Cell Suspension Research Articles

A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane.

The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.

Characterization of melanin from Exophiala mesophila with the prospect of potential biotechnological applications

IntroducionFungal melanin is an underexplored natural biomaterial of great biotechnological interest in different areas. This study investigated the physical, chemical, electrochemical, and metal-binding properties of melanin extracted from the metallotolerant black fungus Exophiala mesophila strain IRTA-M2-F10.Materials and methodsSpecific inhibitory studies with tricyclazole and biochemical profiling of whole cells by synchrotron radiation-based Fourier-transform infrared spectral microscopy (SR-FTIRM) were performed. An optimized extraction protocol was implemented, and purified fungal melanin was characterized using an array of spectrophotometric techniques (UV-Vis, FTIR, and EPR) and by cyclic voltammetry (CV) experiments. The metal-binding capacity of melanin extracts was also assessed by using Cr(VI) as a model heavy metal.ResultsInhibitory studies indicated that 1,8-dihydroxynaphthalene may be the main precursor molecule of E. mesophila melanin (DHN-melanin). The biochemical characterization of fungal melanin extracts were benchmarked against those from two melanins comprising the precursor molecule L-3,4-dihydroxiphenylalanine (DOPA-melanin): extracts from the ink of the cephalopod Sepia officinalis and DOPA-melanin synthesized in the laboratory. The CV results of melanin extracts incubated with and without cell suspensions of the electroconductive bacterium Geobacter sulfurreducens were indicative of novel semiquinone/hydroquinone redox transformations specific for each melanin type. These interactions may play an important role in cation exchange for the adsorption of metals and in microbial interspecies electron transfer processes.DiscussionThe obtained results provided further evidence for the DHN-nature of E. mesophila melanin. The FTIR profiling of melanin extracts exposed to Cr(VI), compared to unexposed melanin, resulted in useful information on the distinct surface-binding properties of fungal melanin. The parameters of the Langmuir and Freundlicht isotherms for the adsorption of Cr(VI) were determined and compared to bibliographic data. Altogether, the inherent properties of fungal melanin suggest its promising potential as a biomaterial for environmental applications.

Noncultured Trypsinized Epidermal Cell Suspension Transplantation After Cryoblebbling Versus Noncultured Nontrypsinized Epidermal Cell Graft Homogenized With Plasma Gel After Dermabrasion for Stable Vitiligo.

Vitiligo treatment is challenging, especially for resistant and stable vitiligo, which requires surgical management. Noncultured epidermal cell suspension has been modified to enhance the treatment outcomes. Comparison of autologous noncultured trypsinized epidermal cell suspension in recipient site prepared by cryoblebbling and noncultured nontrypsinized epidermal cell graft homogenized with plasma gel in recipient site prepared by dermabrasion for stable vitiligo treatment. Interventional comparative study on 30 patients with stable vitiligo, randomly divided into 2 equal groups. Group A: noncultured trypsinized epidermal cell suspension for recipient prepared by cryoblebbling. Group B: noncultured nontrypsinized epidermal cell graft homogenized with plasma gel for recipient prepared by dermabrasion. Afterward, both groups received 3 months of narrow-band ultraviolet B phototherapy. The plasma gel group showed a significantly earlier onset of repigmentation and faster healing (p = .002* and <.001*, respectively). Overall, repigmentation was higher in the plasma gel group (p = .037* at the end of the second month). Color matching and patient satisfaction were higher in the plasma gel group, without statistical significance. The cryobleb group showed more recipient site complications, and the plasma gel procedure was relatively easier and cheaper. Plasma gel modification is cost-effective, less time-consuming, does not require trypsinization, and provides rapid, satisfactory, and uniform repigmentation. Cryoblebbing and trypsinization are effective; however, there are more technical difficulties, delayed healing, and delayed onset of repigmentation.

Anti-4-1BB×PD-L1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD-1 Blockade.

