Cell Genomic DNA Research Articles

Cyclopropenes as Chemical Reporters for Dual Bioorthogonal and Orthogonal Metabolic Labeling of DNA.

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross-reactivity. We developed a pair of differently modified 2'-deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse-electron demand Diels-Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1- and 3-methylcyclopropenyl-modified 2'-deoxynucleosides, two 2'-deoxyuridines were identified to be the fastest-reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2'-deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2'-deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine-styryl tetrazine and a fluorogenic pyrene-tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.

Cyclopropene als Chemische Reporter für die Duale Bioorthogonale und Orthogonale Metabolische Markierung von DNA

AbstractDie duale bioorthogonale Markierung ermöglicht die Untersuchung und das Verständnis von Wechselwirkungen in der biologischen Umgebung, die mit einer einzigen Markierung nicht zugänglich sind. Eine Herausforderung bleibt jedoch die Anwendung von zwei bioorthogonalen Reaktionen in der gleichen Umgebung aufgrund von Kreuzreaktivität. Um dieses Problem für die duale und orthogonale Markierung von DNA zu lösen, haben wir zwei unterschiedlich modifizierte 2′‐Desoxynukleoside entwickelt. Mit Hilfe der Diels‐Alder‐Reaktion mit inversem Elektronenbedarf und Photoklick‐Reaktion wurde genomische DNA in Zellen mit zwei verschiedenen fluorogenen Markern verknüpft. Aus einer kleinen synthetischen Bibliothek aus 1‐ und 3‐methylcyclopropenyl‐modifizierten 2′‐Desoxynukleosiden wurden zwei 2′‐Desoxyuridine identifiziert, die am schnellsten in jeweils einer der beiden bioorthogonalen Reaktionen reagieren. Ihre orthogonale Reaktivität konnte in vitro nachgewiesen werden. Ihre Replikationseigenschaften als Ersatz für Thymidin wurden in Primerverlängerungsexperimenten untersucht, um die anschließenden Markierungsreaktionen an DNA abzuschätzen. Schließlich wurde eine duale, orthogonale und metabolische Fluoreszenzmarkierung von genomischer DNA in HeLa‐Zellen nachgewiesen. Es wurde ein experimentelles Protokoll entwickelt, das den intrazellulären Transport und den metabolischen DNA‐Einbau der beiden 2′‐Desoxyuridine mit der anschließenden dualen bioorthogonalen Markierung unter Verwendung eines fluorogenen Cyanin‐Styryl‐Tetrazins und eines fluorogenen Pyren‐Tetrazols kombiniert. Diese Ergebnisse sind von grundlegender Bedeutung für fortgeschrittene metabolische Markierungsstrategien für Nukleinsäuren in der Zukunft, insbesondere in lebenden Zellen.

Abstract 3175: Dear ABBIE, edit that colon cancer

Abstract Genome editing has exciting potential for gene therapy of cancers. Colorectal cancer is the third most occurring cancer in both men and women. The current treatments for colon cancers include surgery, radiofrequency ablation, chemotherapies and immunotherapies. These either involve removal of intestinal tissue that can alter the quality of life of the patient, or require doses of drugs that generally result in various side effects. ABBIE (A Binding Based Integrase Enzyme) genome editing can insert operational gene sequences into a chosen site in the genomic DNA of cells via its targetability and integrase activity. The ABBIE system offers ease of use, faster time to a readout and decreased time per genome editing experiment. SW480 and Caco-2 colon adenocarcinoma cells were used with the ABBIE system to integrate a GFP donor sequence into a safe harbor locus of the genome. Integration is to be confirmed with fluorescence microscopy and DNA sequencing. The colon cancer cell line will then be edited with the ABBIE genome editing system to include a gene cassette that overexpresses caspase genes into the genomic DNA of these cells. ABBIE-Edited colon cancer cell lines are expected to exhibit increased apoptosis as compared to non-edited controls or controls edited with DNA that do not have the caspase genes. This strategy will help lead to novel and more targetable treatments for colon cancer. Citation Format: David C. Aguilar, Alfred Gallegos. Dear ABBIE, edit that colon cancer [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 3175.

