Cell Titer Research Articles

Modeling the effectiveness of bacillus subtilis strains depending on natural and climatic factors in the soft wheat cultivation

The studies were carried out in 2017-2022 in order to identify natural and climatic factors that can influence the effectiveness of B. subtilis strains in relation to morphometric indicators and soft wheat productivity, the diseases development. The responsiveness of Leningradka 6, k-64900 spring wheat to the microbiological preparation Vitaplan, SP (titer of viable cells ×1011 CFU/g) and cultural liquid (CL) of the B. subtilis VKM B-2604D and B. subtilis VKM B-2605D (at a ratio of 1:1 with the titer of viable cells ×1010 CFU/ml). In the field conditions of the North- West of the Russian Federation, wheat seeds were treated before sowing and plants were sprayed three times during the growing season. The effectiveness of bacterial strains in relation to productivity and especially dangerous wheat diseases depended more on the indicators of moisture availability of the growing season. This was confirmed by the results of multidimensional scaling, correlation, factor analysis of indicators. In the variant «Vitaplan, SP» in F1, high positive factor loads were revealed for the precipitation amount in June, July, August, HTC in July, relative humidity in June (0.73…0.93), plant phase, plant height, area of ag and pre-flag leaves, vegetative part weight (0.71…0.80), and when using CL «B. subtilis VKM V-2604D + B. subtilis VKM B-2605D» - for the precipitation amount in June, August, HTC in July and August (0.71…0.88), productive bushiness, root weight, spike length, spikelets number per spike, grains number, spike grain weight, biological, potential yield (0.71…0.85). The B. subtilis strains application allowed to increase biological and potential yields by 32.5± 7.2 % and 24.3±7.0 %, and to reduce the intensity of diseases development (root rot - by 11.7 ± 1.6 %; powdery mildew, brown and yellow rust - by 7.6 ± 0.7 %), however, the effectiveness of bacterial strains significantly depended on the climatic factors of wheat growing season.

The impact of the duration of wave treatment on the development of yeast populations

Introduction: The scientific literature does not contain research on the influence of monochromatic light treatment on the development of bottom-fermenting beer yeast populations.Purpose: An analysis of the literature data allowed us to suppose the possibility of activating the development of the beer yeast Saccharomyces cerevisiae population through preliminary treatment with light of visible spectrum wavelengths, providing technological and economic efficiency of such an impact method on a production scale.Materials and Methods: The object of research was the process of cultivating a population of bottom-fermenting beer yeast Saflager S-189 (Fermentis). The subject was the influence of preliminary treatment of the seed yeast with monochromatic light with a wavelength of 980 nm on this process. Its effectiveness was assessed by the weight loss of the cultivation medium, the increase in the total cell titre, the proportion of unviable and "fed" cells. A KFK-2 photoelectrocolorimeter was used as a source of monochromatic light with wavelengths of the visible range. Seeding of mediums and sample preparation for analysis were performed in a BAVnp-01-"Laminar-S."-1.2 antibacterial air medium box. The yeast's fermenting activity was assessed by the weight loss of the nutrient medium; the total cell titre was established by counting in a Goryaev's chamber; the percentage of unviable cells was determined using methylene blue dye; the percentage of "fed" yeast cells was established by staining glycogen with iodine solution.Results: Preliminary treatment of the seed yeast with monochromatic light (980 nm) allowed increasing the fermenting activity of bottom-fermenting beer yeast by 10-15% compared to the control, which is consistent with the results of other research groups concerning populations of microorganisms of other genera and species. The values of other determined indicators - the proportion of "fed" and unviable cells, the total yeast cell titre - in the experimental variants were at the level of those in the control samples or slightly inferior to them. Data on the influence of the duration of seed yeast irradiation for 60, 120 or 180 minutes on the listed controlled indicators are presented, and the authors express the opinion about the appropriateness of its conduction for 60 minutes.Conclusion: The principle possibility of activating the development of a population of bottom-fermenting beer yeast by preliminary treatment of seed yeast with light with a wavelength of 980 nm, which can give an economic effect on an industrial scale, is substantiated; the necessity of testing the technological method under study in conditions close to production ones, i.e., for brewing wort with an irradiated suspension of seed yeast, is noted.

