Microbial cancer therapeutics: A promising approach.

The success of conventional cancer therapeutics is hindered by associated dreadful side-effects of antibiotic resistance and the dearth of antitumor drugs' selectivity and specificity. Hence, the conceptual evolution of anti-cancerous therapeutic agents that selectively target cancer cells without impacting the healthy cells or tissues, has led to a new wave of scientific interest in microbial-derived bioactive molecules. Such strategic solutions may pave the way to surmount the shortcomings of conventional therapies and raise the potential and hope for the cure of wide range of cancer in a selective manner. This review aims to provide a comprehensive summary of anti-carcinogenic properties and underlying mechanisms of bioactive molecules of microbial origin, and discuss the current challenges and effective therapeutic application of combinatorial strategies to attain minimal systemic side-effects.

Recycling bread waste into chemical building blocks using a circular biorefining approach.

Food waste is a global problem, causing significant environmental harm and resulting in substantial economic losses globally. Bread is the commonly wasted food item in the developed world and presents a severe problem for the majority of European nations. It is the second most wasted food item in the UK after potatoes, with an equivalent of 20 million slices of bread thrown away daily. Bread is a starchy material and a rich and clean source of easily extractable fermentable sugars - this is in direct contrast to lignocellulosic feedstocks where harsh physical, chemical and/or enzymatic pretreatment processes are required for release of fermentable sugars. Furthermore, these necessary lignocellulosic pretreatment methods often produce sugars contaminated with fermentation inhibitors. Therefore, bread waste presents a clear opportunity as a potential carbon source for novel commercial processes and, to this end, several alternative routes have been developed to utilize bread waste. Possibilities for direct recycling of bread waste within the food industry are limited due to the relatively short material lifetime, stringent process and hygiene requirements. Anaerobic digestion (AD) and incineration are commonly employed methods for the valorisation of bread waste, generating limited amounts of green energy but with little other environmental or economic benefits. Most food wastes and by-products in the UK including bakery waste are treated through AD processes that fail to harness the full potential of the these wastes. This short communication reviews the challenges of handling bread waste, with a focus on a specific UK scenario. The review will consider how bread waste is generated across the supply chain, current practices to deal with the waste and logistics challenges in waste collection. The presence of clean and high-quality fermentable sugars, proteins and other nutrients in bread make it an ideal substrate for generating chemicals, fuels, bioplastics, pharmaceuticals and other renewable products through microbial fermentations. We suggest potential applications for recycling bread waste into its chemical building blocks through a fermentative route where a circular biorefining approach could maximize resource recovery and environmental savings and eliminate waste to as close to zero as possible.

Integrating micro-algae into wastewater treatment: A review.

Improving the ecological status of water sources is a growing focus for many developed and developing nations, in particular with reducing nitrogen and phosphorus in wastewater effluent. In recent years, mixotrophic micro-algae have received increased interest in implementing them as part of wastewater treatment. This is based on their ability to utilise organic and inorganic carbon, as well as inorganic nitrogen (N) and phosphorous (P) in wastewater for their growth, with the desired results of a reduction in the concentration of these substances in the water. The aim of this review is to provide a critical account of micro-algae as an important step in wastewater treatment for enhancing the reduction of N, P and the chemical oxygen demand (COD) in wastewater, whilst utilising a fraction of the energy demand of conventional biological treatment systems. Here, we begin with an overview of the various steps in the treatment process, followed by a review of the cellular and metabolic mechanisms that micro-algae use to reduce N, P and COD of wastewater with identification of when the process may potentially be most effective. We also describe the various abiotic and biotic factors influencing micro-algae wastewater treatment, together with a review of bioreactor configuration and design. Furthermore, a detailed overview is provided of the current state-of-the-art in the use of micro-algae in wastewater treatment.

Correction: Deformability-induced lift force in spiral microchannels for cell separation.

Correction for 'Deformability-induced lift force in spiral microchannels for cell separation' by Ewa Guzniczak et al., Lab Chip, 2020, 20, 614-625.

Deformability-induced lift force in spiral microchannels for cell separation.

Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.

Impact of poloxamer 188 (Pluronic F-68) additive on cell mechanical properties, quantification by real-time deformability cytometry.

Advances in cellular therapies have led to the development of new approaches for cell product purification and formulation, e.g., utilizing cell endogenous properties such as size and deformability as a basis for separation from potentially harmful undesirable by-products. However, commonly used additives such as Pluronic F-68 and other poloxamer macromolecules can change the mechanical properties of cells and consequently alter their processing. In this paper, we quantified the short-term effect of Pluronic F-68 on the mechanotype of three different cell types (Jurkat cells, red blood cells, and human embryonic kidney cells) using real-time deformability cytometry. The impact of the additive concentration was assessed in terms of cell size and deformability. We observed that cells respond progressively to the presence of Pluronic F-68 within first 3 h of incubation and become significantly stiffer (p-value < 0.001) in comparison to a serum-free control and a control containing serum. We also observed that the short-term response manifested as cell stiffening is true (p-value < 0.001) for the concentration reaching 1% (w/v) of the poloxamer additive in tested buffers. Additionally, using flow cytometry, we assessed that changes in cell deformability triggered by addition of Pluronic F-68 are not accompanied by size or viability alterations.

High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.

Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.

A 3D mammalian cell separator biochip.

The dissimilar cytoskeletal architecture in diverse cell types induces a difference in their deformability that presents a viable approach to separate cells in a non-invasive manner. We report on the design and fabrication of a robust and scalable device capable of separating a heterogeneous population of cells with variable degree of deformability into enriched populations with deformability above a certain threshold. The three dimensional device was fabricated in fused silica by femtosecond laser direct writing combined with selective chemical etching. The separator device was evaluated using promyelocytic HL60 cells. Using flow rates as large as 167 µL min(-1), throughputs of up to 2800 cells min(-1) were achieved at the device output. A fluorescence-activated cell sorting (FACS) viability analysis on the cells revealed 81% of the population maintain cellular integrity after passage through the device.

Elasticity of human embryonic stem cells as determined by atomic force microscopy.

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young's modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young's modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.

Mass spectral analyses of corn stover prehydrolysates to assess conditioning processes.

Flow injection electrospray (FIE) and LC-tandem mass spectrometry techniques were used to characterize corn stover acid hydrolysates before and after overliming and ammonia conditioning steps. Analyses were performed on samples without fractionation (dilution only) in an effort provide an inventory of ionizable substances. Statistical evaluation of the results indicates that the ammonia-treated and crude hydrolysates were more similar to one another than any other pairing, with conditioning leading to a decrease in malate levels. LC-tandem mass spectrometry studies were also developed to characterize the oligosaccharides present in each hydrolysate utilizing a hydrophilic interaction chromatographic separation method. Neutral and acidic pentose-based oligosaccharides (xylodextrins) with degrees of polymerization between 2 and 5 were quantified with 4-O-methyl glucuronic acid-containing dimer and trimers predominating. Conditioning had little effect on the quantified oligosaccharide pool.

Large-scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA.

The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.

Adapted ultra scale-down approach for predicting the centrifugal separation behavior of high cell density cultures.

The work presented here describes an ultra scale-down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5- to 10-fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous-flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to approximately 2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell-cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot-scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large-scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale-up, are discussed.