Evaluation of the synergistic potential and mechanisms of action for de novo designed cationic antimicrobial peptides

Antimicrobial peptides (AMPs) have emerged as promising candidates in combating antimicrobial resistance – a growing issue in healthcare. However, to develop AMPs into effective therapeutics, a thorough analysis and extensive investigations are essential. In this study, we employed an in silico approach to design cationic AMPs de novo, followed by their experimental testing. The antibacterial potential of de novo designed cationic AMPs, along with their synergistic properties in combination with conventional antibiotics was examined. Furthermore, the effects of bacterial inoculum density and metabolic state on the antibacterial activity of AMPs were evaluated. Finally, the impact of several potent AMPs on E. coli cell envelope and genomic DNA integrity was determined. Collectively, this comprehensive analysis provides insights into the unique characteristics of cationic AMPs.

Reduced representative methylome profiling of cell-free DNA for breast cancer detection.

Whole-genome methylation sequencing of cfDNA is not cost-effective for tumor detection. Here, we introduce reduced representative methylome profiling (RRMP), which employs restriction enzyme for depletion of AT-rich sequence to achieve enrichment and deep sequencing of CG-rich sequences. We first verified the ability of RRMP to enrich CG-rich sequences using tumor cell genomic DNA and analyzed differential methylation regions between tumor cells and normal whole blood cells. We then analyzed cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals to detect breast cancer by building machine learning models. RRMP captured 81.9% CpG islands and 75.2% gene promoters when sequenced to 10 billion base pairs, with an enrichment efficiency being comparable to RRBS. RRMP allowed us to assess DNA methylation changes between tumor cells and whole blood cells. Applying our approach to cfDNA from 29 breast cancer patients and 27 non-breast cancer individuals, we developed machine learning models that could discriminate between breast cancer and non-breast cancer controls (AUC = 0.85), suggesting possibilities for truly non-invasive cancer detection. We developed a new method to achieve reduced representative methylome profiling of cell-free DNA for tumor detection.

Antibacterial mechanism of kojic acid and tea polyphenols against Escherichia coli O157:H7 through transcriptomic analysis

Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans. The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E. coli O157:H7 than using either alone. This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level. The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E. coli O157:H7 found that acid resistance systems in kojic acid were activated, and the cell membrane and genomic DNA were destructed in the cells, resulting in "oxygen starvation". The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP, which affected the bacteria's life metabolic process. Interestingly, we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA, respiration, tricarboxylic acid cycle. The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E. coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.

Antimicrobial activity of phenyllactic acid against Klebsiella pneumoniae and its effect on cell wall membrane and genomic DNA.

As Klebsiella pneumoniae (KP) has acquired high levels of resistance to multiple antibiotics, it is considered a worldwide pathogen of concern, and substitutes for traditional antibiotics are urgently needed. 3-Phenyllactic acid (PLA) has been reported to have antimicrobial activity against food-borne bacteria. However, there was no experiment evidence for the exact antibacterial effect and mechanism of PLA kills pathogenic KP. In this study, the Oxford cup method indicated that PLA is effective to KP with a minimum inhibitory concentration of 2.5 mg/mL. Furthermore, PLA inhibited the growth and biofilm formation of in a time- and concentration-dependent manner. In vivo, PLA could significantly increase the survival rate of infected mice and reduce the pathological tissue damage. The antibacterial mode of PLA against KP was further explored. Firstly, scanning electron microscopy illustrated the disruption of cellular ultrastructure caused by PLA. Secondly, measurement of leaked alkaline phosphatase demonstrated that PLA disrupted the cell wall integrity of KP and flow cytometry analysis with propidium iodide staining suggested that PLA damaged the cell membrane integrity. Finally, the results of fluorescence spectroscopy and agarose gel electrophoresis demonstrated that PLA bound to genomic DNA and initiated its degradation. The anti-KP mode of action of PLA was attributed to the destruction of the cell wall, membrane, and genomic DNA binding. These findings suggest that PLA has great potential applications as antibiotic substitutes in feed additives against KP infection in animals.

