Expression Of Resistance Genes Research Articles

Relative Expression of Genes Elicited by Clonostachys rosea in Pinus radiata Induces Systemic Resistance

Radiata pine is one of the most commonly planted tree species in Chile due to its fast growth and desirable wood and pulp properties. However, its productivity is hampered by several diseases. Pitch canker disease (PCC) caused by Fusarium circinatum, is considered the most damaging disease to the pine forest industry. Several control measures have been established, with biological control emerging as an environmentally friendly and effective way for F. circinatum control. Previous studies support the value of Clonostachys rosea in reducing PCC damage, with evidence suggesting a potential induced systemic resistance (ISR) triggered in radiata pines by this agent. Ten-month-old radiata pine plants were pre-treated with C. rosea on a substrate at 8 and 1 days before inoculation with F. circinatum on the stem tip, and expression levels were determined for DXS1, LOX, PAL, and PR3 genes 24 h later. Lesion length was 45% lower on plants pre-treated with C. rosea and infected with F. circinatum compared to non-pre-treated and infected plants. Additionally, LOX and PR3 were induced 23 and 62 times more, respectively, in comparison to untreated plants. Our results indicate that C. rosea causes an ISR response in pre-treated plants, significantly increasing the expression of resistance genes and reducing lesion length.

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. NA.

Molecular insights into expression and silencing of resistance determinants in Staphylococcus aureus.

This study aimed to investigate the status of antimicrobial-resistant strains of Staphylococcus aureus in Pakistan, their association in terms of co-occurrence with the biofilm-forming genes, resistance profiling and associated discrepancies in diagnostic methods. A total of 384 milk samples from bovine was collected by using convenient sampling technique and were initially screened for subclinical mastitis, further preceded by isolation and confirmation of S. aureus. The S. aureus isolates were subjected to evaluation of antimicrobial resistance by phenotypic identification using Kirby-Bauer disc diffusion method, while the genotypic estimation was done by polymerase chain reaction to declare isolates as methicillin, beta-lactam, vancomycin, tetracycline, and aminoglycoside resistant S. aureus (MRSA, BRSA, VRSA, TRSA, and ARSA), respectively. The current study revealed an overall prevalence of subclinical mastitis and S. aureus to be 59.11% and 46.69%, respectively. On a phenotypic basis, the prevalence of MRSA, BRSA, VRSA, TRSA, and ARSA was found to be 44.33%, 58.49%, 20.75%, 35.84%, and 30.18%, respectively. The results of PCR analysis showed that 46.80% of the tested isolates were declared as MRSA, 37.09% as BRSA, and 36.36% as VRSA, while the occurrence of TRSA and ARSA was observed in 26.31% and 18.75%, respectively. The current study also reported the existence of biofilm-producing genes (icaA and icaD) in 49.06% and 40.57% isolates, respectively. Lastly, this study also reported a high incidence of discrepancies for both genotypic and phenotypic identification methods of resistance evaluation, with the highest discrepancy ratio for the accA-aphD gene, followed by tetK, vanB, blaZ, and mecA genes. The study concluded that different antibiotic resistance strains of S. aureus are prevalent in study districts with high potential to transmit between human populations. The study also determined that there are multiple resistance determinants and mechanisms that are responsible for the silencing and expression of antibiotic resistance genes.

Expression analysis and characterization of resistance (R) genes in contrasting Fusarium wilt resistant chickpea genotypes

