Cell Strains Research Articles

Chromium-doped ZnO nanoparticles synthesized via auto-combustion: Evaluation of concentration-dependent structural, band gap-narrowing effect, luminescence properties and photocatalytic activity

Herein we report the effect of the Cr3+ content on the distinctive features of the first-ever auto-combustion-synthesis-produced Cr3+ doped ZnO nanoparticles utilizing leucine as the fuel. To assess the impact of fuel and Cr3+ content on the structural, optical, and luminescence characteristics, as-synthesized powders were systematically examined using powder XRD, UV–Visible, and Photoluminescence spectroscopy. Structural studies show that the exothermicity of the redox mixture containing oxidizer and fuel combination leads to the development of polycrystalline, single-phase, and nanosized particles. XRD analysis shows that, reduced crystallite size with Cr3+ content. In contrast, other characteristics like lattice parameters, unit cell volume, strain, and dislocation density, were shown to increase linearly with Cr3+ concentration up to 7 mol%. Additionally, it is seen that the predicted Urbach energy values rise as Cr3+ is added to the ZnO host matrix, indicating that the substitution of Cr3+ for Zn in the host ZnO lattice has increased structural disorder. In contrast to the other samples, the 5 mol% Cr3+ doped ZnO sample exhibits the emission near the white region according to the PL emission measurements. Furthermore, the photocatalytic activity of the as-prepared Cr3+ doped ZnO NPs was examined. It was found that the as-prepared samples exhibit outstanding photocatalytic activity in the visible wavelength region against Malachite Green (MG) dye. Among all, 9 mol% Cr3+ doped ZnO has shown the maximum 95.4% degradation against MG dye. All the investigated results conclude that the produced Cr3+ doped ZnO nanoparticles could be exploited in a variety of upcoming optoelectronic and photocatalytic applications.

Structural insights into interactions of new polymeric (μ–oxo) bridged Cu(II) complexes of taurine with yeast tRNA by spectroscopic and computational approaches and its application towards chemoresistant cancer lines

RNA-targeted drugs are considered as safe treatment option for the cure of many chronic diseases preventing off-targeted delivery and acute toxic manifestations. FDA has approved many such RNA therapies in different phases of clinical trials, validating their use for the treatment of various chronic diseases. We report herein, new water-soluble (μ–oxo) bridged polymeric Cu(II) complexes of taurine (2–aminoethane sulfonic acid) complexes 1 and 2. The therapeutic potency of 1 and 2 was ascertained by studying biophysical interactions with tRNA/ct-DNA. The experimental results demonstrated that the complexes interacted avidly to nucleic acids through intercalation mode depicting a specific preference for tRNA in comparison to ct–DNA and, moreover 2 showed higher binding propensity than 1. The electrophoretic behaviour of the complexes with plasmid pBR322 DNA and tRNA were examined by gel mobility assay that revealed a concentration-dependent activity with complex 2 performing more efficient cleavage as compared to complex 1. Cytotoxicity results on cancer cell strains displayed higher cytotoxicity than complex 1 against treated cancer cells. The synthesized copper(II) taurine complexes have met the basic criteria of anticancer drug design as they are structurally well-characterized, exhibiting good solubility in water, lipophilic in nature with superior intercalating propensity towards tRNA and cytotoxic in nature.

Bif-1c Attenuates Viral Proliferation by Regulating Autophagic Flux Blockade Induced by the Rabies Virus CVS-11 Strain in N2a Cells.

