Types Of DNA Research Articles

Practical Aspects of the Implementation of Screening for the Detection of Malignant Neoplasms of the Cervix during the Medical Examination

Relevance. The global strategy for cervical cancer elimination includes the active implementation of primary, secondary and tertiary prevention programs. In the Russian Federation, screening for the detection of malignant neoplasms of the cervix is carried out while women contact specialists, as well as an organized procedure during preventive medical examinations of the certain groups of adults. The study of the population structure of circulating human papillоmavirus (HPV) types, and evaluation of the effectiveness of the recommended diagnostic models will improve the direction of the fight against cervical cancer and introduce optimal preventive solutions.Aim. Retrospective analysis of screening effectiveness for the detection of malignant neoplasms of the cervix, carried out during the medical examination in one institution of Moscow.Materials and methods. The study examined 1068 women aged 20 to 81 years (M = 37.82, Me = 35, IQR 27–47 years) from one institution in Moscow over a 5-year follow-up period (2017–2021). The screening was based on the co-testing model: liquid-based cytology with Papanicolaou staining and classification according to the Bethesda system and quantitative Real Time HPV-test with determination of the 14 HPV DNA types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).Results. The prevalence of the 14 HPV oncogenic types was 14.87% (95% CI: 12.86–17.13%), the most common were 16 (16.98%), 31 (14.47%), 52 (13.21%). The study determined HPV types with the highest relative risk of persistence: 33, 58, 45, and 52 (p < 0.005). The frequency of HPV detection depended on the age of the examined women and was the highest in the age group of 20–29 years (25.58%). The baseline prevalence was 11.82% (95% CI: 9.98–13.94%). 6 HSIL cases associated with HPV types 16 and 31 were identified. 4/6 were in women under 30 years. The probability of intraepithelial lesions of the cervix increased with an increase of HPV DNA concentration: 23.65% and 66.67% with a viral load of 4.0–6.0 lg copies per 105 human cells and > 6.0 lg copies per 105 human cells, respectively.Conclusions. The study confirmed the great value of HPV- test in screening for the detection of malignant neoplasms of the cervix during the medical examination of the certain groups of adult population, taking into account the advantages of typing and quantitative determination of HPV DNA, as well as the need to consider lowering of the recommended age for the start of HPV-testing.

Carbon Nanotube-Based Sensors for Intelligent Packaging

Aim: The aim of this project is to develop a gas-phase optical sensor for oxygen detection in intelligent packaging. The optical sensor will be based on near-infrared light that can penetrate through visibly opaque materials, including plastics, paper, and cardboard. An automated near-infrared camera will be used to precisely and quickly inform the user of any change in the integrity of packages that are vacuum-sealed or atmospherically modified. Method: The optical sensor is based on near-infrared fluorescence that is emitted from carbon nanotubes (CNTs). CNTs exhibit a sensitive and photostable fluorescence that is well suited for sensing applications. The CNTs are wrapped by one of two types of DNA, AT(15) and GT(15), through sonication. These wrappings control the responsivity of the CNTs to different gases while also solubilizing them for further processing. The resulting solutions are then drop-casted into a film. We subsequently investigate the response of the film in the presence of the target gas, oxygen, at different concentrations. Finally, we evaluate the selectivity of the sensor in the presence other gases including carbon dioxide, ethylene, and ammonia. Results: Our results reveal that the fluorescence intensity of the AT(15) and GT(15)-wrapped CNTs quenches upon the exposure of oxygen. Interestingly, we observe that the different CNT chiralities respond distinctly to the presence of oxygen. In particular, whereas some chiralities undergo an irreversible loss in fluorescence after several oxygen exposure cycles, the (8,6) chirality demonstrates stability over multiple cycles for both AT(15) and GT(15)-wrapped CNTs. Finally, our results show a concentration-dependent and selective response of both the AT(15)- or GT(15)-wrapped CNT sensors to the oxygen, as we observed no significant response to the other studied gases. Conclusion: We developed an optical, gas-phase sensor based on CNT fluorescence for intelligent food packaging. The sensor is selective to oxygen and demonstrates reversibility over multiple oxygen exposure cycles. These sensors can be used to monitor the integrity of packages that are vacuum-sealed or otherwise atmospherically modified. This technology can thus provide consumers and manufactures a means of preventing early spoilage and ensuring food quality.