To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PD-L1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PD-L1 and 4-1BB, and demonstrated strong antitumor T-cell responses without considerable toxicity. Here, we investigated how the combination of ABL503 and anti-PD-1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TILs) and anti-tumor efficacy. Single cell suspensions of hepatocellular carcinoma and ovarian cancer from treatment-naive patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD-1/hPD-L1/h4-1BB triple knock-in mice were used to evaluate the effects of ABL503 and anti-PD-1 blockade in vivo. We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD-1 blockade. Importantly, compared to anti-PD-1 blockade alone, the combination of ABL503 and anti-PD-1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD-1 in vivo significantly alleviated tumor growth, and induced enhanced infiltration and activation of CD8+ TILs. ABL503-a PD-L1 and 4-1BB dual-targeting bispecific antibody-elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anti-cancer effects of anti-PD-1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD-1 inhibitors will likely further enhance therapeutic benefit in clinical trials.

First in vitro measurement of VHEE relative biological effectiveness (RBE) in lung and prostate cancer cells using the ARES linac at DESY

Very high energy electrons (VHEE) are a potential candidate for radiotherapy applications. This includes tumours in inhomogeneous regions such as lung and prostate cancers, due to the insensitivity of VHEE to inhomogeneities. This study explores how electrons in the VHEE range can be used to perform successful in vitro radiobiological studies. The ARES (accelerator research experiment at SINBAD) facility at DESY, Hamburg, Germany was used to deliver 154 MeV electrons to both prostate (PC3) and lung (A549) cancer cells in suspension. Dose was delivered to samples with repeatability and uniformity, quantified with Gafchromic film. Cell survival in response to VHEE was measured using the clonogenic assay to determine the biological effectiveness of VHEE in cancer cells for the first time using this method. Equivalent experiments were performed using 300 kVp X-rays, to enable VHEE irradiated cells to be compared with conventional photons. VHEE irradiated cancer cell survival was fitted to the linear quadratic (LQ) model (R2 = 0.96–0.97). The damage from VHEE and X-ray irradiated cells at doses between 1.41 and 6.33 Gy are comparable, suggesting similar relative biological effectiveness (RBE) between the two modalities. This suggests VHEE is as damaging as photon radiotherapy and therefore could be used to successfully damage cancer cells during radiotherapy. The RBE of VHEE was quantified as the relative doses required for 50% (D0.5) and 10% (D0.1) cell survival. Using these values, VHEE RBE was measured as 0.93 (D0.5) and 0.99 (D0.1) for A549 and 0.74 (D0.5) and 0.93 (D0.1) for PC3 cell lines respectively. For the first time, this study has shown that 154 MeV electrons can be used to effectively kill lung and prostate cancer cells, suggesting that VHEE would be a viable radiotherapy modality. Several studies have shown that VHEE has characteristics that would offer significant improvements over conventional photon radiotherapy for example, electrons are relatively easy to steer and can be used to deliver dose rapidly and with high efficiency. Studies have shown improved dose distribution with VHEE in treatment plans, in comparison to VMAT, indicating that VHEE can offer improved and safer treatment plans with reduced side effects. The biological response of cancer cells to VHEE has not been sufficiently studied as of yet, however this initial study provides some initial insights into cell damage. VHEE offers significant benefits over photon radiotherapy and therefore more studies are required to fully understand the biological effectiveness of VHEE.

Genomic insights into Streptomyces hygroscopicus subsp. hygroscopicus SRF1: a potential biocontrol agent against fusarium wilt with plant growth-promoting abilities in tomatoes

ABSTRACT Streptomyces sp. isolate SRF1 has previously demonstrated promising results in controlling various plant fungi under in vitro conditions. However, further exploration of its genome and its efficacy in disease control and plant growth promotion under in vivo conditions is warranted. This study aimed to utilise whole genome sequencing (WGS) to classify Streptomyces sp. isolate SRF1 at the species level and predict genes associated with antagonistic ability and plant growth promotion, thereby assessing its potential as a biocontrol agent against fusarium wilt and a promoter of plant growth. WGS was employed to elucidate the taxonomic classification of isolate SRF1 and predict genes involved in disease suppression and plant growth promotion. In vitro assays were conducted to evaluate its antagonistic activity against Fusarium oxysporum f. sp. lycopersici (Fol) and its production of cellulase, amylase, indole-3-acetic acid (IAA), and siderophores. Pot experiments were carried out using SRF1 cell suspension and culture filtrate to assess its efficacy against Fol and its impact on tomato growth. Real-time PCR analysis was performed to correlate gene expression levels with observed activities. The results showed that isolate SRF1 was classified as S. hygroscopicus subsp. hygroscopicus and demonstrated antagonism against Fol. Furthermore, WGS revealed genes or gene clusters associated with disease control, including those encoding antifungal agents such as butyrolactol A, nigericin, and hygromycin A. Additionally, genes responsible for auxin biosynthesis, siderophore production, and carbohydrate-digesting enzymes such as chitinase, cellulase, and amylase were identified, suggesting potential roles in disease suppression and plant growth promotion.