The ATR inhibitor VE-821 increases the sensitivity of gastric cancer cells to cisplatin

BackgroundChemoresistance is a common event after cancer chemotherapy, including gastric cancer (GC). Cisplatin has been reported to induce the DNA damage response (DDR), thus leading to chemoresistance. VE-821, a specific inhibitor of ATR, has been proven to suppress a variety of solid malignancies effectively. Our study aimed to explore the effect of VE-821 on enhancing the chemical sensitivity to cisplatin and clarify the potential molecular mechanisms. MethodsCell viability and apoptosis of MKN-45 and AGS were measured by CCK8 and flow cytometry assay respectively. Western blotting was used to detect the expression of target proteins. TCGA database was used to analyze the correlation between the ATR expression with the prognosis of GC patients. The viability of GC organoids was detected by Cell Titer Glo (CTG) through luminescence. ResultsCisplatin inhibited the proliferation and induced apoptosis of GC cells with a relatively high IC50 value, and increased the phosphorylation levels of ATR-CHK1 and H2AX. VE-821 achieved the same effects but by downregulating the phosphorylation levels of the ATR-CHK1 pathway. Besides, higher ATR expression in GC tissues was positively correlated with higher pathological stage in GC patients. Interestingly, ATR inhibition reversed cisplatin-induced STAT3 activation and enhanced H2AX levels. Moreover, VE-821 significantly sensitized GC cells to cisplatin, and these two drugs had synergistic effects in GC cell lines, organoids, and in vivo. ConclusionOur results suggested VE-821 sensitized GC cells to cisplatin via reversing DDR activation. And VE-821 treatment may be a promising therapeutic strategy for GC patients with cisplatin resistance.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

SARS-CoV-2 utilization of ACE2 from different bat species allows for virus entry and replication in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to have a zoonotic origin with bats suspected as a natural host. In this work, we individually express the ACE2 of seven bat species including, little brown, great roundleaf, Pearson's horseshoe, greater horseshoe, Brazilian free-tailed, Egyptian rousette, and Chinese rufous horseshoe in DF1 cells and determine their ability to support attachment and replication of SARS-CoV-2 viruses. We demonstrate that the ACE2 receptor of all seven species made DF1 cells permissible to SARS-CoV-2. The level of virus replication differed between bat species and variants tested. The Wuhan lineage SARS-CoV-2 virus replicated to higher titers than either variant virus tested. All viruses tested grew to higher titers in cells expressing the human ACE2 gene compared to a bat ACE2. This study provides a practical in vitromethod for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.

In Vitro Evaluation of Antibacterial and Antibiofilm Activity of Different Chlorhexidine-Containing Mouthwash Formulations against Streptococcus mutans

Daily use of mouthwash is generally recommended to control dental plaque development and limit further adhesion of oral bacteria. To this purpose, Chlorhexidine di-gluconate (CHG)-containing products are still the most used antiseptic in oral health. The aim of this study was to compare the antimicrobial and antibiofilm activity of three different mouthwash commercial formulations containing CHG at the same concentration (0.2%) on the main etiological agent of dental plaque, Streptococcus mutans (S. mutans). Liquid Chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to confirm the CHG concentration in the commercial formulations. The Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal concentration (MBC) were measured to evaluate the antimicrobial activity of mouthwashes on planktonic cells. The biofilm prevention concentration (BPC) and CHG formulation activity towards mature biofilm were investigated. Both total cell titer and viability in S. mutans pregrown biofilms were evaluated after treatment with formulations at different time points and concentrations, using Crystal Violet (CV) and tetrazolium dye assay (MTT). The Shapiro–Wilk and Levene tests were used to evaluate the normality and overall homogeneity of the datasets, respectively. The chi-square test, Fischer’s exact test and Student’s t-test were used to evaluate the inhibitory capacity of the commercial formulations in CV and MTT assays. Results showed that even though only slight differences in MIC and MBC values were found on planktonic bacteria, the antibiofilm effect of the different formulations exhibited wide variation when mature biofilms were treated. In addition, results were discussed based on the different compositions of the three formulations, suggesting that herbal extracts, when present in mouthwash formulations, may not always have a beneficial effect, especially when mature biofilms are concerned.