Bax-interacting factor-1 (Bif-1) is a multifunctional protein involved in apoptosis, autophagy, and mitochondrial morphology. However, the associations between Bif-1 and viruses are poorly understood. As discrete Bif-1 isoforms are selectively expressed and exert corresponding effects, we evaluated the effects of neuron-specific/ubiquitous Bif-1 isoforms on rabies virus (RABV) proliferation. First, infection with the RABV CVS-11 strain significantly altered Bif-1 expression in mouse neuroblastoma (N2a) cells, and Bif-1 knockdown in turn promoted RABV replication. Overexpression of neuron-specific Bif-1 isoforms (Bif-1b/c/e) suppressed RABV replication. Moreover, our study showed that Bif-1c colocalized with LC3 and partially alleviated the incomplete autophagic flux induced by RABV. Taken together, our data reveal that neuron-specific Bif-1 isoforms impair the RABV replication process by abolishing autophagosome accumulation and blocking autophagic flux induced by the RABV CVS-11 strain in N2a cells. IMPORTANCE Autophagy can be triggered by viral infection and replication. Autophagosomes are generated and affect RABV replication, which differs by viral strain and infected cell type. Bax-interacting factor-1 (Bif-1) mainly has a proapoptotic function but is also involved in autophagosome formation. However, the association between Bif-1-involved autophagy and RABV infection remains unclear. In this study, our data reveal that a neuron-specific Bif-1 isoform, Bif-1c, impaired viral replication by unchoking autophagosome accumulation induced by RABV in N2a cells to a certain extent. Our study reveals for the first time that Bif-1 is involved in modulating autophagic flux and plays a crucial role in RABV replication, establishing Bif-1 as a potential therapeutic target for rabies.

Abstract 5073: NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy

Abstract While the advent of immune checkpoint blockade (ICB) has dramatically improved the prognosis of many immune-infiltrated cancers, for others, unfortunately, these benefits have yet to be realized. The major challenge before the field, then, is to identify combination therapies that act both to combat evolved resistance. NT219 is a novel dual inhibitor of insulin receptor substrates 1 and 2 (IRS) and STAT3. NT219 demonstrated antitumor effects against both in situ and metastatic human melanoma models in mice as a stand-alone treatment and in combination with mutated BRAF and MEK inhibitors. The potential of NT219 to overcome resistance and increase efficacy was demonstrated in PDX models with multiple drug classes. Collectively, these findings provided preclinical proof-of-concept NT219 as a promising novel cancer therapy. Given this data, the goal of our study was to assess the efficacy of combining NT219 with anti-PD-1 and anti-CTLA-4 ICB, and test capacity of the combination to overcome immune resistance. To that end, we have examined PD-L1 expression levels in vitro following NT219 treatment in two different strains of melanoma cells, B16-tdTomato (TMT) and B16 3I-F4, ICB sensitive and ICB resistant derivative lines, respectively. In addition, to evaluate the combination effect of NT219 with ICB therapy in ICB-resistant PDX model, we used a humanized PDX model of pembrolizumab-resistant gastroesophageal tumor (GEJ). These mice were injected with PBMCs from the same patient. Using multiple syngeneic immunocompetent models of melanoma (TMT and 3I-F4 cells) and the humanized PDX model, we investigated the potential of each ICB with NT219. Our findings showed that NT219 increased PD-L1 expression levels on both melanoma cell lines in vitro, however, the PD-L1 expression levels were much more elevated 3I-F4 than TMT cells. Interestingly, the levels of both IRS and activated pSTAT3, the targets of NT219, which consist well known resistance mechanisms, were higher in the resistant cells as compared to the sensitive line. In accordance we found that NT219 was able to restore anti-PD-1 sensitivity in 3I-F4 model (TGI = 58%) in the syngeneic model, promoting tumor rejection and increasing survival rates. The combined approach between NT219 and anti-CTLA-4 displayed a moderate effect (TGI = 37%). In the TMT, NT219 combined with anti-PD-1 showed greater efficacy compared to anti-PD-1 alone (TGI = 63% and TGI = 47%, respectively). These results were confirmed using the humanized ICB-resistant PDX model, where impressive synergy between NT219 and the anti-PD-1 (TGI = 98%) was demonstrated. To summarize, we found a significant synergistic effect of NT219 combined with anti-PD-1 therapy, supported by a mechanism of PDL-1 induction making ICB resistant tumors amenable to ICB treatment. Collectively, these findings demonstrated that NT219 has the potential to reverse ICB resistance in both human PDX and murine syngeneic tumor model systems Citation Format: Ricardo Alexandre de Azevedo, Hadas Reuveni, Menashe Bar-Eli, Michael Curran. NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5073.