Development of a label‐free, impedance‐based biosensor to identify harmful effects of pesticides on insect cells

AbstractInsects are a major part of the planet's ecosystem and their vital role as pollinators for agriculture is undisputed. Alongside factors as climate change or loss of habitats, rising use of pesticides emerges as a key threat to insect populations. For fighting this man‐made problem, development of an easy, fast, sensitive, and non‐invasive biosensor for determining pesticide toxicity may help to ban harmful substances and formulations. Here, a biosensor based on Sf21 (Spodoptera frugiperda) insect cells as sensors and electric cell‐substrate impedance sensing (ECIS) as physical transducer is described. Sf21 cell suspensions and well‐defined pesticide solutions were mixed immediately before seeding on planar gold‐film electrodes. The capacitance at 20 kHz was recorded as a function of time as a measurand for cell adhesion providing dose–response profiles of pesticide impact. For future in‐field applications, decoupling of the cell culture routines from the actual cytotoxicity assay is mandatory. Thus, suspensions of Sf21 cells were cryopreserved at –80°C in the wells of multielectrode arrays and thawed anytime for conducting the assays. Five pesticides were tested for their concentration‐dependent cytotoxicity expressed as EC50 values by ECIS and validated using the well‐established WST‐1 cell viability assay. Results were found to be in good agreement. Our studies revealed cytotoxic effects of some pesticides sold for home usage far below the recommended concentration and were found to be more toxic than formulations sold for agricultural industry only.

Analysis of microcystins in alum water treatment sludges: holding times, temperatures, linearity of response, and sensitivity to pre-coagulation cell titers

ABSTRACT ELISA assays are a potential tool to screen for dissolved or cell bound microcystins in drinking water treatment sludges. In order to evaluate this potential more thoroughly, experiments were performed in alum sludges to: (1) evaluate the impacts of sample storage times, temperatures, and sludge composition on spiked microcystin-LR recovery by ELISA; (2) examine the linearity of ELISA responses to spiked microcystin-LR as a function of sludge composition; and (3) examine the sensitivity ELISA and LC/MS/MS to five different concentrations of microcystin-producing cyanobacteria entrained in sludges of two different compositions. During storage experiments, microcystin recovery efficiencies ranged from 85% to 125% across the range of 12 storage time and temperature combinations with recovery efficiencies in 7 of the 12 combinations falling into the 90% to 110% range. During the linearity experiments, linear models fit ELISA responses in all sludge compositions with R2 values ≥ 0.95. During the sensitivity studies, simple freeze/thaw/centrifugation processing combined with ELISA or LC/MS/MS analyses resulted in detection of microcystins in thickened sludges derived from pre-coagulation cell suspensions of 102–106 cells/mL. In addition, the relationships between toxin concentrations in sludges and the original cell titers were linear regardless of analytical method.

Retinal microglia express more MHC class I and promote greater T-cell-driven inflammation than brain microglia

IntroductionMacrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments.MethodsEyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results in vivo.ResultsCluster analysis of integrated gene expression data from eye and brain identified 6 Tmem119+P2ry12+ microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, H2-D1 and H2-K1) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8+ T-cell density was greater in the retina than the brain.DiscussionOur data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8+ T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8+ T-cell driven autoimmune disease.

Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.

Melatonin Mitigates Lead-Induced Oxidative Stress and Modifies Phospholipid Profile in Tobacco BY-2 Suspension Cells

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin’s potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium—control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth—just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.

Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.

Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection

Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection

Thermal treatment impact on the evolution of active phases in layered double hydroxide-based ZnCr photocatalysts: Photodegradation and antibacterial performance

Abstract This study investigated the influence of thermal treatment on the photocatalytic performance of ZnCr layered double hydroxide-based mixed metal oxides in the degradation of methylene blue and brilliant cresyl blue organic dyes under simulated solar light irradiation. The photocatalysts were synthesized using a simple coprecipitation method and subjected to thermal treatment at temperatures ranging from 100°C to 900°C. Additionally, the study explored the antibacterial activity against Escherichia coli and Staphylococcus aureus using a novel antibacterial experimental setup. It not only involved the introduction of ZnCr samples into BioPeptone/prepared cell suspension to enhance photocatalyst–bacteria cell contact but also included research on antibacterial activity induced by solar irradiation and also in the absence of light, providing crucial insights into photocatalytic antibacterial activity of ZnCr photocatalysts. Despite satisfactory efficiencies observed for all thermally treated ZnCr samples (removal efficiency ranging from 40% to 90%), ZnCr 900 (thermally treated at 900°C) exhibited exceptional performance, achieving nearly 100% removal efficiency and complete growth inhibition for both bacteria. Integrating these findings with structural and textural characterization data, as well as kinetic studies, our comprehensive analysis enhances the understanding of structure-dependent photocatalytic activities. These insights open possibilities for the application of ZnCr photocatalysts in water purification and environmental remediation.

Graft cell expansion from hiPSC-RPE strip after transplantation in primate eyes with or without RPE damage

Clinical studies using suspensions or sheets of human pluripotent cell-derived retinal pigment epithelial cells (hiPSC-RPE) have been conducted globally for diseases such as age-related macular degeneration. Despite being minimally invasive, cell suspension transplantation faces challenges in targeted cell delivery and frequent cell leakage. Conversely, although the RPE sheet ensures targeted delivery with correct cell polarity, it requires invasive surgery, and graft preparation is time-consuming. We previously reported hiPSC-RPE strips as a form of quick cell aggregate that allows for reliable cell delivery to the target area with minimal invasiveness. In this study, we used a microsecond pulse laser to create a local RPE ablation model in cynomolgus monkey eyes. The hiPSC-RPE strips were transplanted into the RPE-ablated and intact sites. The hiPSC-RPE strip stably survived in all transplanted monkey eyes. The expansion area of the RPE from the engrafted strip was larger at the RPE injury site than at the intact site with no tumorigenic growth. Histological observation showed a monolayer expansion of the transplanted RPE cells with the expression of MERTK apically and collagen type 4 basally. The hiPSC-RPE strip is considered a beneficial transplantation option for RPE cell therapy.

Abstract PO1-26-06: The effect of localized radiotherapy in combination with chemoimmunotherapy in a humanized triple-negative breast cancer mouse model