DNA-Binding Protein Dps Protects Escherichia coli Cells against Multiple Stresses during Desiccation.

Gradual dehydration is one of the frequent lethal yet poorly understood stresses that bacterial cells constantly face in the environment when their micro ecotopes dry out, as well as in industrial processes. Bacteria successfully survive extreme desiccation through complex rearrangements at the structural, physiological, and molecular levels, in which proteins are involved. The DNA-binding protein Dps has previously been shown to protect bacterial cells from many adverse effects. In our work, using engineered genetic models of E. coli to produce bacterial cells with overproduction of Dps protein, the protective function of Dps protein under multiple desiccation stresses was demonstrated for the first time. It was shown that the titer of viable cells after rehydration in the experimental variants with Dps protein overexpression was 1.5-8.5 times higher. Scanning electron microscopy was used to show a change in cell morphology upon rehydration. It was also proved that immobilization in the extracellular matrix, which is greater when the Dps protein is overexpressed, helps the cells survive. Transmission electron microscopy revealed disruption of the crystal structure of DNA-Dps crystals in E. coli cells that underwent desiccation stress and subsequent watering. Coarse-grained molecular dynamics simulations showed the protective function of Dps in DNA-Dps co-crystals during desiccation. The data obtained are important for improving biotechnological processes in which bacterial cells undergo desiccation.

CBFβ is induced by spring viremia of carp virus and promotes virus replication in zebrafish

The core binding factor subunit beta (CBFβ) is a transcription factor that forms a complex with virial proteins to promote viral infection. In this study, we identified a CBFβ homolog from zebrafish (zfCBFβ) and characterized the biological activity. The deduced zfCBFβ protein was highly similar to orthologs from other species. The zfcbfβ gene was constitutively expressed in tissues and was induced in immune tissues after infection with spring viremia carp virus (SVCV) and stimulation with poly(I:C). Interestingly, zfcbfβ is not induced by type I interferons. Overexpression of zfcbfβ induced tnfα expression but inhibited isg15 expression. Also, overexpression of zfcbfβ significantly increased SVCV titer in the EPC cells. Co-immunoprecipitation assay revealed that zfCBFβ interacts with SVCV phosphoprotein (SVCVP) and host p53, resulting in the increased stability of zfCBFβ. Our results provide evidence that CBFβ is targeted by virus to suppress host antiviral response.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

INFLUENCE OF BIOLOGICALLY ACTIVE SUBSTANCES AND LACTULOSE PREBIOTICS ON THE FERMENTATION PROCESSES WITH THE PARTICIPATION OF LACTIC ACID BACTERIA