A rapid and flexible microneutralization assay for serological assessment of influenza viruses.

Serological responses from influenza vaccination or infection are typically measured by hemagglutinin inhibition (HAI) or microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability to recent strains. We developed a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay. Sera were obtained from the HIVE study, a Michigan household cohort. Reverse genetics was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from an A/WSN/1933 backbone, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in light emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. Three hundred thirty-three sera were collected from 259 participants between May 2016 and July 2018. Sampled participants were 7-68 years of age, and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/Hong Kong/4801/2014 (ρ= 0.52, p≤ 0.01) and A/Singapore/INFIMH-16-0019/2016 (ρ= 0.79, p≤ 0.01). LMN titers were lower for cell strains compared to egg strains (A/Hong Kong/4801/2014 mean log2 fold change = -2.66, p≤ 0.01 and A/Singapore/INFIMH-16-0019/2016 mean log2 fold change = -3.15, p≤ 0.01). The LMN assay was feasible using limited sample volumes and able to differentiate small antigenic differences between egg-adapted and cell-derived strains. The correspondence of these results with the commonly used HAI confirms the utility of this assay for high-throughput studies of correlates of protection and vaccine response.

In-line monitoring of Bordetella pertussis cultivation using fluorescence spectroscopy.

Fluorescence spectroscopy is a non-invasive and highly sensitive method for bioprocess monitoring. The use of fluorescence spectroscopy is not very well established in the industry for in-line monitoring. In the present work, a 2-D fluorometer with two excitation lights (365 and 405 nm) and emission spectra in the range of 350-850 nm were used for in-line monitoring of two strains of Bordetella pertussis cultivation operated in batch and fed batch. A Partial Least Squares (PLS) based regression model was used for the estimation of cell biomass, amino acids (glutamate and proline) and antigen (Pertactin)produced. It was observed that accurate predictions were achieved when models were calibrated separately for each cell strain and nutrient media formulation. Also, prediction accuracy was improved when dissolved oxygen, agitation and culture volume are added as additional features in the regression model. The proposed approach of combining in-line fluorescence and other online measurements is shown to have good potential for in-line monitoring of bioprocesses.

Methyltransferase K-D-K-E motif influences the intercellular transmission of Newcastle disease virus

ABSTRACT We previously demonstrated that two methyltransferase motifs, K-D-K-E and G-G-D, affect the pathogenicity of Newcastle disease virus (NDV) by regulating mRNA translation and virus transmission. Here, we compared the infectious centre area produced by the NDV strain, rSG10, and methyltransferase motifs mutant rSG10 strains in DF-1 cells. The results show that intercellular transmission was attenuated by methyltransferase motif mutations. We further determined the ability of mutant viruses to spread in cell-free and cell-to-cell situations. Cell-free transmission of rSG10-K1756A was not reduced, indicating that cell-to-cell transmission of rSG10-K1756A was decreased. Using a donor and target system, we demonstrated that NDV can spread from cell-to-cell directly. Furthermore, by comparing the protein distribution area of three strains when treated with 2% agar overlay, we found that rSG10-K1756A was defective in cell-to-cell transmission. Tunnelling nanotubes (TNTs) are an important mode for cell-to-cell transmission. Treatment of cells with cytochalasin D (CytoD) or nocodazole to inhibit the formation of TNTs, reduced protein levels in all strains, but rSG10-K1756A was the least affected. These results indicate that mutation of the K-D-K-E motif is likely to restricted the spread of NDV via TNTs. Finally, we observed that matrix protein (M) and fusion protein (F) promoted the formation of cellular extensions, which may be involved in the cell-to-cell spread of NDV. Our research reveals a novel mechanism by which methyltransferase motifs affect the cell-to-cell spread of NDV and provides insight into dissemination of paramyxoviruses.