Abstract Background: Metastatic triple-negative breast cancer (TNBC) is an aggressive sub-type of breast cancer that is challenging to treat. While the addition of immunotherapy (IT) with pembrolizumab (pembro) to chemotherapy prolongs overall survival in patients with programmed death-ligand 1 positive (PD-L1+) TNBC, few patients obtain long-term benefit. Radiotherapy (RT) can stimulate cellular damage, release of tumor antigens and promote the T cell anti-tumor response including at distant sites (abscopal response) when used with IT. The purpose of this study was to investigate the combination of RT with pembro and paclitaxel (pac) in a TNBC humanized mouse model. Methods: Human Immune System BRGS (BALB/c, Rag2-/-, IL2RγC-/-, NODSIRPa ) mice were engrafted with TNBC MDA-MB-231 cells in bilateral flanks. Treatments included: vehicle, pac 10 mg/kg intra-peritoneal (IP) weekly, pembro 15 mg/kg IP weekly, and RT 8 Gy x 3 fractions every other day the first week of treatment to one-sided tumors (contralateral tumors received no RT). Mice were treated and analyzed in the following groups: vehicle, pac + pembro, RT + pac, and RT + pac + pembro. Treatments began when tumors reached 100-300 mm3 and tumors were measured twice weekly. At the end of study, sera, lymph nodes (LNs), spleen, and tumor tissue were collected for immunohistochemistry, single cell suspensions, and flow cytometry analysis for immune cell populations. Results: All treatment arms resulted in a decrease in tumor volume when compared to vehicle with the greatest reduction in the RT + pac and RT + pac + pembro arms. Combination therapy with RT + paclitaxel (p=0.0002) and RT + pac + pembro (p=0.008) resulted in a statistically significant decrease in specific growth rate (SGR) when compared to vehicle. An abscopal effect was not observed in the non-RT tumors compared to RT-tumors in either group. RT + pembro was associated with an increase in hCD45 T cells in LNs and spleens but not in tumors. In mice that received RT, a decrease was noted in HLA-ABC class I and Intermediate Class II major histocompatibility complex (MHC). In addition, RT-tumors had reduced expression of PD-L1 and polio virus receptor (PVR) and increased expression of PD-1. The percentage of interferon gamma (IFNg+), tumor necrosis factor alpha (TNFa+) CD8+ T cells increased with radiation. This percentage was the highest in the triplet therapy with RT+ pembro + pac. Conclusion: Combination treatment with RT + immunochemotherapy resulted in a significant increase in anti-tumor activity when compared to immunochemotherapy alone, however, an abscopal effect was not observed in contralateral non-RT tumors. RT resulted in down-regulation of immune inhibitory receptors and tumor MHC. Citation Format: Anna Schreiber, Stacey Bagby, Stephen Smoots, Adrian Dominguez, Christine Fisher, Julie Lang, Todd Pitts, Jennifer Diamond. The effect of localized radiotherapy in combination with chemoimmunotherapy in a humanized triple-negative breast cancer mouse model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-06.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

An automated approach for hemocytometer cell counting based on image-processing method

Evaluating cell viability plays an important role in routine cell culture, toxicology, and drug development. Trypan blue stain is one of the simplest assays that are used to determine the number of viable cells in a cell suspension. The stained cell suspension can then be counted under a microscope using a hemocytometer. Hemocytometers have a fixed volume, making it possible to calculate the concentration of cells in the sample and measure viability. Most of the related studies are conducted using manual counting methods. However, manual counting is time-consuming and laborious. While various automated cell counting devices have been proposed, they tend to be too expensive for widespread deployment. Although low-cost automated cell counting methods presented in the literatures provide high accuracy and cost-effective alternatives, only cells in the field of view of microscope with unknown volume are counted. This will make the concentration inestimable. Accordingly, the present study proposes a low-cost but accurate method for hemocytometer cell counting using an image-processing approach. The accuracy of the proposed method is demonstrated by comparing the cell counting results obtained for human cervical carcinoma (HeLa) cells with the manual counts. It is shown that the proposed approach has a mean absolute percentage error of less than 6.26 % for samples containing a mixture of live and dead cells. Moreover, the proposed system is equipped with a simple and intuitive human machine interface such that the counting task can be performed by individuals with no prior experience in the image-processing field.

The Enhancement of Tumor Ablation Effect by the Combination of High-Frequency and Low-Voltage Bipolar Electroporation Pulses.

Recently, combining short high-voltage IRE monopolar pulses with long low-voltage IRE monopolar pulses was shown to enlarge the ablation region. This finding indicates that combining HFBPs with low-voltage bipolar pulses (LVBPs), which are called composited bipolar pulses (CBPs), may enhance the ablation effect. This study designed a pulse generator by modifying a full-bridge inverter. The cell suspension and 3D tumor mimic experiments (U251 cells) were performed to examine the enhancement of the ablation effect. The generator outputs HFBPs with 0-±2.5 kV and LVBPs with 0-±0.3 kV in one period. The pulse parameters are adjustable by programming on a human-computer interface. The cell suspension experiments showed that CBPs could enhance cytotoxicity, as compared to HFBPs with no cell-killing effect. Even at lower electric energy, the cell viability by CBPs was significantly lower than that of the HFBPs protocol. The ablation experiments on the 3D tumor mimic showed that the CBPs could create a larger connected ablation area. In contrast, the HFBPs protocol with a similar dose generated a nonconnected ablation area. Results indicate that the CBPs protocol can enhance the ablation effect of HFBPs protocol. This proposed generator that uses the CBPs principle may be a useful tool for tumor ablation.

Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by Wall-associated kinase gene MbWAK1.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.