Effects of cuminaldehyde combined with mild heat against Salmonella enterica serovar Typhimurium in powdered infant formula

The study investigated the influence of cuminaldehyde (CUM) on cell membrane and genomic DNA of Salmonella enterica serovar Typhimurium (S. Typhimurium), and revealed its antibacterial mechanism. The minimum inhibitory concentration (MIC) of CUM for S. Typhimurium was determined to be 0.6 μL/mL. CUM could bind to bacterial membrane proteins and change its structure and conformation, leading to the leakage of intracellular substances and the destruction of cell integrity. DNA interaction analysis showed that CUM bound to genomic DNA through groove mode, affecting the normal functioning of S. Typhimurium. In addition, CUM at sub-MIC decreased bacterial motility, prevent cell attachment, restrain metabolic activity, and thus inhibiting S. Typhimurium biofilm formation. Furthermore, mild heat treatment combined with CUM at 1, 2 and 4 MIC could completely kill S. Typhimurium in powdered infant formula. Besides, the combination at 1 MIC of CUM had no obvious influence on the aroma of the powdered infant formula. The combined application of CUM has broad prospects in the control of food-borne pathogens.

Phenotypic Evaluation of TREX1 Null Mice as a Model for Lupus-Like Autoimmune Disease

Abstract TREX1 (DNase III) is the major mammalian 3’-5’ DNA exonuclease and thought to play a major role in cell death and genomic DNA degradation. Dysfunction of Trex1 has been suggested to activate immune response by self DNA, as TREX1 null mice develop inflammatory myocarditis similar to autoimmune cardiomyopathy and produce type 1 IFN. TREX1 D18N causes a monogenic cutaneous form of lupus called familial chilblain lupus and exhibits dysfunctioned dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune diseases. Using CRISPR-Cas9, we generated a Trex1 −/−mouse by knocking out exon 2 and performed comprehensive phenotypic analyses to evaluate the loss of function of TREX1 and its potential application in SLE research. Both female and male TREX1 null mice showed dramatically increased post-wean morbidity and significantly increased serum ALT, AST and LDH levels, compared to wildtype and heterozygotes from 6 to 24 weeks of age, indicating liver and heart injuries. Pathohistological analysis showed inflammatory immune cell infiltration in liver, heart, spleen, kidney, lung and skin among TREX1 null mice. Serum anti-dsDNA antibody levels were significantly increased and remained high from 7 to 25 weeks of age, consistent with Trex1’s being the major DNA exonuclease. Urine protein to creatinine ratio was dramatically increased at 28 weeks of age among TREX1 null mice. In conclusion, the Trex1 −/−mice we generated in this work showed lupus-like inflammatory autoimmune responses in multiple organs, providing an urgently needed tool to study Trex1-mediated autoimmunity and to evaluate potential therapeutic treatments for lupus.

A clinical prognostic model for patients with esophageal squamous cell carcinoma based on circulating tumor DNA mutation features.