Enhancing the Efficiency of Doxorubicin -Conjugated with gold nanocomposites for Active Targeting and Laser Treatment for Human Esophagus Cancer Cells

Nanotechnology is being used in cancer treatment to deliver drugs to tumors, improve imaging, and detect cancer cells. Nanoparticles are being used to deliver drugs more effectively to tumors, while also reducing the side effects of the drugs. The current context shows the conceivable biomedical utility of gold nanoparticles (AuNPs) covered with doxorubicin (DOX) for double chemotherapy and photothermal therapy (PTT) of throat cancers (esophagus) in vitro. DOX as FDA-authorized anti-cancer drug was added to AuNPs to enhance the chemotherapy impact. The blended DOX - AuNPs showed exorbitant cytotoxicity at the cancer cells and strong light absorption for temperature expansion in vitro. In the wake of treating DOX– Au nanocomposites with inside the esophagus cell line, PTT-helped DOX-Au nanocomposites involved the fantastical cytotoxicity of the cell strains for 15 min after the laser treatment. Exceptionally cytotoxicity impact in esophagus cell line turned out to be more attractive due to specific light absorption with the aid of using the directed DOX-Au nanocomposites. Thusly, the proposed DOX-Au nanocomposites might be an ability nano-theranostic material for treating throat cancers. The gene expression levels withinside the esophagus cell line from every group with or without laser exposure have been compared individually with control group. Bax has been overexpressed in esophagus cell line organization post 15 min laser irradiation. Conversely, BCL-2 gene has been down regulated with the aid of using RT-PCR analysis post 15 min laser irradiation, while in comparison to control cell line.

Proposed Mechanism of Long-Term Intraocular Pressure Lowering With the Bimatoprost Implant.

The purpose of this study was to evaluate the effects of pharmacologically relevant bimatoprost and bimatoprost free acid (BFA) concentrations on matrix metalloproteinase (MMP) gene expression in cells from human aqueous outflow tissues. MMP gene expression by human trabecular meshwork (TM), scleral fibroblast (SF), and ciliary muscle (CM) cells exposed to 10 to 1000 µM bimatoprost or 0.1 to 10 µM BFA (intraocular concentrations after intracameral bimatoprost implant and topical bimatoprost dosing, respectively) was measured by polymerase chain reaction array. Bimatoprost dose-dependently upregulated MMP1 and MMP14 mRNA in all cell types and MMP10 and MMP11 mRNA in TM and CM cells; in TM cells from normal eyes, mean MMP1 mRNA levels were 62.9-fold control levels at 1000 µM bimatoprost. BFA upregulated MMP1 mRNA only in TM and SF cells, to two- to three-fold control levels. The largest changes in extracellular matrix (ECM)-related gene expression by TM cells derived from normal (n = 6) or primary open-angle glaucoma (n = 3) eyes occurred with 1000 µM bimatoprost (statistically significant, ≥50% change for 9-11 of 84 genes on the array, versus 1 gene with 10 µM BFA). Bimatoprost and BFA had differential effects on MMP/ECM gene expression. Dramatic upregulation in MMP1 and downregulation of fibronectin, which occurred only with bimatoprost at high concentrations observed in bimatoprost implant-treated eyes, may promote sustained outflow tissue remodeling and long-term intraocular pressure reduction beyond the duration of intraocular drug bioavailability. Variability in bimatoprost-stimulated MMP upregulation among cell strains from different donors may help explain differential long-term responses of patients to bimatoprost implant.

Label-free multiphoton imaging flow cytometry.