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

Sperm dna bridges versus genetic variability: an editorial

Sperm DNA fragmentation is recognized to compromise male fertility. The data entail that sperm DNA damage can be competently treated with oral antioxidant administration during a comparatively short time period. It is an understanding that the 2 types of DNA appear to be different and autonomously packaged molecules; though, research has demonstrated the interdependent nature of these conformations in contributing to male infertility. Data must endure to be congregated to establish strong associations between traditional semen analysis parameters and sperm DNA integrity; this information remnants controversial and useless in clinical practice until novel techniques for the diagnosis and treatment of sperm DNA can be established. More modern technology must be employed to correlate such information into valuable clinical awareness. This editorial is the collation of reports relating to research on sperm DNA biochemistry and molecular biology to establish an opinion revealing sperm DNA integrity likely to be both tremendously fragile and remarkably significant for male fertility.

Research Progress on the Development of Porcine Reproductive and Respiratory Syndrome Vaccines.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in the pig industry, but its pathogenesis is not yet fully understood. The disease is caused by the PRRS virus (PRRSV), which primarily infects porcine alveolar macrophages and disrupts the immune system. Unfortunately, there is no specific drug to cure PRRS, so vaccination is crucial for controlling the disease. There are various types of single and combined vaccines available, including live, inactivated, subunit, DNA, and vector vaccines. Among them, live vaccines provide better protection, but cross-protection is weak. Inactivated vaccines are safe but have poor immune efficacy. Subunit vaccines can be used in the third trimester of pregnancy, and DNA vaccines can enhance the protective effect of live vaccines. However, vector vaccines only confer partial protection and have not been widely used in practice. A PRRS vaccine that meets new-generation international standards is still needed. This manuscript provides a comprehensive review of the advantages, disadvantages, and applicability of live-attenuated, inactivated, subunit, live vector, DNA, gene-deletion, synthetic peptide, virus-like particle, and other types of vaccines for the prevention and control of PRRS. The aim is to provide a theoretical basis for vaccine research and development.

Development of non-electrically controlled SalivaDirect LAMP (NEC-SD-LAMP), a new nonelectrical infectious disease testing method

In this study, non-electrically controlled SalivaDirect loop-mediated isothermal amplification (NEC-SD-LAMP), which can detect infections by amplifying viral DNA expression in saliva without using electrical control systems, was developed. By this method, only by adding water to the device, viral DNA was extracted from saliva using SalivaDirect, the extracted DNA was amplified via loop-mediated isothermal amplification (LAMP), and the results were visually confirmed. Melting palmitic acid maintained the optimal temperature for the LAMP reaction, as the temperature of palmitic acid is maintained at 62.9 °C, its melting point. By exploiting the proximity of the melting point to the optimal temperature for LAMP, LAMP can be performed without electricity. We detected several viruses in the saliva using this method. NEC-SD-LAMP could clearly distinguish 3 types of viral DNA, indicating the high specificity of this reaction. Furthermore, the viral concentration detection limit of the device was 2 copies per µL, indicating that it is possible to detect DNA viral infections in saliva even before the onset of viral infection.

Kangaroo endogenous retrovirus (KERV) forms megasatellite DNA with a simple repetition pattern in which the provirus structure is retained

The kangaroo endogenous retrovirus (KERV) was previously reported to have undergone a rapid copy number increase in the red-necked wallaby; however, the mode of amplification was left to be clarified. The present study revealed that the long terminal repeat (LTR) (0.6 kb) and internal region (2.0 kb) of a provirus are repeated alternately, forming megasatellite DNA which we named kervRep. This repetition pattern was the same as that observed for walbRep, megasatellite DNA originating from another endogenous retrovirus. Their formation process can be explained using a simple model: pairing slippage followed by homologous recombination. This model features that the initial step is triggered by the presence of two identical sequences within a short distance; the possession of LTRs by endogenous retroviruses fulfills this condition. The discovery of two cases suggests that formation of this type of satellite DNA is one of non-negligible effects of endogenous retroviruses on their host genomes.

Effect of various types of extracellular DNA on V. hyugaensis biofilm formation.

Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, another Vibrio spp. gDNA, and an oligomer of 61-80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCE Bacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of a Vibrio hyugaensis biofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.