Few predictive models have included circulating tumor DNA (ctDNA) indicators to predict prognosis of esophageal squamous cell carcinoma (ESCC) patients. Here, we aimed to explore whether ctDNA can be used as a predictive biomarker in nomogram models to predict the prognosis of patients with ESCC. We included 57 patients who underwent surgery and completed a 5-year follow-up. With next-generation sequencing, a 61-gene panel was used to evaluate plasma cell-free DNA and white blood cell genomic DNA from patients with ESCC. We analyzed the relationship between the mutation features of ctDNA and the prognosis of patients with ESCC, identified candidate risk predictors by Cox analysis, and developed nomogram models to predict the 2- and 5-year disease-free survival (DFS) and overall survival (OS). The area under the curve of the receiver operating characteristic (ROC) curve, concordance index (C-index), calibration plot, and integrated discrimination improvement (IDI) were used to evaluate the performance of the nomogram model. The model was compared with the traditional tumor-nodes-metastasis (TNM) staging system. The ROC curve showed that the average mutant allele frequency (MAF) of ctDNA variants and the number of ctDNA variants were potential biomarkers for predicting the prognosis of patients with ESCC. The predictors included in the models were common candidate predictors of ESCC, such as lymph node stage, angiolymphatic invasion, drinking history, and ctDNA characteristics. The calibration curve demonstrated consistency between the observed and predicted results. Moreover, our nomogram models showed clear prognostic superiority over the traditional TNM staging system (based on C-index, 2-year DFS: 0.82 vs. 0.64; 5-year DFS: 0.78 vs. 0.65; 2-year OS: 0.80 vs. 0.66; 5-year OS: 0.77 vs. 0.66; based on IDI, 2-year DFS: 0.33, p <0.001; 5-year DFS: 0.18, p = 0.04; 2-year OS: 0.28, p <0.001; 5-year OS: 0.15, p = 0.04). The comprehensive scores of the nomogram models could be used to stratify patients with ESCC. The novel nomogram incorporating ctDNA features may help predict the prognosis of patients with resectable ESCC. This model can potentially be used to guide the postoperative management of ESCC patients in the future, such as adjuvant therapy and follow-up.

Metabolic labelling of DNA in cells by means of the "photoclick" reaction triggered by visible light.

Two pyrene-tetrazole conjugates were synthesized as photoreactive chromophores that allow for the first time the combination of metabolic labelling of DNA in cells and subsequent bioorthogonal "photoclick" modification triggered by visible light. Two strained alkenes and three alkene-modified nucleosides were used as reactive counterparts and revealed no major differences in their "photoclick" reactivity. This is a significant advantage because it allows 5-vinyl-2'-deoxyuridine to be applied as the smallest possible alkene-modified nucleoside for metabolic labelling of DNA in cells. Both pyrene-tetrazole conjugates show fluorogenicity during the "photoclick" reactions, which is a second advantage for cellular imaging. Living HeLa cells were incubated with 5-vinyl-2'-deoxyuridine for 48 h to ensure one cell division. After fixation, the newly synthesized genomic DNA was successfully labelled by irradiation with visible light at 405 nm and 450 nm. This method is an attractive tool for the visualization of genomic DNA in cells with full spatiotemporal control by the use of visible light as a reaction trigger.

A Non-Canonical Teleost NK-Lysin: Antimicrobial Activity via Multiple Mechanisms.

NK-lysin (NKL) is a family of antimicrobial proteins with an important role in innate and adaptive immunity. In this study, a non-canonical NK-lysin (NKLnc) was identified in the Japanese flounder (Paralichthys olivaceus), which shares low sequence identities (15.8-20.6%) with previously reported fish NKLs and was phylogenetically separated from the canonical NKLs in teleost. NKLnc expression was upregulated in flounder tissues during bacterial infection, and interference with NKLnc expression impaired the ability of flounder cells to eliminate invading bacteria. When expressed in Escherichia coli, NKLnc was detrimental to the host cells. P35, a peptide derived from the saposin B domain (SapB) of NKLnc, bound major bacterial surface molecules and killed both Gram-negative and Gram-positive bacteria by inflicting damage to bacterial cell structure and genomic DNA. The bactericidal activity, but not the bacteria-binding capacity, of P35 required the structural integrity of the alpha 2/3 helices in SapB. Furthermore, P35 induced the migration of flounder peripheral blood leukocytes, inhibited bacterial dissemination in fish tissues, and facilitated fish survival after bacterial challenge. Together our study reveals that NKLnc plays an important part in flounder immune defense, and that NKLnc peptide exerts an antimicrobial effect via multiple mechanisms by targeting both bacteria and fish cells.

Antimicrobial activities and mechanism of sturgeon spermary protein extracts against Escherichia coli.