A study of the influence of biologically active substances (fat-soluble vitamins, omega-3 fatty acids and vitamin D, as well as water-soluble vitamin C), prebiotic lactulose on the titer of viable cells of lactic acid bacteria was carried out for the further use of the obtained research results in the biotechnology of functional food products, obtained thanks to the successful course fermentation processes. Experiments have proven the expediency of using biologically active substances as stimulators of the growth of lactic acid bacteria, which contribute to the increase of biomass and support of their vital activity for 192 hours. Research has established that at the end of the experiment (192 hours), the highest titer of lactic acid bacteria was recorded in samples 1, 2, 3, 4, to which the prebiotic lactulose was added in the amount of 1, 3, 5 and 7 %, respectively. This is due to the fact that the prebiotic acts as a specific substrate that is used by the symbiosis of pure cultures of lactic acid bacteria for a long time, unlike milk sugar. Analyzing the obtained data, it is indicated that thanks to the addition of fat-soluble nutrients omega-3 fatty acids and vitamin D, as well as water-soluble vitamin C, there is a significant increase in the titer of viable cells of probiotic cultures at 6 and 24 hours of the experiment, although at the end of the experiment (by 192 hour) in these samples, an insignificant decrease of this indicator was recorded — from 10 to 8.9 log cells/ml. In the control sample (without the addition of biologically active substances and lactulose prebiotic), a significant decrease in the titer of viable cells of lactic acid bacteria was noted, compared to the test samples to which nutrients were added. The titer of lactic acid bacteria cells in the control sample was reduced by 192 hours from 8.5 to 5.5 log cells/ml. It is recommended to use the studied nutrients in the indicated concentrations to stimulate the growth and maintain the viability of lactic acid bacteria cultures in the technology of production of functional food products.

Characterization of an Aedes ADP-Ribosylation Protein Domain and Role of Post-Translational Modification during Chikungunya Virus Infection.

Poly ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation, a subclass of post-translational modification (PTM). Mono-ADP-ribose (MAR) moieties bind to target molecules such as proteins and nucleic acids, and are added as part of the process which also leads to formation of polymer chains of ADP-ribose. ADP-ribosylation is reversible; its removal is carried out by ribosyl hydrolases such as PARG (poly ADP-ribose glycohydrolase), TARG (terminal ADP-ribose protein glycohydrolase), macrodomain, etc. In this study, the catalytic domain of Aedes aegypti tankyrase was expressed in bacteria and purified. The tankyrase PARP catalytic domain was found to be enzymatically active, as demonstrated by an in vitro poly ADP-ribosylation (PARylation) experiment. Using in vitro ADP-ribosylation assay, we further demonstrate that the chikungunya virus (CHIKV) nsp3 (non-structural protein 3) macrodomain inhibits ADP-ribosylation in a time-dependent way. We have also demonstrated that transfection of the CHIKV nsP3 macrodomain increases the CHIKV viral titer in mosquito cells, suggesting that ADP-ribosylation may play a significant role in viral replication.

Cell-intrinsic genomic reassortment of pandemic H1N1 2009 and Eurasian avian-like swine influenza viruses results in potentially zoonotic variants

ABSTRACT Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.

Development of an Inactivated Camelpox Vaccine from Attenuated Camelpox Virus Strain: Safety and Protection in Camels.

This article describes the preparation of an inactivated vaccine from an attenuated strain of camelpox. The attenuated camelpox virus (CMLV) was grown in lamb kidney cells and in Vero cells. CMLV was accumulated to a significantly higher (p ≤ 0.05) titer in lamb kidney cells (7.75 ± 0.08 log TCID50/mL) than in Vero cells (4.00 ± 0.14 log TCID50/mL). During virus inactivation, a concentration of 0.05% beta-propiolactone (BPL) completely inactivated the virus in 6 h at a temperature of 22 ± 1 °C, while a concentration of 0.2% formaldehyde inactivated the virus in 8 h. However, a viral antigen inactivated by BPL was used for vaccine preparation. The inactivated viral antigen was adsorbed with aluminum hydroxide gel, and as a result, an inactivated candidate vaccine was prepared. While the safety of the candidate vaccine was tested in camels and white mice, the protective efficacy of the vaccine was tested only in camels. In the safety evaluation of the inactivated vaccine, the vaccine was not observed to cause any adverse effects in mice and camels. During the immunogenicity study in camels, antibody formation started (0.2 ± 0.16 log2) at Day 21 post-vaccination (PV), and the antibody titer peaked (1.33 ± 0.21 log2) at Day 60 PV and decreased at Day 90 PV (0.50 ± 0.22 log2). Furthermore, no antibodies were detected in vaccinated camels from Days 180 to 365 PV. Camels that received vaccination and were subsequently exposed to wild-type virus evinced a healthy state despite lacking antibodies. In contrast, unvaccinated camels exhibited susceptibility to camelpox upon challenge.