Fusarium wilt is one of the most notorious diseases of crop plants. This disease leads to huge crop losses in chickpea with substantial monetary value worldwide. The objective of the current study was to characterise Fusarium oxysporum f. sp. cicris (Foc), the causal agent of chickpea wilt, on morphological basis followed by the assessment of resistance of thirty chickpea germplasm accessions to Foc in a pot experiment. The second objective of this research was to study gene expression and characterisation of specific resistance (R) genes in different Fusarium wilt-resistant chickpea genotypes. The morphological identification and characterization of Foc was done by culturing the fungus potato dextrose agar (PDA) medium. Fusarium wild resistance was assessed by challenging 30 chickpea genotypes to artificial inoculum of Foc by using a pre-defined disease rating scale. Different resistance levels were showed by different chickpea genotypes ranging from highly resistant to highly susceptible. Two accessions demonstrated resistance, 15 were moderately resistant and the rest of the genotypes were moderately susceptible to susceptible. The in-silico expression analysis of R genes showed that two R genes, LOC101499430 and LOC101499568 were upregulated in chickpea roots, indicating their probable role in Fusarium wilt resistance. The genes were further assessed for their expression in response to Fusarium inoculation in contrasting Fusarium wilt resistant and susceptible chickpea genotypes. The resistant genotypes showed significantly higher expression levels of the selected R genes in comparison with susceptible genotypes. This study provides valuable insights into evaluating chickpea germplasm for Fusarium wilt resistance and identification of candidate resistance genes which could be employed for enhancement of Fusarium wilt resistance in chickpea.

Metatranscriptome and Resistome of the Endodontic Microbiome

IntroductionIn this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. MethodsRoot canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. ResultsProteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT−286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. ConclusionsMetatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

Artificial diets can manipulate the reproductive development status of predatory ladybird beetles and hold promise for their shelf-life management

With the development of biological control methods, the predatory ladybird beetle Harmonia axyridis Pallas (Coleoptera: Coccinellidae) has been widely used for pest control in agricultural production. Appropriate shelf-life management strategies could synchronize H. axyridis production with pest outbreaks, finally improving the effectiveness of biological control. Herein, we preliminarily explored whether an artificial diet could optimize the shelf-life management of H. axyridis. We compared the survival rate, nutrition accumulation, reproductive development, juvenile hormone (JH) related-gene expression levels, and stress resistance gene expression levels between aphid-fed and artificial diet-fed H. axyridis females. The results revealed that H. axyridis females maintained a high survival rate after being fed an artificial diet for 60 days, whereas the survival rate of aphid-fed females decreased. Continuous feeding of the artificial diet caused H. axyridis females to enter a diapause-like state, which was characterized by low JH levels, high triglycerides and trehalose accumulation, ovarian development inhibition, decreased Vgs expression levels, and increased stress resistance gene expression levels. This diapause-like state could be promptly recovered upon transferring to an aphid diet. These results indicate that the artificial diet could manipulate the reproductive development status of H. axyridis and lay the foundation for its shelf-life management.

Circular RNA ath-circ032768, a competing endogenous RNA, response the drought stress by targeting miR472-RPS5 module.

CircRNAs (circular RNAs) reduce the abundance of miRNAs through ceRNA (competing endogenous RNA), to regulate many physiological processes and stress responses in plants. However, the role of circRNA in drought stress is poorly understood. Through ring identification and sequencing verification of ath-circ032768, bioinformatics analysis predicted the interaction of ath-circ032768-miR472-RPS5, and further obtained transgenic plants overexpressing ath-circ032768 and silencing STTM-miR472. The change in drought stress was analysed using biochemical and molecular biological methods. Sequencing and biological analysis confirmed that ath-circ032768, miR472 and RPS5 were responsive to drought stress, and changes in gene expression were consistent with the prediction of ceRNA. The silencing vectors ath-circ032768 and STTM-miR472 were constructed using molecular biology techniques, and stable transgenic plants with drought tolerance obtained. Further physiological and biochemical studies showed that ath-circ032768 could bind to miR472, and that miR472 could bind to the RPS5 gene, resulting in decreased expression of RPS5. Hence, ath-circ032768 can competitively inhibit degradation of RPS5 by miR472 through ceRNA. This process is accompanied by increased expression of DREB2A, RD29A and RD29B genes. Through the ath-circ032768-miR472-RPS5 pathway, the RPS5 stress resistance protein interacts with DREB2A protein to enhance expression of downstream drought resistance genes, RD29A and RD29B, and participate in the regulation mechanism of plant drought resistance, thereby improving drought tolerance of plants.

ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Dynamical model of antibiotic responses linking expression of resistance genes to metabolism explains emergence of heterogeneity during drug exposures

Antibiotic responses in bacteria are highly dynamic and heterogeneous, with sudden exposure of bacterial colonies to high drug doses resulting in the coexistence of recovered and arrested cells. The dynamics of the response is determined by regulatory circuits controlling the expression of resistance genes, which are in turn modulated by the drug’s action on cell growth and metabolism. Despite advances in understanding gene regulation at the molecular level, we still lack a framework to describe how feedback mechanisms resulting from the interdependence between expression of resistance and cell metabolism can amplify naturally occurring noise and create heterogeneity at the population level. To understand how this interplay affects cell survival upon exposure, we constructed a mathematical model of the dynamics of antibiotic responses that links metabolism and regulation of gene expression, based on the tetracycline resistance tet operon in E. coli. We use this model to interpret measurements of growth and expression of resistance in microfluidic experiments, both in single cells and in biofilms. We also implemented a stochastic model of the drug response, to show that exposure to high drug levels results in large variations of recovery times and heterogeneity at the population level. We show that stochasticity is important to determine how nutrient quality affects cell survival during exposure to high drug concentrations. A quantitative description of how microbes respond to antibiotics in dynamical environments is crucial to understand population-level behaviors such as biofilms and pathogenesis.

Effect of High-Intensity Interval Training on TCF7L2 Gene Expression in Hepatocytes of Obese Rats

&#x0D; &#x0D; &#x0D; Objective: Hepatic glucose release is greatly increased in the presence of obesity and related diseases. The research objective was to explore the impact of high intensity interval training (HIIT) on TCF7L2 gene expression in hepatocytes of obese rats.&#x0D; Materials and Methods: Out of 21 male Wistar rats aged 10 weeks years (220±10 g), obesity was induced in 14 rats by 8-week high-fat diet. The rats were then divided into normal (n=7), obese control (n=7), and HIIT obese (n=7) groups. Rats in the HIIT group completed 8 weeks of HIIT/5 days weekly, whereas the other groups were inactive. After intervention, TCF7L2 gene expression in hepatocytes, insulin resistance and glucose compared using ANOVA /Tukey’s post hoc test between groups by SPSS-22.&#x0D; Results: Obesity induction led to a significant decrease in TCF7L2 gene expression (P: 0.011) and an increase in blood glucose (P: 0.009) and insulin resistance (P: 0.019) compared with the normal group (P&lt; 0.001). On the other hand, interval training led to a significant increase in TCF7L2 gene expression (P: 0.029) and a decrease in blood glucose (P&lt; 0.001) and insulin resistance (P&lt; 0.001) in the obese group compared with the control group.&#x0D; Conclusion: The observed enhancement in fasting blood glucose levels among obese rats could be linked to increased TCF7L2 gene expression in liver cells, which appears to be a response to interval training intervention. Nevertheless, understanding the main mechanisms responsible for observed changes requires further studies in this field.&#x0D; &#x0D; &#x0D;

Abstract 4557: Overexpressed CPAP promotes cancer stemness and enhances chemo-resistance in HCC

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is highly resistant to chemotherapy and targeted therapy. It was reported that HCC cancer stem cells (CSCs) are involved in chemo-drug resistance by regulating multidrug resistance (MDR) gene expression, such as ABC transporters. Our previous reports indicated that CPAP, a centrosomal protein, can promote HCC progression by up-regulating NF-κB and STAT3 transcriptional activities. Here, we demonstrated that overexpressed CPAP can increase cancer stemness features and CSCs-derived sorafenib resistance in HCC by up-regulating the transcriptional activities of NF-κB and β-catenin. Importantly, CPAP stably expressed HCC spheroids are significantly resistance to sorafenib in vitro and in vivo. Our results suggested that CD24 and ABCG2 are two potential downstream effectors in CPAP overexpressed HCC. CPAP can transcriptionally up-regulate CD24 and ABCG2 expression via Wnt/β-catenin and NF-κB signaling pathways, respectively; high CPAP/ABCG2/CD24 expression levels are negatively correlated with disease-specific survival rates in HCC patients. Combined treatment with sorafenib and Wnt/β-catenin pathway inhibitor PRI-724 significantly inhibited CPAP-overexpressing Hep3B spheroids-derived tumor growth in a xenograft mouse model. Our study highlights the oncogenic role of CPAP in promoting CSCs-induced sorafenib resistance in HCC and provides insights into potential therapeutic strategies. Citation Format: Liang-Yi Hung. Overexpressed CPAP promotes cancer stemness and enhances chemo-resistance in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4557.

