reoperation and length of hospital stay. Logistic regression was used to calculate odds ratios with 95 per cent confidence intervals of reoperation within 90 days and prolonged hospital stay (4 days or more). 
Results: Of 8947 included patients, 5306 (59.3 per cent) were men and 551 (6.2 per cent) had significant co-morbidity (Charlson score above 0). Median age at surgery was 48 years, and median hospital stay was 2 days. The annual rate of laparoscopic fundoplication decreased from 15.3 to 2.4 patients per 100 000 population during the study period, whereas the proportion of patients with co-morbidity increased more than twofold. All-cause 90-and 30-day mortality rates were 0.08 per cent (7 patients) and 0.03 per cent (3 patients) respectively. Only one death (0.01 per cent) was directly surgery-related. The 90-day reoperation rate was 0.4 per cent (39 patients). Co-morbidity and older age were associated with an increased risk of prolonged hospital stay, but not reoperation. 
Conclusion: This population-based study revealed very low mortality and reoperation rates following primary laparoscopic fundoplication in the working-age population. The findings may influence clinical decision-making in the treatment of severe GORD
Cost analysis of laparoscopic lavage compared with sigmoid resection for perforated diverticulitis in the Ladies trial

Background: Laparoscopic peritoneal lavage is an alternative to sigmoid resection in selected patients presenting with purulent peritonitis from perforated diverticulitis. Although recent trials have lacked superiority for lavage in terms of morbidity, mortality was not compromised, and beneficial secondary outcomes were shown. These included shorter duration of surgery, less stoma formation and less surgical reintervention (including stoma reversal) for laparoscopic lavage versus sigmoid resection respectively. The cost analysis of laparoscopic lavage for perforated diverticulitis in the Ladies RCT was assessed in the present study. Methods: This study involved an economic evaluation of the randomized LOLA (LaparOscopic LAvage) arm of the Ladies trial (comparing laparoscopic lavage with sigmoid resection in patients with purulent peritonitis due to perforated diverticulitis). The actual resource use per individual patient was documented prospectively and analysed (according to intention-to-treat) for up to 1 year after randomization. Results: Eighty-eight patients were randomized to either laparoscopic lavage (46) or sigmoid resection (42). The total medical costs for lavage were lower (mean difference € − 3512, 95 per cent bias-corrected and accelerated c.i. −16 020 to 8149). Surgical reintervention increased costs in the lavage group, whereas stoma reversal increased costs in the sigmoid resection group. Differences in favour of laparoscopy were robust when costs were varied by ±20 per cent in a sensitivity analysis (mean cost difference € − 2509 to −4438). Conclusion: Laparoscopic lavage for perforated diverticulitis is more cost-effective than sigmoid resection
Economic case for intraoperative interventions to prevent  surgical-site infection

Background:

Surgical-site infection (SSI) occurs in 1–10 per cent of all patients undergoing surgery; rates can be higher depending on the type of surgery. The aim of this review was to establish whether (or not) surgical hand asepsis, intraoperative skin antisepsis and selected surgical dressings are cost-effective in SSI prevention, and to examine the quality of reporting.
Methods:

The authors searched MEDLINE via Ovid, CINAHL via EBSCO, Cochrane Central and Scopus databases systematically from 1990 to 2016. Included were RCTs and quasi-experimental studies published in English, evaluating the economic impact of interventions to prevent SSI relative to surgical hand and skin antisepsis, and wound dressings. Characteristics and results of included studies were extracted using a standard data collection tool. Study and reporting quality were assessed using SIGN and CHEERS checklists.
Results:

Across the three areas of SSI prevention, the combined searches identified 1214 articles. Of these, five health economic studies evaluating the cost-effectiveness of selected surgical dressings were eligible. Study authors concluded that the interventions being assessed were cost-effective, or were potentially cost-saving. Still, there is high uncertainty around the decision to adopt these dressings/devices in practice. The studies' reporting quality was reasonable; three reported at least 15 of the 24 CHEERS items appropriately. Assessment of methodological quality found that two studies were considered to be of high quality.
Conclusion:

With few economic studies undertaken in this area, the cost-effectiveness of these strategies is unclear. Incorporating economic evaluations alongside RCTs will help towards evidence-informed decisions.Griffith Health, School of Nursing and MidwiferyFull Tex