Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

Nature of Novel 2D van der Waals Heterostructures for Superior Potassium Ion Batteries

Novel 2D van der Waals heterostructures with innovative bimetallic oxychloride (Bi‐ and Sb‐based oxychloride) nanosheets that are well dispersed on reduced graphene oxide nanosheets, are established through element engineering for superior potassium ion battery (PIBs) anodes. This material displays an exceptional electrochemical performance, obtaining a discharge capacity as high as 360 mAh g^?1 at 100 mA g^?1 after running 1000 cycles for over 9 months with a capacity preservation percentage of 88.5% and achieving a discharge capacity as high as 319 mAh g^?1 at 1000 mA g^?1, in addition to the low charge/discharge plateaus for anodes and promising full cell performance. More significantly, the nature of such 2D van der Waals heterostructures, including the element engineering for morphology control, the function of each component of heterostructures, the mechanism of potassium ion storage, and the process of K⁺ intercalation accompanied with the lattice distortion and chemical bond breakages, is explored in depth. This study is critical for not only paving the way for the practical application of PIBs but also shedding light on fundamentals of potassium ion storage in 2D van der Waals heterostructures.     

Shewanella biofilm development and engineering for environmental and bioenergy applications

The genus Shewanella comprises about 70 species of Gram-negative, facultative anaerobic bacteria inhabiting various environments, which have shown great potential in various biotechnological applications ranging from environmental bioremediation, metal(loid) recovery and material synthesis to bioenergy generation. Most environmental and energy applications of Shewanella involve the biofilm mode of growth on surfaces of solid minerals or electrodes. In this article, we first provide an overview of Shewanella biofilm biology with the focus on biofilm dynamics, biofilm matrix, and key signalling systems involved in Shewanella biofilm development. Then we review strategies recently exploited to engineer Shewanella biofilms to improve biofilm-mediated bioprocesses.     

Study of accumulation behaviour of tungsten based composite using electron probe micro analyser for the application in bone tissue engineering

In this research, a proto-type study we have conducted, where we have synthesized tungsten based composite materials which are tungsten along with combined oxides of other elements like calcium, scandium, barium, and aluminium in the form of powder with bones powder of mice devised by high energy ball mill and later on fabricating high dense pellets by sintering by spark plasma. The particle sizes of the composite materials are found to be 1–2 µm, as evidenced by the electron microscope, suggesting synthesized materials are of micron size. The quantitative and qualitative analysis of sintered pellets are well confirmed by electron probe micro analyzer (EPMA) and energy dispersive X-ray spectrometer (EDS) which illustrate the greater percentage of tungsten presents in the profound scan areas with other elements of the composite. The absence of pores across the 3D geometry suggesting dense sample, which is quite revealed by the X-ray tomography inspection. The prepared sintered pellets from the tungsten based composites are found to be ≈ 99.5% density with the observation of tungsten to be accumulated uniformly across the scan regions along with focussed hot spots as implied by EPMA. This study paves the way, to examine how the tungsten accumulation and the distribution with the other elements for future understanding in bone tissue engineering application and the in vivo specification of tungsten.     

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Cryogenic 3D printing for tissue engineering

We describe a new cryogenic 3D printing technology for freezing hydrogels, with a potential impact to tissue engineering. We show that complex frozen hydrogel structures can be generated when the 3D object is printed immersed in a liquid coolant (liquid nitrogen), whose upper surface is maintained at the same level as the highest deposited layer of the object. This novel approach ensures that the process of freezing is controlled precisely, and that already printed frozen layers remain at a constant temperature. We describe the device and present results which illustrate the potential of the new technology.