Absorbance biosensors-based hybrid mathbf {MoS_{2}} nanosheets for Escherichia coli detection

Detecting Escherichia coli is essential in biomedical, environmental, and food safety applications. In this paper, we have developed a simple, rapid, sensitive, and selective E. coli DNA sensor based on the novel hybrid-type text {MoS}_text {2} and mathrm {(NH_4)_6Mo_7O_{24}} nanosheets. The sensor uses the absorbance measurement to distinguish among the DNA of E. coli, Vibrio proteolyticus, and Bacillus subtilis when implemented in conjunction with text {NH}_text {2}-probes. Our experiments showed that the absorbance increased when sensors detected E. coli DNA, whereas it decreased when sensors detected V. proteolyticus and B. subtilis DNA. To the best of authors’ knowledge, there are no reports using the novel hybrid-mathrm {MoS_2} and mathrm {(NH_4)_6Mo_7O_{24}} materials for differentiating three types of DNA using cost-effective and rapid absorbance measurements. In addition, the label-free E. coli DNA biosensor exhibited a linear response in the range of 0 fM to 11.65 fM with a limit of detection of 2 fM. The effect of text {NH}_text {2}-probes on our sensors’ working performance is also investigated. Our results will facilitate further research in pathogen detection applications, which have not been fully developed yet.

Electrochemical Detection of Different Foodborne Bacteria for Point-of-Care Applications.

Bacterial infections resulting from foodborne pathogenic bacteria cause millions of infections that greatly threaten human health and are one of the leading causes of mortality around the world. To counter this, the early, rapid, and accurate detection of bacterial infections is very important to address serious health issue concerns. We, therefore, present an electrochemical biosensor based on aptamers that selectively bind with the DNA of specific bacteria for the accurate and rapid detection of various foodborne bacteria for the selective determination of bacterial infection types. Different aptamers were synthesized and immobilized on Au electrodes for selective bindings of different types of bacterial DNA (Escherichia coli, Salmonella enterica, and Staphylococcus aureus) for the accurate detection and quantification of bacterial concentrations from 101 to 107 CFU/mL without using any labeling methods. Under optimized conditions, the sensor showed a good response to the various concentrations of bacteria, and a robust calibration curve was obtained. The sensor could detect the bacterial concentration at meager quantities and possessed an LOD of 4.2 × 101, 6.1 × 101, and 4.4 × 101 CFU/mL for S. Typhimurium, E. Coli, and S. aureus, respectively, with a linear range from 100 to 104 CFU/mL for the total bacteria probe and 100 to 103 CFU/mL for individual probes, respectively. The proposed biosensor is simple and rapid and has shown a good response to bacterial DNA detections and thus can be applied in clinical applications and food safety monitoring.

Multi-dimensional cell-free DNA-based liquid biopsy and early detection of gastric cancer.

4060 Background: Gastric cancer is one of the most common cancer types. Most patients were diagnosed at advanced stages and experienced poor prognosis. A non-invasive assay for the detection of early-stage gastric cancer is highly desirable for reducing the gastric cancer-associated mortality. Methods: We prospectively collected a study cohort of 249 participants including 110 pathologically confirmed with stage I-II gastric cancer and 139 with non-cancerous conditions. A plasma sample was collected from each patient before conventional screening modalities for gastric cancer. We performed whole-genome sequencing with plasma samples and profiled four types of cell-free DNA (cfDNA) characteristics, fragment size pattern, copy number variation, nucleosome coverage pattern, and single nucleotide substitution. We used these differential profiles to develop an ensemble model to identify gastric cancer patients from non-cancer individuals. We further collected a validation cohort consisting of 73 gastric cancer patients and 94 non-cancer individuals to validate the cfDNA-based assay. Additionally, we compared the performance of our assay and conventional gastroscopy in a hypothetical 100,000 screening population by Monte Carlo simulation. Results: The liquid biopsy assay that incorporated four types of cfDNA characteristics was able to identify early-stage gastric cancer patients from non-cancer individuals at an AUROC of 0.962 in the study cohort and 0.972 in the validation cohort. At a specificity of 92.1% (128/139), it achieved a sensitivity of 88.2% (97/110) in the study cohort. With this threshold, in the validation cohort, 91.5% (86/94) of healthy individuals and 91.8% (67/73) of gastric cancer patients were correctly identified. In both study and validation cohorts, the inferred probabilities of cancer showed consistent trends to increase with pathological stages in the cancer group and disease statuses in the non-cancer group. Of note, our approach detected all gastric tumors located in the cardia and fundus (100.0%, 19/19), which could be challenging for gastroscopic examination. Additionally, through in-silico simulations, we showed that our cfDNA-based non-invasive assay may detect 96.3% more gastric cancer cases than conventional gastroscopy owing to higher sensitivity and anticipated better participant compliance in a large hypothetical screening population. Conclusions: We introduced a liquid biopsy assay using multiple dimensions of cfDNA characteristics that could accurately identify early-stage gastric cancer from non-cancerous conditions. As a cost-effective non-invasive approach, it may provide population-wide benefits for the early detection of gastric cancer.