Ways of Long-Term Survival of Hydrocarbon-Oxidizing Bacteria in a New Biocomposite Material-Silanol-Humate Gel.

Immobilized bacterial cells are presently widely used in the development of bacterial preparations for the bioremediation of contaminated environmental objects. Oil hydrocarbons are among the most abundant pollutants. We have previously described a new biocomposite material containing hydrocarbon-oxidizing bacteria (HOB) embedded in silanol-humate gels (SHG) based on humates and aminopropyltriethoxysilane (APTES); high viable cell titer was maintained in this material for at least 12 months. The goal of the work was to describe the ways of long-term HOB survival in SHG and the relevant morphotypes using the techniques of microbiology, instrumental analytical chemistry and biochemistry, and electron microscopy. Bacteria surviving in SHG were characterized by: (1) capacity for rapid reactivation (growth and hydrocarbon oxidation) in fresh medium; (2) ability to synthesize surface-active compounds, which was not observed in the cultures stored without SHG); (3) elevated stress resistance (ability to grow at high Cu2+ and NaCl concentrations); (4) physiological heterogeneity of the populations, which contained the stationary hypometabolic cells, cystlike anabiotic dormant forms (DF), and ultrasmall cells; (5) occurrence of piles in many cells, which were probably used to exchange genetic material; (6) modification of the phase variants spectrum in the population growing after long-term storage in SHG; and (7) oxidation of ethanol and acetate by HOB populations stored in SHG. The combination of the physiological and cytomorphological properties of the cells surviving in SHG for long periods may indicate a new type of long-term bacterial survival, i.e., in a hypometabolic state.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Concordance of adenosine deaminase with immunoglobulins and lymphocyte subsets in EBV-related diseases

BackgroundClinical manifestations of Epstein–Barr virus (EBV) infection are diverse. This study aimed to explore the immune response in EBV-related diseases and the correlation between immune cells and adenosine deaminase (ADA) levels.MethodsThis study was conducted at the Children’s Hospital of Soochow University. In total, 104 patients with EBV-associated respiratory tract infection (EBV-RTI), 32 patients with atypical EBV infection, 54 patients with EBV-associated infectious mononucleosis (IM1, with normal alanine aminotransferase [ALT] levels), 50 patients with EBV-IM2 (with elevated ALT levels), 50 patients with acute respiratory infection (AURI, with other pathogens), and 30 healthy controls were enrolled in this study. Indicators of ADA, immunoglobulins (Igs), and lymphocyte subsets were analyzed for EBV-related diseases.ResultsDifferences in the white blood cell, lymphocyte counts, ADA levels, IgA, IgG and IgM titers, percentage of CD3+, CD3+CD4+, CD3+CD8+, CD16+CD56+, CD3−CD19+, and CD19+CD23+ lymphocytes, and CD4+/CD8+ ratio between EBV-related disease groups were all statistically significant (P < 0.01). ADA levels in the EBV-related disease groups were significantly higher than those in the control group (P < 0.01). The lymphocyte count, ADA levels, IgA and IgG titers, and percentage of CD3+ and CD3+CD8 + lymphocytes in the atypical EBV infection, EBV-IM1, and EBV-IM2 groups were significantly higher than those in the EBV-RTI, AUTI, and control groups (P < 0.01), whereas the percentage of CD3+CD4+, CD3−CD19+, and CD19+CD23+ lymphocytes and CD4+/CD8+ ratio showed the opposite trend. ADA levels were consistent with and closely related to the viral load and cellular and humoral immunity in EBV-related diseases.ConclusionsADA levels, humoral immunity, and cellular immunity were diverse in EBV-related diseases, and ADA was closely related to Igs and lymphocyte subsets.

