Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

A loadless 4T SRAM powered by gate leakage current with a high tolerance for fluctuations in device parameters

A novel loadless four-transistor static random access memory cell is proposed that consists of two N-type driver MOSFETs and two P-type access ones whose gate leakage currents from word-line are used for holding data in the cell. It is shown that the proposed cell has a higher tolerance for manufacturing device fluctuations compared with the conventional loadless 4T SRAM. Furthermore, it is free from bit-line disturb in contrast to the conventional cell. It is confirmed by simulation in 32nm technology node that the read static noise margin of the proposed cell reaches 138.7% of the six-transistor SRAM cell and that the hold static noise margin can be acceptable when the gate insulator thickness of the P-type access MOSFETs is made thinner than the N-type driver MOSFETs. The retention current for the proposed cell decreases to 66.7% of the 6TSRAM and the data rate in read increases to 125%.

Albumin Microspheres as “Trans-Ferry-Beads” for Easy Cell Passaging in Cell Culture Technology

Protein hydrogels represent ideal materials for advanced cell culture applications, including 3D-cultivation of even fastidious cells. Key properties of fully functional and, at the same time, economically successful cell culture materials are excellent biocompatibility and advanced fabrication processes allowing their easy production even on a large scale based on affordable compounds. Chemical crosslinking of bovine serum albumin (BSA) with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) in a water-in-oil emulsion with isoparaffinic oil as the continuous phase and sorbitan monooleate as surfactant generates micro-meter-scale spherical particles. They allow a significant simplification of an indispensable and laborious step in traditional cell culture workflows. This cell passaging (or splitting) to fresh culture vessels/flasks conventionally requires harsh trypsinization, which can be omitted by using the “trans-ferry-beads” presented here. When added to different pre-cultivated adherent cell lines, the beads are efficiently boarded by cells as passengers and can be easily transferred afterward for the embarkment of novel flasks. After this procedure, cells are perfectly viable and show normal growth behavior. Thus, the trans-ferry-beads not only may become extremely affordable as a final product but also may generally replace trypsinization in conventional cell culture, thereby opening new routes for the establishment of optimized and resource-efficient workflows in biological and medical cell culture laboratories.

Syringe cell to avoid crevice corrosion on stainless steels during potentiodynamic polarization testing

In this study, an electrochemical syringe droplet cell was developed and used to evaluate the pitting corrosion resistance of AISI 304 stainless steel. The cell was also adapted to perform measurements in de-aerated environment, by means of continuous N2 injection. The results obtained with the syringe cells, in both conditions (aerated and de-aerated), were reproducible and free of crevice corrosion, since the cell configuration does not allow obtaining this condition. The proposed cell demonstrates coherency with data acquired using conventional cell arrangements, being a potential alternative to study pitting corrosion of passive metals.

Implication of ferroptosis in aging

Life is indeed continuously going through the irreversible and inevitable process of aging. The rate of aging process depends on various factors and varies individually. These factors include various environmental stimuli including exposure to toxic chemicals, psychological stress whereas suffering with various illnesses specially the chronic diseases serve as endogenous triggers. The basic underlying mechanism for all kinds of stresses is now known to be manifested as production of excessive ROS, exhaustion of ROS neutralizing antioxidant enzymes and proteins leading to imbalance in oxidation and antioxidant processes with subsequent oxidative stress induced inflammation affecting the cells, tissues, organs and the whole body. All these factors lead to conventional cell death either through necrosis, apoptosis, or autophagy. Currently, a newly identified mechanism of iron dependent regulated cell death called ferroptosis, is of special interest for its implication in pathogenesis of various diseases such as cardiovascular disease, neurological disorders, cancers, and various other age-related disorders (ARD). In ferroptosis, the cell death occur neither by conventional apoptosis, necrosis nor by autophagy, rather dysregulated iron in the cell mediates excessive lipid peroxidation of accumulated lethal lipids. It is not surprising to assume its role in aging as previous research have identified some solid cues on the subject. In this review, we will highlight the factual evidences to support the possible role and implication of ferroptosis in aging in order to declare the need to identify and explore the interventions to prevent excessive ferroptosis leading to accelerated aging and associated liabilities of aging.

The discriminatory power of the T cell receptor

T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity non-self pMHC antigens. Although the discriminatory power of the TCR is widely believed to be near-perfect, technical difficulties have hampered efforts to precisely quantify it. Here, we describe a method for measuring very low TCR/pMHC affinities, and use it to measure the discriminatory power of the TCR, and the factors affecting it. We find that TCR discrimination, although enhanced compared with conventional cell-surface receptors, is imperfect: primary human T cells can respond to pMHC with affinities as low as KD ~1 mM. The kinetic proofreading mechanism fit our data, providing the first estimates of both the time delay (2.8 s) and number of biochemical steps (2.67) that are consistent with the extraordinary sensitivity of antigen recognition. Our findings explain why self pMHC frequently induce autoimmune diseases and anti-tumour responses, and suggest ways to modify TCR discrimination.

Extremely High-Throughput Parallel Microfluidic Vortex-Actuated Cell Sorting

We demonstrate extremely high-throughput microfluidic cell sorting by making a parallel version of the vortex-actuated cell sorter (VACS). The set-up includes a parallel microfluidic sorter chip and parallel cytometry instrumentation: optics, electronics and control software. The result is capable of sorting lymphocyte-sized particles at 16 times the rate of our single-stream VACS devices, and approximately 10 times the rate of commercial cell sorters for an equivalent procedure. We believe this opens the potential to scale cell sorting for applications requiring the processing of much greater cell numbers than currently possible with conventional cell sorting.

Investigating heterogeneities of live mesenchymal stromal cells using AI-based label-free imaging

Mesenchymal stromal cells (MSCs) are multipotent cells that have great potential for regenerative medicine, tissue repair, and immunotherapy. Unfortunately, the outcomes of MSC-based research and therapies can be highly inconsistent and difficult to reproduce, largely due to the inherently significant heterogeneity in MSCs, which has not been well investigated. To quantify cell heterogeneity, a standard approach is to measure marker expression on the protein level via immunochemistry assays. Performing such measurements non-invasively and at scale has remained challenging as conventional methods such as flow cytometry and immunofluorescence microscopy typically require cell fixation and laborious sample preparation. Here, we developed an artificial intelligence (AI)-based method that converts transmitted light microscopy images of MSCs into quantitative measurements of protein expression levels. By training a U-Net+ conditional generative adversarial network (cGAN) model that accurately (mean $$r_s$$ r s = 0.77) predicts expression of 8 MSC-specific markers, we showed that expression of surface markers provides a heterogeneity characterization that is complementary to conventional cell-level morphological analyses. Using this label-free imaging method, we also observed a multi-marker temporal-spatial fluctuation of protein distributions in live MSCs. These demonstrations suggest that our AI-based microscopy can be utilized to perform quantitative, non-invasive, single-cell, and multi-marker characterizations of heterogeneous live MSC culture. Our method provides a foundational step toward the instant integrative assessment of MSC properties, which is critical for high-throughput screening and quality control in cellular therapies.

Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate

The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5′ and 3′ rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5′ and 3′ NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5′ and 3′ non-coding regions (NCR) highlighted that the terminal 3′ end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5′ and 3′ NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5′ and 3′ NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.

Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers

Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.