Investigating causality in the association between DNA methylation and type 2 diabetes using bidirectional two-sample Mendelian randomisation

Aims/hypothesisSeveral studies have identified associations between type 2 diabetes and DNA methylation (DNAm). However, the causal role of these associations remains unclear. This study aimed to provide evidence for a causal relationship between DNAm and type 2 diabetes.MethodsWe used bidirectional two-sample Mendelian randomisation (2SMR) to evaluate causality at 58 CpG sites previously detected in a meta-analysis of epigenome-wide association studies (meta-EWAS) of prevalent type 2 diabetes in European populations. We retrieved genetic proxies for type 2 diabetes and DNAm from the largest genome-wide association study (GWAS) available. We also used data from the Avon Longitudinal Study of Parents and Children (ALSPAC, UK) when associations of interest were not available in the larger datasets. We identified 62 independent SNPs as proxies for type 2 diabetes, and 39 methylation quantitative trait loci as proxies for 30 of the 58 type 2 diabetes-related CpGs. We applied the Bonferroni correction for multiple testing and inferred causality based on p<0.001 for the type 2 diabetes to DNAm direction and p<0.002 for the opposing DNAm to type 2 diabetes direction in the 2SMR analysis.ResultsWe found strong evidence of a causal effect of DNAm at cg25536676 (DHCR24) on type 2 diabetes. An increase in transformed residuals of DNAm at this site was associated with a 43% (OR 1.43, 95% CI 1.15, 1.78, p=0.001) higher risk of type 2 diabetes. We inferred a likely causal direction for the remaining CpG sites assessed. In silico analyses showed that the CpGs analysed were enriched for expression quantitative trait methylation sites (eQTMs) and for specific traits, dependent on the direction of causality predicted by the 2SMR analysis.Conclusions/interpretationWe identified one CpG mapping to a gene related to the metabolism of lipids (DHCR24) as a novel causal biomarker for risk of type 2 diabetes. CpGs within the same gene region have previously been associated with type 2 diabetes-related traits in observational studies (BMI, waist circumference, HDL-cholesterol, insulin) and in Mendelian randomisation analyses (LDL-cholesterol). Thus, we hypothesise that our candidate CpG in DHCR24 may be a causal mediator of the association between known modifiable risk factors and type 2 diabetes. Formal causal mediation analysis should be implemented to further validate this assumption.Graphical

Abstract LB059: DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer

Abstract Ultraviolet A (UVA) rays are long wavelengths that can penetrate the deeper layer of the skin and are responsible for the immediate tanning effect. A multitude of natural and artificial sources of UV exposure exist, including UV-emitting lamps that are commonly used in dentistry, cosmetics, and medical laboratories. Recently, several reports suggested that long-term use of UV-nail polish dryers, used for curating nail polish formulas, known as gel, may increase the risk for developing skin cancer. However, no experimental evaluation has been conducted to reveal the effect of radiation released by UV-nail polish dryers on mammalian cells. Here, we address the continuous exposure of clients and employees to UVA light by employing both murine and human primary cell models for irradiation, in an acute and chronic manner, using a UV-dryer machine. We, then, subject the cells to DNA sequencing either by bulk whole-genome sequencing after clonal expansion or by single-molecule duplex sequencing without clonal expansion. Our findings show that UVA-exposed samples manifest an increase of C:G&amp;gt;A:T transversions compared to control cells. The patterns of mutations found in these cells can be recapitulated by mutational signatures previously attributed to reactive oxygen species (ROS), namely COSMIC signatures SBS18/36. These results are further corroborated by the presence of high levels of ROS in irradiated samples, consistent with 8-oxo-7,8-dihydroguanine damage and mitochondrial dysfunction. Importantly, we show that in contrast to UVB exposure, UVA does not induce an increase of C:G&amp;gt;T:A transitions, indicating that different types of ultraviolet light radiation affect DNA through distinctive molecular mechanisms. Finally, re-examination of somatic mutations from skin cancers reported by the International Cancer Genome Consortium (ICGC) reveals that SBS18/36 are ubiquitously present in melanoma and account for 12% of the previously annotated driver mutations. In summary, this study demonstrates that radiation emitted by UV-nail polish dryers can both damage DNA and permanently engrave mutations on the genomes of mammalian cells. Citation Format: Maria Zhivagui, Areebah Hoda, Noelia Valenzuela, Yi-yu Yeh, Jason Dai, Yudou He, Shuvro P. Nandi, Burcak Otlu, Bennett Van Houten, Ludmil Alexandrov. DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB059.