Label-free imaging flow cytometry is a powerful tool for biological and medical research as it overcomes technical challenges in conventional fluorescence-based imaging flow cytometry that predominantly relies on fluorescent labeling. To date, two distinct types of label-free imaging flow cytometry have been developed, namely optofluidic time-stretch quantitative phase imaging flow cytometry and stimulated Raman scattering (SRS) imaging flow cytometry. Unfortunately, these two methods are incapable of probing some important molecules such as starch and collagen. Here, we present another type of label-free imaging flow cytometry, namely multiphoton imaging flow cytometry, for visualizing starch and collagen in live cells with high throughput. Our multiphoton imaging flow cytometer is based on nonlinear optical imaging whose image contrast is provided by two optical nonlinear effects: four-wave mixing (FWM) and second-harmonic generation (SHG). It is composed of a microfluidic chip with an acoustic focuser, a lab-made laser scanning SHG-FWM microscope, and a high-speed image acquisition circuit to simultaneously acquire FWM and SHG images of flowing cells. As a result, it acquires FWM and SHG images (100 × 100 pixels) with a spatial resolution of 500 nm and a field of view of 50 μm × 50 μm at a high event rate of four to five events per second, corresponding to a high throughput of 560-700 kb/s, where the event is defined by the passage of a cell or a cell-like particle. To show the utility of our multiphoton imaging flow cytometer, we used it to characterize Chromochloris zofingiensis (NIES-2175), a unicellular green alga that has recently attracted attention from the industrial sector for its ability to efficiently produce valuable materials for bioplastics, food, and biofuel. Our statistical image analysis found that starch was distributed at the center of the cells at the early cell cycle stage and became delocalized at the later stage. Multiphoton imaging flow cytometry is expected to be an effective tool for statistical high-content studies of biological functions and optimizing the evolution of highly productive cell strains.

L-kynurenine induces NK cell loss in gastric cancer microenvironment via promoting ferroptosis

BackgroundNatural killer (NK) cells play a major role in body’s fighting against various types of cancers. Their infiltration in the tumor microenvironment (TME) of gastric cancer (GC) are significantly decreased, which has been reported as a robust prognostic marker. However, the causes leading to NK cells loss in GC TME remains poorly understood.MethodsWe constructed a non-contact co-culturing system and humanized xenograft tumor mice model to detect the influence of GC microenvironment on NK-92 or primary human NK cells viability by flow cytometry. Then through using the specific inhibitors for different types of cell death and examining the surrogate markers, we confirmed ferroptosis in NK cells. Inspired by the accidental discoveries, we constructed a NK-92 cell strain with high expression of GPX4 and treated the humanized xenograft tumor mice model with the NK-92 cells.ResultsWe found L-KYN, mainly generated through indoleamine 2, 3-dioxygenase (IDO) from GC cells, impaired NK cells viability in TME. Further analysis revealed L-KYN induced ferroptosis in NK cells via an AHR-independent way. Moreover, we found NK cells with higher GPX4 expression showed resistance to L-KYN induced ferroptosis. Based on this, we generated GPX4 over-expressed NK-92 cells, and found these cells showed therapeutic potential towards GC.ConclusionsOur study revealed a novel mechanism to explain the decline of NK cell number in GC TME. Notably, we also developed a potential immunotherapy strategy, which might be beneficial in clinical treatment in the future.

The joint impact of crystal-cell thickness and biaxial ([110]) strain on the ferroelectricity of KNbO[formula omitted] in bulk/thin-film form

Due to robust spontaneous Polarization (P), ferroelectric materials are extensively used in numerous applications such as power consumption, exhaustion-free capacitor, and energy harvesting. Herein, we focus on the possibility of controlling the ferroelectric properties of the bulk/thin films of KNbO3 perovskite oxide via unit cell thickness and strain engineering using ab-initio calculations. First, the thermodynamic stability of the bulk and thin film structures is determined by computing the formation enthalpies and surface energies using Convex Hull analysis, respectively, which affirms their experimental realization. It is demonstrated that bulk structure is stable against its competing KO and NbO2 phases, while NbO2-NbO2 symmetric film is more energetically stable to grow in the lab as compared to others. Moreover, mechanical stability of the structures is confirmed by calculating the elastic constants. The estimated value of (P) for the bulk motif is 37.4 μC cm−2, which is in a good agreement with experimentally observed value. Thin films with larger slab thickness display more structural distortion, which enhances the P magnitude. For possible [KO-NbO2] (asymmetric) and [KO-KO]/[NbO2-NbO2] (symmetric) slabs, P gradually increases from 11.77 to 26.87 μC cm−2 and 8.59 to 16.43/23.38 to 27.42 μC cm−2 as the KNO unit cell thickness increases from 1 to 5 (1.86 nm) and 1.5 to 5.5 (2.05 nm), respectively. Interestingly, it is found that -5% compressive strain increases the P value of bulk KNO up to 31% than that of unstrained one. Similarly, the P amplitude in KO-NbO2 (1.86 nm thick) and KO-KO/NbO2-NbO2 (2.05 nm thick) slabs also improve up to 10.6% and 10.8%/11% as compared to unstrained ones for −5% compressive strain, respectively. Hence, the present work yields deep observations into the combined effect of crystal cell thickness and strain on the ferroelectricity of KNO, which provides a possible way to tune the P to enhance its potential realization in the oxide industry.