MYB alternative promoter activity is increased in adenoid cystic carcinoma metastases and is associated with a specific gene expression signature

ObjectiveAdenoid cystic carcinoma (ACC) is a head and neck cancer with a poor long-term prognosis that shows frequent local recurrences and distant metastases. The tumors are characterized by MYB oncogene activation and are notoriously unresponsive to systemic therapies. The biological underpinnings behind therapy resistance of disseminated ACC are largely unknown. Here, we have studied the molecular and clinical significance of MYB alternative promoter (TSS2) usage in ACC metastases. Materials and methodsMYB TSS2 activity was investigated in primary tumors and metastases from 26 ACC patients using RNA-sequencing and quantitative real-time PCR analysis. Differences in global gene expression between MYB TSS2 high and low cases were studied, and pathway analyses were performed. ResultsMYB TSS2 activity was significantly higher in ACC metastases than in primary tumors (median activity 15.1 vs 3.0, P = 0.0003). MYB TSS2 high ACC metastases showed a specific gene expression signature, including increased expression of multi-drug resistance genes and canonical MYB target genes, and suppression of the p53 and NOTCH pathways. ConclusionsCollectively, our findings indicate that elevated MYB TSS2 activity is associated with metastases, potential drug resistance, and augmented MYB-driven gene expression in ACC. Our study advocates the need for new therapies that specifically target MYB and drug resistance mechanisms in disseminated ACC.

Acquired resistance or tolerance? – in search of mechanisms underlying changes in the resistance profile of Candida albicans and Candida parapsilosis as a result of exposure to methotrexate

The increasing prevalence of fungal strains showing acquired resistance and multidrug resistance is an increasing therapeutic problem, especially in patients with a severely weakened immune system and undergoing chemotherapy. What is also extremely disturbing is the similarity of the resistance mechanisms of fungal cells and other eukaryotic cells, including human cells, which may contribute to the development of cross-resistance in fungi in response to substances used in e.g. anticancer treatment. An example of such a drug is methotrexate, which is pumped out of eukaryotic cells by ABC transmembrane transporters - in fungi, used to remove azoles from fungal cells.For this reason, the aim of the study was to analyze the expression levels of genes: ERG11, MDR1 and CDR1, potentially responsible for the occurrence of cross-resistance in Candida albicans and Candida parapsilosis as a result of fungal exposure to methotrexate (MTX).In vitro exposure of C. albicans and C. parapsilosis strains to methotrexate showed a high increase in resistance to fluconazole and a partial increase in resistance to voriconazole. Analysis of the expression of resistance genes showed varied responses of the tested strains depending on the species. In the case of C. albicans, an increase in the expression of the MDR1 gene was observed, and a decrease in ERG11 and CDR1. However, for C. parapsilosis there was an increase in the expression of the CDR1 gene and a decrease in ERG11 and MDR1.We noted the relationship between the level of resistance to voriconazole and the level of ERG11 gene expression in C. albicans. This indicates that this type of relationship is different for each species. Our research confirms that the mechanisms by which fungi acquire resistance and develop cross-resistance are highly complex and most likely involve several pathways simultaneously. The emergence of multidrug resistance may be related to the possibility of developing tolerance to antimycotics by fungi.

Deep model predictive control of gene expression in thousands of single cells

Gene expression is inherently dynamic, due to complex regulation and stochastic biochemical events. However, the effects of these dynamics on cell phenotypes can be difficult to determine. Researchers have historically been limited to passive observations of natural dynamics, which can preclude studies of elusive and noisy cellular events where large amounts of data are required to reveal statistically significant effects. Here, using recent advances in the fields of machine learning and control theory, we train a deep neural network to accurately predict the response of an optogenetic system in Escherichia coli cells. We then use the network in a deep model predictive control framework to impose arbitrary and cell-specific gene expression dynamics on thousands of single cells in real time, applying the framework to generate complex time-varying patterns. We also showcase the framework’s ability to link expression patterns to dynamic functional outcomes by controlling expression of the tetA antibiotic resistance gene. This study highlights how deep learning-enabled feedback control can be used to tailor distributions of gene expression dynamics with high accuracy and throughput without expert knowledge of the biological system.