Evaluation of the accuracy of hTERT gene aberrant methylation using electrochemical hybridization assay and liquid‐based cytology in screening for oral squamous cell carcinoma

AbstractIn order to improve the survival rate of oral squamous cell carcinoma (OSCC) patients, a reliable diagnostic method for early OSCC detection is required that is minimally invasive, less burdensome to the patient, and has high sensitivity and specificity. Therefore, we performed the detection of abnormal methylation at three locations in the hTERT promoter region of oral exfoliated cells by employing the ferrocenylnaphthalene diimide (FND)‐based electrochemical hybridization assay (FND‐EHA) using three types of DNA probe‐immobilized electrodes. We also performed liquid cytology using oral exfoliated cells and compared these obtained data to evaluate whether FND‐ EHA can be used as an OSCC screening system. The results showed a good correlation between this method and conventional OSCC screening, and cytology. In addition, FND‐EHA was also able to determine samples that had been ambiguously determined by liquid cytology. This indicates that FND‐EHA may be useful as an OSCC screening system.

Abstract 5173: BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most malignant neoplasm of the pancreas, and is refractory to most therapeutic strategies, including immunotherapy. We have previously demonstrated that loss of CDKN2A in PDAC tumor cells promotes an immunologically-cold tumor microenvironment, which can be counteracted through the use of bromodomain and extraterminal domain inhibitors (BETi). The immunosuppressive PDAC tumor microenvironment is also characterized by a dense desmoplastic stroma, rich in cancer associated fibroblasts (CAFs). These CAFs are a highly plastic population of cells, comprised of myofibroblastic, inflammatory, and antigen-presenting CAFs (myCAFs, iCAFs, and apCAFs, respectively). Previously research has suggested BETi can alter the function of CAFs, suggesting this therapeutic strategy may be effective at targeting both the tumor and stromal compartments of PDAC. Here we hypothesize that BETi enhances anti-tumor immunity in CDKN2A-deficient PDAC by modulating the phenotype and function of PDAC CAFs. METHODS: Isogenic wild-type and CDKN2A-deficient KPCluc pancreatic cancer cell lines were used in conjunction with KPCluc-derived murine CAF cells. CAF spheroids were used to evaluate the effects of BETi on CAF phenotype (as measured by flow cytometry) and function (as measured by NanoString analysis, checkpoint ligand expression by flow cytometry, and T cell migration assays). Additionally, the impact of BETi-induced modulation of CAF phenotype and function on wild-type and CDKN2A-deficient tumor cell spheroids was measured by flow cytometry. RESULTS: Treatment of pancreatic CAFs with BETi induced expression of genes associated with DNA damage responses and type I IFN expression. Additionally, while CAF supernatants suppressed T cell migration in vitro, treatment with BETi restored T cell migration across a transwell. This was due in part to DNA damage/STING/NF-κB signaling induced in PDAC CAF following BETi. Supernatants from BETi-treated PDAC CAFs were found to modulate PD-L1 expression on PDAC tumor cells. Direct treatment of CAFs with BETi induced a dramatic dose- and time-dependent polarization of CAF spheroids towards a myCAF phenotype. However, while BETi induced DNA damage/STING/NF-κB signaling in CAF cells, this signaling did not contribute towards this myCAF polarization, as treatment of CAF spheroids with BETi and STING or NF-κB inhibitors did not impact myCAF polarization. Additional mechanisms of polarization are currently being evaluated and will be updated at presentation. CONCLUSIONS: BET inhibition modulates CAF cell phenotype and function to promote anti-tumor immune responses. Taken together with the impact of BET inhibition on CDKN2A-deficient PDAC tumor cells, these data suggest BETi is a rational treatment approach to promote anti-tumor immunity in this genetically defined immunotherapy resistant patient population. Citation Format: Trisha Minocha, Alison Thomas, Brian M. Olson. BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity. [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 5173.

Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design-build-test-learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field.