Exploitation of renewable energy sources for water desalination using biological tools.

The emerging impacts of climate change and the growing world population are driving the demand for more food resources and creating an urgent need for new water resources. About 93% of Earth's surface is made up of water bodies, mainly oceans. Seawater attracted a lot of attention in order to be used as a sustainable source of usable water. However, an essential step in harnessing this source of water is desalination. Utilizing renewable sources of energy, biology offers several tools for removal of salts. This article for the first time reviews all currently available biological water desalination tools and compares their efficiency with industrial systems. Bacteria are employed as electrical power generators to provide the energy needed for desalination in microbial desalination cells. Its salt removal efficiency varied from 0.8 to 30g/L/d. Many strains of algal cells can grow in high concentrations of salts, adsorb and accumulate it inside the cell, and therefore could be used without prior treatment for seawater desalination. This biological tool can yield salt removal efficiency of 0.4-5g/L/d. Biopolymers are also used for treatment of seawater through enhancing water evaporation as a component of solar steam generators. Despite significant advances in biological water desalination, further modifications and improvements are still needed to make its use sustainable and cost-effective.

Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line.

Chalcone-3 has been shown to be cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of epidermal growth factor receptors (EGFR) inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with triple negative breast cancer (TNBC), where EGFR expression is known to increase. This study aimed to identify the role of chalcone-3 in one of the downstream targets of EGFR as an antiproliferative agent. Chalcone-3 was examined for its effect on proliferation in human breast cancer MDA-MB-231 cell lines. The percentage of proliferation inhibition was analyzed using methyl-thiazol tetrazolium assay. Flow cytometry was used to analyze the population of cell cycle distribution and the expression of cyclin-D1 and pEGFR. Chalcone-3 inhibited the proliferation of MDA-MB-231 cells in a dose and time-dependent manner with an IC50 value of 17.98±6.36 µg/mL by inducing cell cycle arrest at the G2/M phase. Flow cytometry assays showed that chalcone-3 significantly reduced the expression of pEGFR and cyclin-D1, contributing to cell cycle arrest. Chalcone-3 might have potential as an anti-proliferative drug to treat TNBC.

Identifying the role of the FACT histone chaperone in epigenetics through single-molecule fluorescence imaging.

Modifications of histones in eukaryotic cells lead to reversible changes in gene expression without altering the genetic code itself. Such epigenetic modifications are necessary for the proper functioning of cellular processes like gene expression, DNA repair, and cell division. Modified histones directly affect chromatin dynamics and promote other regulation factors such as chromatin remodelers, including histone chaperones. Histone chaperones make functional DNA templates that maintain epigenetic information by remodeling the nucleosome during essential life processes, such as transcription and replication, and by avoiding histone aggregation. The facilitates chromatin transcription (FACT) histone chaperone includes two essential proteins, Pob3 and Spt16. Previously, we found that deleting Pob3 suppresses epigenetic inheritance, indicating that FACT plays an important role in epigenetic maintenance. Therefore, we engineered Schizosaccharomyces pombe (fission yeast) cell strains that express fusions of Spt16 or Pob3 to the fluorescent protein PAmCherry and investigated the dynamics of single proteins in the FACT complex in living S. pombe cells to identify the mechanism for this epigenetic inheritance suppression, including measuring changes in interactions during transcription and replication. By analyzing the motion of Spt16-PAmCherry single-molecule trajectories with nonparametric Bayesian statistics, we were able to distinguish three types of single-molecule motion: a fast (free) mobility state, an intermediate (transiently bound) mobility state, and a slow (bound) mobility state. We are assigning these biophysical states to specific biochemical processes by constructing Spt16 and Pob3 mutants with impaired histone protein binding properties, as well as drug-susceptible strains in which we can inhibit transcription and replication to identify changes in the FACT protein mobility. Overall, our live-cell single-molecule experiments reveal how the FACT complex interacts with histones and other chaperones and indicate a mechanism for epigenetic maintenance.

