Metagenomic insights into the effects of benzyl dodecyl dimethyl ammonium bromide (BDAB) shock on bacteria-driven nitrogen removal in a moving-bed biofilm reactor (MBBR).

The use of disinfectants made from quaternary ammonium compounds (QACs) has greatly increased since the outbreak of SARS-CoV-2. However, the effect of QACs on wastewater treatment performance is still unclear. In this study, a commonly used QAC, i.e., benzyl dodecyl dimethyl ammonium bromide (BDAB), was added to a moving-bed biofilm reactor (MBBR) to investigate BDAB's effect on nutrient removal. When the BDAB concentration was increased to 50 mg L-1, the ammonia removal efficiency (ARE) greatly decreased, as did the nitrate production rate constants (NPR). This inhibition was partly recovered by decreasing the BDAB concentration to 30 mg L-1. Metagenomic sequencing revealed the functional genera present during different stages of the control (Rc) and BDAB-added reactors (Re). The enriched genera (Rudaea, Nitrosospira, Sphingomonas, and Rhodanobacter) in Rc mainly related to the nitrogen metabolism, while the enriched genera in Re was BDAB-concentration dependent. Functional genes analysis suggested that a lack of ammonia oxidase-encoding genes (amoABC) may have caused a decrease in ARE in Re, while the efflux pump-encoding genes emrE, mdfA, and oprM and a gene encoding BAC oxygenase (oxyBAC) were responsible for BDAB resistance. The increase in the total abundance of antibiotic resistance genes (ARGs) in Re revealed a potential risk arising from BDAB. Overall, this study revealed the potential effect and ecological risks of BDAB introduction in WWTPs.

New Vaccine-Manufacturing Methods Are Moving Away From the Egg.

With seasonal influenza, Ebola, shingles, pneumonia, human papillomavirus, and other pathogens-combined now with the novel coronavirus (SARS-CoV-2)-the world's demand for vaccines is on a steep incline. New vaccine development is progressing rapidly, as seen with recent announcements of coronavirus options [1], [2], but what about their manufacture?

Mesenchymal stromal cell delivery via an ex vivo bioreactor preclinical test system attenuates clot formation for intravascular application.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).

Bone marrow in orthopaedics (part II): a three hundred and seventy million-year saga from the Devonian to the coronavirus disease 2019 pandemic-osteonecrosis; transplantation; "human chimera"; stem cells, bioreactors, and coronavirus disease.

PURPOSE: Three hundred seventy million years ago, bone marrow appeared in skeleton of a fish. More than one hundred years ago, the concept of bone marrow transplantation was proposed to treat human diseases. During the last five decades, this concept became a reality first in hematology and later for orthopaedic diseases. MATERIAL AND METHODS: These advances were possible due to the comprehension of the three major components of bone marrow: the fat part, the haematologic part, and the stroma part. Each part has a different history, but the three parts are linked in physiology as in history. RESULTS: During many centuries, bone marrow was considered just as food; however, one hundred years ago, the concept of bone marrow transplantation to treat humans was proposed by the French physician Brown-Séquard. During the last five decades, this concept became a reality first in haematology and later for orthopaedic diseases. Transferring what was known from experimental animal models to humans was met with many challenges, the atomic bomb research, and many deaths. Yet through the recognition and subsequent understanding of fundamental processes, medical resiliency, and the determination of a few pioneers, local bone marrow transplantation in orthopaedic surgery became a therapeutic option first for a limited number of diseases and patients. Over the last two decades, mesenchymal stromal cells (MSCs) have been the focus of intense research by acadaemia and industry due to their unique features. MSCs can be easily isolated and expanded through in vitro culture by taking full advantage of their self-renewing capacity. In addition, MSCs exert immunomodulatory effects and can be differentiated into various lineages, which makes them highly attractive for clinical applications in cell-based therapies. CONCLUSION: In this review, we attempted to provide a historical overview of bone marrow history, MSC discovery, characterization, and the first clinical studies conducted.

Decay of SARS-CoV-2 RNA along the wastewater treatment outfitted with Upflow Anaerobic Sludge Blanket (UASB) system evaluated through two sample concentration techniques.

For the first time, we present, i) an account of decay in the genetic material loading of SARS-CoV-2 during Upflow Anaerobic Sludge Blanket (UASB) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (PEG), and ultrafiltration as virus concentration methods from wastewater for the quantification of SARS-CoV-2 genes. The objectives were achieved through tracking of SARS-CoV-2 genetic loadings i.e. ORF1ab, N and S protein genes on 8th and 27th May 2020 along the wastewater treatment plant (106000 m3 million liters per day) equipped with UASB system in Ahmedabad, India. PEG method performed better in removing materials inhibiting RT-qPCR for SARS-CoV-2 gene detection from the samples, as evident from constant and lower CT values of control (MS2). Using the PEG method, we found a reduction >1.3 log10 reduction in SARS-CoV-2 RNA abundance during UASB treatment, and the RNA was not detected at all in the final effluent. The study implies that i) conventional wastewater treatment systems is effective in SARS-CoV-2 RNA removal, and ii) UASB system significantly reduces SARS-CoV-2 genetic loadings. Finally, PEG method is recommended for better sensitivity and inhibition removal during SARS-CoV-2 RNA quantification in wastewater.

Shattering barriers toward clinically meaningful MSC therapies.

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.

Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.