Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

The Effect of Substrate Temperature on the Evaporative Behaviour and Desiccation Patterns of Foetal Bovine Serum Drops

The drying of bio-fluid drops results in the formation of complex patterns, which are morphologically and topographically affected by environmental conditions including temperature. We examine the effect of substrate temperatures between 20 °C and 40 °C, on the evaporative dynamics and dried deposits of foetal bovine serum (FBS) drops. The deposits consist of four zones: a peripheral protein ring, a zone of protein structures, a protein gel, and a central crystalline zone. We investigate the link between the evaporative behaviour, final deposit volume, and cracking. Drops dried at higher substrate temperatures in the range of 20 °C to 35 °C produce deposits of lower final volume. We attribute this to a lower water content and a more brittle gel in the deposits formed at higher temperatures. However, the average deposit volume is higher for drops dried at 40 °C compared to drops dried at 35 °C, indicating protein denaturation. Focusing on the protein ring, we show that the ring volume decreases with increasing temperature from 20 °C to 35 °C, whereas the number of cracks increases due to faster water evaporation. Interestingly, for deposits of drops dried at 40 °C, the ring volume increases, but the number of cracks also increases, suggesting an interplay between water evaporation and increasing strain in the deposits due to protein denaturation.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research.

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Unlike many other candidates being investigated, tizoxanide (the active metabolite of nitazoxanide) plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbeccos Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Therefore, a robustly validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6ng/mL to 1000ng/mL. Accuracy and precision ranged between 102.2% and 113.5%, 100.1% and 105.4%, respectively. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient’s quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes.

Measuring the density and viscosity of culture media for optimized computational fluid dynamics analysis of in vitro devices

Culture medium is frequently modelled as water in computational fluid dynamics (CFD) analysis of in vitro culture systems involving flow, such as bioreactors and organ-on-chips. However, culture medium can be expected to have different properties to water due to its higher solute content. Furthermore, cellular activities such as metabolism and secretion of ECM proteins alter the composition of culture medium and therefore its properties during culture. As these properties directly determine the hydromechanical stimuli exerted on cells in vitro, these, along with any changes during culture must be known for CFD modelling accuracy and meaningful interpretation of cellular responses. In this study, the density and dynamic viscosity of DMEM and RPMI-1640 media supplemented with typical concentrations of foetal bovine serum (0, 5, 10 and 20% v/v) were measured to serve as a reference for computational design analysis. Any changes in the properties of medium during culture were also investigated with NCI-H460 and HN6 cell lines. The density and dynamic viscosity of the media increased proportional to the % volume of added foetal bovine serum (FBS). Importantly, the viscosity of 5% FBS-supplemented RPMI-1640 was found to increase significantly after 3 days of culture of NCI-H460 and HN6 cell lines, with distinct differences between magnitude of change for each cell line. Finally, these experimentally-derived values were applied in CFD analysis of a simple microfluidic device, which demonstrated clear differences in maximum wall shear stress and pressure between fluid models. Overall, these results highlight the importance of characterizing model-specific properties for CFD design analysis of cell culture systems.

Complete Virus Inactivation Using a Combined Heat and Chemical Treatment

Validated inactivation protocols are required for the safe handling and disposal of virus samples. This is particularly important for hazard group 3 agents and above. Various methods are employed for virus inactivation, some of which include the use of heat or chemical chaotropic agents such as guanidine hydrochloride. It is generally accepted that these processes are sufficient to denature all viruses. While inactivation of certain viruses with such methods have been reported, validation of their activity against a wide of range of viruses is required. Here, we examined the inactivation of a panel of hazard group 2 viruses (Rhabdoviridae, Togaviridae, Peribunyaviridae, and Flaviviridae) using a combination of heat, guanidine hydrochloride-containing buffer, and ethanol. Viruses were treated with proteinase K and guanidine hydrochloride-containing buffer, and heated at 56 °C for 30 min. This was followed by additional treatment with absolute ethanol. Resulting virus–buffer–ethanol mixtures were column-purified to remove residual ethanol and other toxic reagents, before being introduced to cells. Column purification was confirmed to be insufficient for virus removal. Cultures were incubated at 37 °C for 1 h, after which media supplemented with 2% foetal bovine serum was added. Cultures were then observed daily for cytopathic effects. Samples that showed no evidence of cytopathic effects were passaged thrice to confirm absence of cytopathic effects and complete inactivation of viruses. Cultures infected with control viruses that had not been treated with buffer, heat, and ethanol but column-purified developed cytopathic effects, while cultures infected with treated viruses showed no cytopathic effects even after three passages, thus confirming complete virus inactivation. Results from this study provide evidence of the use of a combination of heat, guanidine hydrochloride-containing buffer, and ethanol for the complete inactivation of all members of the four families investigated.

The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses

During the 2020 SARS-Cov-2 pandemic, there has been an acute shortage of viral transport medium. Many different products have been used to meet the demands of large-scale diagnostic and surveillance testing. The stability of SARS-Cov-2 RNA was assessed in several commercially produced transport media and an in-house solution. Coronavirus RNA was rapidly destroyed in the commercial transport media though the deleterious effects on intact virus were limited. Similar results were obtained for a Type A influenza virus. There was reduced detection of both virus and nucleic acid when a herpesvirus sample and purified DNA were tested. Collectively these data showed that the commercial viral transport media contained nucleases or similar substances and may seriously compromise diagnostic and epidemiological investigations.Recommendations to include foetal bovine serum as a source of protein to enhance the stabilising properties of viral transport media are contraindicated. Almost all commercial batches of foetal bovine serum contain pestiviruses and at times other bovine viruses. In addition to the potential for there to be nucleases in the transport medium, the presence of these viruses and other extraneous nucleic acid in samples may compromise the interpretation of sequence data. The inclusion of foetal bovine serum presents a biosecurity risk for the movement of animal pathogens and renders these transport media unsuitable for animal disease diagnostic applications. While these transport media may be suitable for virus culture purposes, there could be misleading results if used for nucleic acid-based tests. Therefore, these products should be evaluated to ensure fitness for purpose.

Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss)

The lymphocyte proliferation assay is a valuable method used for the evaluation of the fish immune system. However, there are many variations and optimal results are not always obtained. Unification is necessary to ensure the comparability between different studies. The aim of this study was to optimise the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). This goal included the determination of the optimal incubation length, serum type, incubation temperature, type of mitogen and its concentration, and anticoagulant. The peripheral blood and head kidney lymphocytes were isolated by density gradient centrifugation. Subsequently, the cells were incubated for 3–8 days with different mitogens (pokeweed mitogen 5, 10 and 50 µg/ml, concanavalin A 1, 10 and 20 µg/ml, phytohaemagglutinin 25, 50 and 100 µg/ml, lipopolysaccharide 1, 50 and 100 µg/ml). The use of the different serum types (foetal bovine serum, trout serum), incubation temperatures (10–20 °C) and anticoagulants (heparin, EDTA) was compared. Labelled thymidine was used to evaluate the assay. The best results were obtained after seven days of incubation at 15 °C with foetal bovine serum (FBS). The head kidney lymphocytes showed the highest proliferative response with 50 µg/ml phytohaemagglutinin. With the peripheral blood lymphocytes (heparin and EDTA), the best results were obtained with 50 µg/ml pokeweed mitogen. The highest proliferation levels were detected with heparinised blood. In conclusion, optimisation of this assay contributes to the improved assessment of the rainbow trout immune function.