Abstract 383: Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors

Abstract Activating ERBB2 (HER2) mutations occur across multiple malignancies, and although mutational hotpots vary by disease, HER2 mutations most frequently occur in the tyrosine kinase domain. Among lung cancer patients, the most common HER2 mutation is the exon 20 insertion mutations, Y772dupYVMA, which accounts for over 30% of all HER2 mutations in lung cancer. Recent clinical testing of trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), against HER2 mutant non-small cell lung cancer (NSCLC) demonstrated a confirmed objective response of 55%, a median duration of response of 9.3 months, and a median overall survival of 17.8 months, which ultimately led to the recent approval of T-DXd for HER2 mutant NSCLC. Acquired resistance to targeted agents remains a major clinical challenge, and HER2-mutant patients that initially respond to T-DXd will eventually develop progressive disease. Thus, understanding mechanism of T-DXd resistance and identifying targeted agents that remain effective in cells with acquired T-DXd resistance is of great clinical importance. We generated Ba/F3 cells expressing the HER2 YVMA mutation and assessed sensitivity to HER2 targeting agents by Cell Titer Glo assay. HER2 YVMA cells were sensitive to the EGFR/HER2 tyrosine kinase inhibitor (TKI) poziotinib and the HER2 ADC T-DXd. To generate HER2 ADC resistant cells, we cultured Ba/F3 HER2 YVMA cells in T-DXd continuously for 8 weeks at which point cells resumed rapid proliferation. Cell Titer Glo assay confirmed that Ba/F3 HER2 YVMA cells were sensitive to T-DXd with an IC50 of 195 μg/ml, whereas Ba/F3 HER2 YVMA T-DXd-resistant cells had an IC50 of greater than 1000 μg/ml. Notably, T-DXd resistant cells remained highly sensitive to the HER2 TKI poziotinib with an IC50 of 14 nM and 7.5 nM for T-DXd-resistant and parental cells, respectively. Moreover, T-DXd-resistant cells demonstrated reduced sensitivity to the topoisomerase inhibitor topotecan with an IC50 of 200 nM whereas parental cells had an IC50 of 66 nM, suggesting that acquired T-DXd resistance may be mediated by loss of sensitivity to the ADC payload. Collectively, these data demonstrate that HER2 TKIs such as poziotinib may retain anti-tumor cell activity in HER2 mutant tumor cells with acquired resistance to HER2 ADCs. Citation Format: Monique B. Nilsson, Alissa Poteete, Hibiki Udagawa, Xiaoxing Yu, Junqin He, Xiuning Le, John Heymach. Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors [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 383.

Abstract 150: A 515 tumor specimens collection from surgery to benchtop: the WFORCE organoid experience in personalized research