Antiviral activity of dandelion aqueous extract against pseudorabies virus both in vitro and in vivo.

Pseudorabies virus (PRV) is one of the most significant pathogens of swine. In recent years, the continual emergence of novel PRV variants has caused substantial economic losses in the global pig industry. PRV can infect humans leading to symptoms of acute encephalitis with implications for public health. Thus, new measures are urgently needed to prevent PRV infection. This study evaluated the anti-PRV capability of dandelion aqueous extract (DAE) in vitro and in vivo. DAE was found to inhibit the multiplication of the PRV TJ strain in PK15 cells in a concentration-dependent manner, with a 50% inhibitory concentration (IC50) of 0.2559 mg/mL and a selectivity index (SI) of 14.4. DAE inhibited the adsorption and replication stages of the PRV life cycle in vitro, and the expression of IE180, EP0, UL29, UL44, and UL52 was inhibited in the presence of DAE. In vivo experiment results of mice show that a 0.5 g/kg dose of DAE injected intraperitoneally protected 28.6% of the mice from the lethal challenge; decreased the viral load in the liver, lung, brain, heart, and kidney of PRV-infected mice; and attenuated brain damage caused by PRV infection. Furthermore, DAE could also ameliorate viral infection through regulation of the levels of cytokines (IFN-γ, TNF-α, and IL-4) in PRV-infected mouse serum. These results demonstrated that DAE exhibited potent inhibitory capability against PRV infection in vitro and in vivo; DAE is therefore expected to be a candidate TCM herb for use against PRV infection.

Obtaining and investigating immunochemical properties of monoclonal antibodies against rCTLA-4 protein

Monoclonal antibodies are used to block control points of the tumor development of many oncological pathologies. One of the critical control points of tumor development of several oncological pathologies is the receptor for cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Monoclonal antibodies against the CTLA-4 receptor are laboratory-derived humanized antibodies. An essential step in the humanization of antibodies is the production of murine hybrid cells producing monoclonal antibodies. This article describes studies of mice monoclonal antibodies against a recombinant human CTLA-4 receptor (rCTLA-4) expressed in Escherichia coli. To obtain strains of hybrid cells producing monoclonal antibodies were used methods of hybridoma technology. As a result, hybrid cells producing monoclonal antibodies to CTLA-4 were obtained. Strains of hybrid cells have high productive activity in vitro and in vivo. Monoclonal antibodies react with rCTLA-4 protein, belong to the class of IgG1, and have a high binding constant. They efficiently bind to the rCTLA-4 receptor and block the interaction of rCTLA-4 with the commercial recombinant human B7-1 Fc and rhesus monkey PD-1 hFc proteins. These monoclonal antibodies to rCTLA-4 can be used to obtain recombinant humanized monoclonal antibodies to the human CTLA-4 receptor.