From "resistance genes expression" to "horizontal migration" as well as over secretion of Extracellular Polymeric Substances: Sludge microorganism’s response to the increasing of long-term disinfectant stress

Glutaraldehyde-Didecyldimethylammonium bromides (GDs) has been frequently and widely employed in livestock and poultry breeding farms to avoid epidemics such as African swine fever, but its long-term effect on the active sludge microorganisms of the receiving wastewater treatment plant was keep unclear. Four simulation systems were built here to explore the performance of aerobic activated sludge with the long-term exposure of GDs and its mechanism by analyzing water qualities, resistance genes, extracellular polymeric substances and microbial community structure. The results showed that the removal rates of CODCr and ammonia nitrogen decreased with the exposure concentration of GDs increasing. It is worth noting that long-term exposure to GDs can induce the horizontal transfer and coordinated expression of a large number of resistance genes, such as qacE, sul1, tetx, and int1, in drug-resistant microorganisms. Additionally, it promotes the secretion of more extracellular proteins, including arginine, forming a "barrier-like" protection. Therefore, long-term exposure to disinfectants can alter the treatment capacity of activated sludge receiving systems, and the abundance of resistance genes generated through horizontal transfer and coordinated expression by drug-resistant microorganisms does pose a significant threat to ecosystems and health. It is recommended to develop effective pretreatment methods to eliminate disinfectants.

Abstract A028: Investigating the role of SOX2 in promoting anchorage-independent survival of ovarian cancer cells

Abstract Ovarian cancer (OVCA) is the most lethal gynecologic malignancy, often detected at an advanced stage. OVCA progression involves transcoelomic metastasis, where tumor cells disseminate into the peritoneal ascites. Malignant cells in the ascites adapt to survive under Anchorage-Independent (A-I) conditions by altering the expression of genes escalating anoikis (A-I cell death) resistance and metastasis. Our lab has shown that OVCA cells manipulate their transcriptomic profile to promote the expression of anoikis resistance genes in A-I. Expression profiling of genes upregulated in A-I revealed the transcription factor SRY-Box Transcription Factor 2 (SOX2) to be highly upregulated in response to OVCA cell detachment. While we have previously demonstrated that SOX2 is necessary for the A-I survival of OVCA cells, the specific mechanism underlying SOX2-dependent A-I survival remains unknown. RNA-sequencing studies performed in the high-grade serous ovarian cancer cell line, OVCAR3, revealed that the Leucine-rich repeat-containing G-protein coupled receptor 5, LGR5, is the most significantly upregulated gene in A-I compared to attached conditions. Importantly, amongst those genes that are upregulated in A-I, LGR5 is significantly downregulated following SOX2 knockdown in A-I. LGR5 is an ‘orphan’ G-protein coupled receptor known to modulate canonical Wnt signaling upon interacting with its ligand R-spondin1 (RSPO1). Time-course experiments in OVCAR3 cells revealed a striking increase in LGR5 expression that peaked after 24 hours in A-I. Furthermore, siRNA mediated knockdown of SOX2 downregulated LGR5 expression only in A-I, suggesting that LGR5 is a potential SOX2 target under A-I conditions. Interestingly, bioinformatics analysis of the existing OVCA datasets from the Cancer Genome Atlas showed increased expression of RSPO1, which correlated with a significant decrease in the overall survival of OVCA patients. Interaction of LGR5 with its ligand RSPO1 is crucial for the receptor to signal through the Wnt pathway to promote cell survival and cancer stemness. Although SOX2 and LGR5 are established markers of cancer stemness in various tumor types, their direct relationship in promoting tumor progression and regulation of OVCA A-I cell survival remains unknown. Our data suggests that increased SOX2 and LGR5 expression could drive OVCA cell survival under A-I conditions. Our current studies focus on understanding the molecular mechanisms behind SOX2-driven regulation of LGR5 and the role of LGR5 in driving OVCA A-I cell survival. Citation Format: Shriya Kamlapurkar, Coryn Stump, Mythreye Karthikeyan, Nadine Hempel. Investigating the role of SOX2 in promoting anchorage-independent survival of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A028.

IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers.

SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.

Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.

Vitis vinifera L. seed standardized extract; a promising therapeutic against metabolic syndrome induced by high-fat/high-carbohydrate diet and streptozotocin in rats

Metabolic syndrome (MetS) is a group of abnormal disorders; hypertension, glucose intolerance, dyslipidemia, proinflammatory, and prothrombotic states, affecting approximately 14 % of the world's population. The potential of grape seeds extract (GSE) supplementation to improve the metabolic disturbances and their related conditions like obesity, type 2 diabetes, and nonalcoholic fatty liver disease was investigated in this study. In-vitro metabolic syndrome assays were investigated through α-amylase, α-glucosidase, lipase, lipoxygenase (LOX), cyclooxygenases (COX-1 and COX-2), renin and angiotensin converting enzyme (ACE) inhibition assays. Additionally, in-vivo rat model of high-fat-high-carbohydrate diet (HFD)-induced MetS was established, where metformin (MT) (200 mg/kg) was used as a reference drug and GSE was given at 100 and 200 mg/kg by oral gavage. GSE was standardized using HPLC analysis for the major reported compound; catechin which should be ≥ 160.76 ± 5.52 μg/mL. Total phenolic content of GSE was 21.12±0.61 µg gallic acid equivalent/1 mg extract and total flavonoid content was 23.81±0.64 µg rutin equivalent/1 mg extract. In-vitro assays revealed the potential of GSE to manage the metabolic syndrome, besides its strong antioxidant capacity. Treatment with GSE (100 mg/kg and 200 mg/kg) markedly (P < 0.05) controlled the weight gain, improved the metabolic pathways (total glucose, cholesterol, triglycerides, AST, and ALT), oxidative stress parameters (MDA, GSH, and catalase) and inflammatory biomarkers in HFD fed rats. GSE downregulated the expression of insulin resistance gene (IR) and some inflammatory related genes (TNF-α and NF-κB), additionally it improved the pathological features of metabolic conditions and upgraded the expression of Nrf2 compared to HFD group. The superior effects were owned to the high dose of GSE, 200 mg/kg b.wt. (P < 0.05, P < 0.001). All results sustenance the beneficial effects of the standardized GSE in the management of metabolic syndrome.

Brain-Decellularized ECM-Based 3D Myeloid Sarcoma Platform: Mimicking Adaptive Phenotypic Alterations in the Brain.

Leukemia circulates in the bloodstream and induces various symptoms and complications. Occasionally, these cells accumulate in non-marrow tissues, forming a tumor-like myeloid sarcoma (MS). When the blast-stage leukemia cells invade the brain parenchyma, intracranial MS occurs, leading to a challenging prognosis owing to the limited penetration of cytostatic drugs into the brain and the development of drug resistance. The scarcity of tissue samples from MS makes understanding the phenotypic changes occurring in leukemia cells within the brain environment challenging, thereby hindering development of effective treatment strategies for intracranial MS. This study presents a novel 3D in vitro model mimicking intracranial MS, employing a hydrogel scaffold derived from the brain-decellularized extracellular matrix in which suspended leukemia cells are embedded, simulating the formation of tumor masses in the brain parenchyma. This model reveals marked phenotypic changes in leukemia cells, including altered survival, proliferation, differentiation, and cell cycle regulation. Notably, proportion of dormant leukemia stem cells increases and expression of multidrug resistance genes is upregulated, leading to imatinib resistance, mirroring the pathological features of in vivo MS tissue. Furthermore, suppression of ferroptosis is identified as an important characteristic of intracranial MS, providing valuable insights for the development of targeted therapeutic strategies.