Abstract Introduction: Patient tumor organoids (PTOs) are a new and developing preclinical model in cancer research. However, there is variation in protocols between centers and lack of consistency in important variables, impacting PTO culture success. In this study, we analyzed factors in clinical data and tumor tissue processing to determine optimal conditions for cell isolation and PTO success. Methods: Under IRB approval, tumor tissues from primary and metastatic sites were procured in clinically indicated surgeries then dissociated using a standardized protocol. PTOs were fabricated using collagen-hyaluronic acid ECM and underwent drug screening at 10 days post fabrication, where viability was assessed. Clinical and tissue processing inputs such as type of primary, peritoneal carcinomatosis index (PCI), prior surgery or neoadjuvant chemotherapy, presence of mucin, tissue digestion time, specimen mass, digestion time, and the diameter of isolated cells were analyzed. These factors were assessed by calculating the Pearson Correlation Coefficient (r) and best fit modeling to determine the effect on outputs including tissue viability as measured by % of viable cells using an automated cellular counter, viable cells per mg tissue, and PTO log10 viability by Promega 3D Cell Titer Glo assay. Results: From December 2018-July 2022, 515 tumors from 286 patients with 25 primary types were processed for cellular isolation and PTO fabrication. 407 (79%) provided PTOs for therapeutic testing, with 354 (69%) providing sufficiently viable PTOs for therapeutic interrogation. The most common primary tumor type accrued was appendiceal cancer (n=223, 43%). Tissues processed at 24 hours did not differ in viability outputs when compared to tissues processed immediately (p&amp;gt;0.05). Tissue % viability was better maintained in larger specimens (r=.18, p&amp;lt;0.001), resulting in more viable cells per mg of tissue (r=.12, p=0.009) while it was negatively related to larger cell diameter (r=-.32, p&amp;lt;0.001). The number of viable cells per mg of tumor correlated to higher post-processing cell counts (r=.49, p&amp;lt;0.001) while it was decreased by longer digestion times (r=-.11, p=0.017). Finally, PTO log10 viability, correlated with higher burden of disease as measured by PCI scores (r=.35, p&amp;lt;0.001), larger specimens (r=.19, p&amp;lt;0.001), mucinous tumors (PTO log10 viability 5.46 vs 5.17, p=0.01), and tissues with higher cellular viability (r=.31, p&amp;lt;0.001), while it was negatively impacted by neoadjuvant chemotherapy (5.06 vs 5.41, p&amp;lt;0.001). Conclusions: Higher rates of successful PTO fabrication are obtained in patients with untreated tumors, more aggressive biologic behavior, and increased tumor burden. Creating a unified protocol for tissue processing, agnostic to tumor type, will facilitate incorporation of tissue from the operating room to research protocols with wider translational implications. Citation Format: Steven Donald Forsythe, Richard A. Erali, Preston Laney, William Meeker, Salomat Abdulkhuseynova, Nadeem Wajih, Nicholas Edenhoffer, Cecilia R. Schaaf, Hemamyammal Sivakumar, Aleksander Skardal, Roy E. Strowd, Stephen B. Tatter, Clancy J. Clark, Reese W. Randle, Perry Shen, Edward A. Levine, Ralph B. D'Agostino, Shay Soker, Konstantinos I. Votanopoulos. A 515 tumor specimens collection from surgery to benchtop: the WFORCE organoid experience in personalized research [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 150.

Abstract 6735: Identifying the role of novel driver TREX subcomplex in medulloblastoma growth and progression

Abstract Background: Medulloblastoma (MB) is the most common malignant brain cancer in children. Despite the progress made in treating MB, the 5-year survival rate for high-risk tumors remains poor and risk of recurrence within 2 years of treatment is still high. Almost all MB deaths are attributed to leptomeningeal dissemination (LMD) as MB spreads exclusively through cerebrospinal fluid (CSF) to spinal and intracranial leptomeninges. Unfortunately, patients who do survive have reduced quality of life because of the highly toxic side effects of radiation and chemotherapy. Those facts underline the importance of identifying new drivers of medulloblastoma and understanding the mechanism by which those drivers may promote medulloblastoma. Towards that goal, I aim to identify novel drivers of MB and characterize novel therapies for treating (LMD-MB). Using an unbiased genome-wide screen, we discovered THO complex as one of the highly differentially expressed genes that may provide survival advantage to MB cells. THO is a subcomplex of the TREX complex, is known to couple mRNA transcription, processing and nuclear export associated with spliced mRNA. Methods: Recent work in our lab utilizing various genomes and proteomics identified THO complex as a possible target. Transfection agents were used to knockdown part of the TREX subcomplex expression in several MB cells. Western blot and qPCR verified the knockdown. The cell viability was measured using Cell Titer Glo, and cell proliferation was evaluated with an IncuCyte. Cells were seeded at a low density and cultured until colonies formed then were fixed and stained with crystal violet. In addition, cells were seeded in a serum-free medium and allowed to migrate to complete media before being fixed and stained. Flow cytometry was used to examine cells for apoptosis and cell cycle. For apoptosis, cells were stained with propidium iodide (PI) and Annexin FTIC, and for the cell cycle, cells were stained and followed by PI. Results: The Cancer Dependency Map (Dep-Map) meta-analysis revealed that MB strongly depends on The TREX subcomplex. Significantly, increased THO complex expression was associated with a worse overall survival rate in MB patients. Our findings indicated that part of the TREX subcomplex depletion decreased MB cell survival in the short- and long-term proliferation, migration, and invasion. Furthermore, part of TREX subcomplex depletion promoted apoptosis in MB cells. I propose that THO complex may be a new driver of MB and that strategies aiming to reduce part of TREX subcomplex levels and activity might benefit MB patients. Conclusion: TREX subcomplex parts depletion has been demonstrated to reduce MB cells in vitro in various experiments. Future research, including CRISPR-CAS9-based knockout models, single-cell sequencing, and metabolomics, will further elucidate the mechanism(s) and utility of the TREX subcomplex axis in LMD-MB. Citation Format: Shahad M. Abdulsahib, Santosh Timilsina, Manjeet Rao. Identifying the role of novel driver TREX subcomplex in medulloblastoma growth and progression [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 6735.