Two-way strategy for enhanced pectinase production: Random mutagenesis and utilization of a halophytic biomass

Strain improvement through mutagenesis has remained an effective strategy for the cost-effective production of industrially important enzymes. Here, we focused on random mutagenesis of a pectinolytic yeast, Geotrichum candidum AA15, and on utilization of halophytic biomass along with apple waste as substrate for pectinase production to make the process cost effective. G. candidum AA15 was treated with a physical (UV) and a chemical (Ethidium bromide) mutagen separately and in a sequential way. After mutagenic treatments, a total of eight mutants (AHU0 to AHU5, AHC1 and AHUC) were obtained and assessed for pectinase production but a mutant strain (AHC1) yielded the highest pectinase production of 12.03 IU mL−1 which was >2 folds higher than the wildtype strain. The reason for positive effect of mutagenesis on pectinase production was investigated, however, the gene encoding polygalacturonase in the mutant strains was found similar to that of the wildtype strain. The purified pectinase from the mutant strains also shared molecular mass of 60–66 kDa with the wildtype. The mutant strain's cell density was higher than the wildtype and it can be linked with the higher productivity of pectinase reported in this study. The mutant strain, AHC1, yielded 8.32 IU mL−1 and 6.35 IU mL−1 of pectinase after the fermentation of apple peels and biomass from a halophytic plant, Cressa cretica, respectively. The utilization of the substrate was observed through Scanning Electron Microscopy. Thus, the study established a path to proceed for cost effective pectinase production through strain improvement and utilization of crude substrates.

A new hyperelastic lookup table for RT-DC.

Real-time deformability cytometry (RT-DC) is an established method that quantifies features like size, shape, and stiffness for whole cell populations on a single-cell level in real-time. A lookup table (LUT) disentangles the experimentally derived steady-state cell deformation and the projected area to extract the cell stiffness in the form of the Young's modulus. So far, two lookup tables exist but are limited to simple linear material models and cylindrical channel geometries. Here, we present two new lookup tables for RT-DC based on a neo-Hookean hyperelastic material numerically derived by simulations based on the finite element method in square and cylindrical channel geometries. At the same time, we quantify the influence of the shear-thinning behavior of the surrounding medium on the stationary deformation of cells in RT-DC and discuss the applicability and impact of the proposed LUTs regarding past and future RT-DC data analysis. Additionally, we provide insights about the cell strain and stresses, as well as the influence resulting from the rotational symmetric assumption on the cell deformation and volume estimation. The new lookup tables and the numerical cell shapes are made freely available.

Cold-adapted SARS-CoV-2 variants with different temperature sensitivity exhibit an attenuated phenotype and confer protective immunity.

As novel SARS-CoV-2 Variants of Concern emerge, the efficacy of existing vaccines against COVID-19 is declining. A possible solution to this problem lies in the development of a live attenuated vaccine potentially able of providing cross-protective activity against a wide range of SARS-CoV-2 antigenic variants. Cold-adapted (ca) SARS-CoV-2 variants, Dubrovka-ca-B4 (D-B4) and Dubrovka-ca-D2 (D-D2), were obtained after long-term passaging of the Dubrovka (D) strain in Vero cells at reduced temperatures. Virulence, immunogenicity, and protective activity of SARS-CoV-2 variants were evaluated in experiments on intranasal infection of Syrian golden hamsters (Mesocricetus auratus). In animal model infecting with ca variants, the absence of body weight loss, the significantly lower viral titer and viral RNA concentration in animal tissues, the less pronounced inflammatory lesions in animal lungs as compared with the D strain indicated the reduced virulence of the virus variant. Single intranasal immunization with D-B4 and D-D2 variants induced the production of neutralizing antibodies in hamsters and protected them from infection with the D strain and the development of severe pneumonia. It was shown that for ca SARS-CoV-2 variants, the temperature-sensitive (ts) phenotype was not obligate for virulence reduction. Indeed, the D-B4 variant, which did not possess the ts phenotype but had lost the ability to infect human lung cells Calu-3, exhibited reduced virulence in hamsters. Consequently, the potential phenotypic markers of attenuation of ca SARS-CoV-2 variants are the ca phenotype, the ts phenotype, and the change in species specificity of the virus. This study demonstrates the great potential of SARS-CoV-2 cold adaptation as a strategy to develop a live attenuated COVID-19 vaccine.