Nomenclatural standards and genetic passports of potato cultivars bred by the Omsk Agrarian Research Center

In accordance with the International Code of Nomenclature for Cultivated Plants, five nomenclature standards were prepared for five potato сultivars* bred in the Omsk Agrarian Research Center: ‘Alena’, ‘Bylina Sibiri’, ‘Večernij Omsk’, ‘Triumf’, ‘Hozâûška’. Genetic passport of these five cultivars were carried out according to the new integrated strategy developed at VIR. According to the strategy, the plant material donated by the author of the cultivars to the VIR Herbarium was used for molecular genotyping. Genetic passports included data of allelic composition of eight chromosome specific microsatellite loci, markers of different types of organelle DNA, as well as data about diagnostic fragments of 15 markers of 11 R-genes conferring resistance to the most dangerous diseases and pests of potato: late blight, nematodes, potato X and Y viruses. Data from the developed genetic passports of five Omsk cultivars were compared to the results of genotyping of samples of the same cultivars obtained from different sources. Based on the analysis of pedigrees and genetic passports of these five cultivars we established the origin of their resistance to harmful organisms.

Interaction of dpyatriz and Cu/Zn-dpyatriz complexes with human telomere DNA: The role of G-quadruplex formation and its effect on antitumor and antitelomerase activity

1,3,5-Triazine derivative of dipyridyl amine (dpyatriz) and the Cu(II), Zn(II) complexes have been prepared, characterized by CHN, IR, NMR and mass measurements and interacted with HTelo8 and HTelo20, the two types of human telomere repeat DNA (TTAGGG)n; n = 8, 20. The telomere DNAs have been treated with all three compounds (dpyatriz, Cu-dpyatriz and Zn-dpyatriz) under parallel/antiparallel and random coiled conditions. The interactions are followed by circular dichrosim measurements, fluorescent intercalator displacement (FID) assays and molecular docking studies through MOE program. The free ligand and its Cu(II) and Zn(II) complexes stabilize predominantly antiparallel G-quadruplex form under antiparallel and no salt conditions. The binding constant (Kb) values calculated for the ligand and complexes are in the range of 1.9 x 105 M-1 to 4.2 x 107 M-1 under various conditions show the higher affinity of G-quadruplex conformations. The FID assay using thiazole orange with HTelo20 clearly depicts the G-quadruplex stabilization through strong binding mode. Also, all the three compounds dpyatriz, Cu-dpyatriz and Zn-dpyatriz show an intercalative mode of interaction with antiparallel G quadruplex DNA in molecular docking studies. The compounds show cytotoxicity with the IC50 values in the range of 50 – 90 nM, and antitelomerase activity in the range of 5 to 10 μM.

Is it inappropriate to ask for your age? Evaluating parameter impact on tree dating in a challenging clade (Macroscelidea)

Sengis (order Macroscelidea) are small mammals endemic to Africa. The taxonomy and phylogeny of sengis has been difficult to resolve due to a lack of clear morphological apomorphies. Molecular phylogenies have already significantly revised sengi systematics, but until now no molecular phylogeny has included all 20 extant species. In addition, the age of origin of the sengi crown clade and the divergence age of its two extant families remain unclear. Two recently published studies based on different datasets and age-calibration parameters (DNA type, outgroup selection, fossil calibration points) proposed highly different divergent age estimates and evolutionary scenarios. We obtained nuclear and mitochondrial DNA from mainly museum specimens using target enrichment of single-stranded DNA libraries to generate the first phylogeny of all extant macroscelidean species. We then explored the effects of different parameters (type of DNA, ratio of ingroup to outgroup sampling, number and type of fossil calibration points) and their resulting impacts on age estimates for the origin and initial diversification of Macroscelidea. We show that, even after correcting for substitution saturation, both using mitochondrial DNA in conjunction with nuclear DNA or alone results in much older ages and different branch lengths than when using nuclear DNA alone. We further show that the former effect can be attributed to insufficient amounts of nuclear data. If multiple calibration points are included, the age of the sengi crown group fossil prior has minimal impact on the estimated time frame of sengi evolution. In contrast, the inclusion or exclusion of outgroup fossil priors has a major effect on the resulting node ages. We also find that a reduced sampling of ingroup species does not significantly affect overall age estimates and that terminal specific substitution rates can serve as a means to evaluate the biological likeliness of the produced temporal estimates. Our study demonstrates how commonly varied parameters in temporal calibration of phylogenies affect age estimates. Dated phylogenies should therefore always be seen in the context of the dataset which was used to produce them.