Abstract 207: Ex vivo modeling of multiple myeloma: A novel drug sensitivity screening platform

Abstract Multiple Myeloma (MM) is a heterogeneous malignancy characterized by abnormal clonal plasma cell infiltration in the bone marrow. It is the second most common hematologic malignancy, after non-Hodgkin’s Lymphoma. This deadly disease globally affects 1 to 5 people in every 100,000 people each year. After initial diagnosis, the 5-year survival rate is 44% because there is no curative therapy, and most patients will eventually experience relapse, and some become refractory. Large inter-patient and intra-patient genetic heterogeneity limit the identification of universal drivers of MM. However, several oncogenic dependencies such as primary events related to driver gene mutations and primary translocations can be targeted for better treatment strategies for R/R multiple myeloma. To establish a robust drug sensitivity screening platform, both MM cell lines and cryopreserved primary MM patient samples were used. MM cell lines screen with H929, MM1R and RPMI-8226 was performed to narrow down optimal concentrations of investigative test agents. During assay development, a panel of FDA approved agents for relapsed MM including panobinostat, bortezomib, melphalan, omacetaxine, and selinexor were tested. The cells were seeded at 50,000 cells per well in triplicates in a 96-well plate on day 0. The test agents were added in 9 concentrations with the top concentration being 25 uM with 2-fold dilutions. After 6- days of incubation, a luminescent cell viability assay was performed to calculate relative viability with drug treatment and IC50 was computed by fitting data using a standard four-parameter logistic model. For primary multiple myeloma samples, bone marrow aspirates were collected from Multiple Myeloma patients procured from different providers. BM mononuclear (BMMNC) cells were separated by Ficoll-Hypaque density sedimentation. Unfractionated BMMNC cells were used in these assays to preserve intra-tumor heterogeneity. Primary cells were seeded in triplicates per dose point on day 0 in Champions’ proprietary media. The test agents were also added on day 0 and were incubated for 6 days. On Day 6, the relative cell viability was measured based on Cell Titer Glo readout. The cryopreserved primary MM patient samples were viable in culture for the entire assay duration validating assay feasibility. A panel of FDA approved, standard of care drugs were tested in numerous clinically well-annotated and diverse MM patient models. Primary model selection was based on disease burden including plasma cell count from Core Biopsy and BM aspirates. Multiparametric flow cytometric immunophenotyping was also performed in these primary samples using monoclonal antibodies against CD56, CD19, CD117, CD27, CD138, and CD38. For the first time, we demonstrate the feasibility of a novel, ex-vivo drug sensitivity screening platform with cryopreserved, primary multiple myeloma cells from diverse, clinically well-annotated patient samples. Citation Format: Vaishnavi Sambandam, Sharvari Inamdar, Haoting Hsu, Brandon Walling, Paolo Schiviani, Abhay Andar, Marianna Zipeto, Michael Ritchie, Karin Abarca Heidemann, Maria Mancini. Ex vivo modeling of multiple myeloma: A novel drug sensitivity screening platform [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 207.