This study aimed to evaluate the antimicrobial activities and mechanism of sturgeon spermary protein extracts (SSPE) against Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Cell structural change was analyzed using scanning electron microscopy-energy dispersive X-ray spectrometry and transmission electron microscope. Moreover, pH, zeta potential, membrane potential, intracellular ATP concentrations and the interaction of SSPE with genomic DNA were analyzed. Results showed that molecular weight of SSPE is 13.4 kDa, the content of basic amino acids is the highest, in which arginine accounts for 73.2%. The MIC and MBC of SSPE for E. coli were 0.05 and 5 mg/mL, respectively. After SSPE treatment, cell membrane permeability changes, zeta potential decrease and genomic DNA lysis occurred in E. coli, which indicated it exerted bacteriostatic effects either independently or simultaneously by destroying the cell membrane and genomic DNA. These findings indicated that SSPE has potential to be a natural antiseptic.

MiR-205 Regulates LRRK2 Expression in Dopamine Neurons in Parkinson's Disease through Methylation Modification.

Background:We explored the methylation modification in miR-205 promoter during the pathological changes of Parkinson’s disease (PD) and its regulation on Leucine-Rich Repeat Kinase 2 (LRRK2), clarified the important role of methylation in miR-205 promoter region in PD, explained the role of miR-205 methylation in the pathological changes of PD, and looked for new targets for PD.Methods:Methylation of miR-205 promoter regions was determined by cell genomic DNA, with model bisulfite treatment, and the transcription of miR-205 and LRRK2 in PD model cells was determined by qPCR, and LRRK2 expression was determined by Western blot. The binding sites of miRNAs in the non-coding region of LRRK2 were analyzed by the targetscan database, and miR-205 expression in 293T cells was controlled. The correlation between miR-205 expression and LRRK2 was determined to clarify the regulation mode of miR-205 on LRRK2.Results:The level of miR-205 were reduced in the SH-SY5Y Parkinson model cells, and its promoter region was highly methylated, while LRRK2 expression decreased in the model cells after 5-Azacytidine inhibition of methylation in miR-205 promoter region. According to the target scan database analysis, LRRK2 non-coding region is a miR-205-specific binding site. After further miR-205 overexpression in 293T cells, the transcription and translation of LRRK2 decreased in cells, which increased after the treatment of miR-205 inhibitor on LRRK2.Conclusion:The methylation modification of miR-205 promoter region could regulate the transcription and translation of LRRK2 in dopaminergic neurons, so miR-205 methylation regulation can serve as a new potential target for the treatment of PD.

Perinatal hypoxia aggravates occlusive pulmonary vasculopathy in SU5416/hypoxia-treated rats later in life.

Pulmonary arterial hypertension (PAH) is a fatal disease, which is characterized by occlusive pulmonary vascular disease (PVD) in small pulmonary arteries. It remains unknown whether perinatal insults aggravate occlusive PVD later in life. We tested the hypothesis that perinatal hypoxia aggravates PVD and survival in rats. PVD was induced in rats with/without perinatal hypoxia (embryonic day 14 to postnatal day 3) by injecting SU5416 at 7 wk of age and subsequent exposure to hypoxia for 3 wk (SU5416/hypoxia). Hemodynamic and morphological analyses were performed in rats with/without perinatal hypoxia at 7 wk of age (baseline rats, n = 12) and at 15 wk of age in 4 groups of rats: SU5416/hypoxia or control rats with/without perinatal hypoxia (n = 40). Pulmonary artery smooth muscle cells (PASMCs) from the baseline rats with/without perinatal hypoxia were used to assess cell proliferation, inflammation, and genomic DNA methylation profile. Although perinatal hypoxia alone did not affect survival, physiological, or pathological parameters at baseline or at the end of the experimental period in controls, perinatal hypoxia decreased weight gain and survival rate and increased right ventricular systolic pressure, right ventricular hypertrophy, and indices of PVD in SU5416/hypoxia rats. Perinatal hypoxia alone accelerated the proliferation and inflammation of cultured PASMCs from baseline rats, which was associated with DNA methylation. In conclusion, we established the first fatal animal model of PAH with worsening hemodynamics and occlusive PVD elicited by perinatal hypoxia, which was associated with hyperproliferative, proinflammatory, and epigenetic changes in cultured PASMCs. These findings provide insights into the treatment and prevention of occlusive PVD.

Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro

Abstract Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic. Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 13.

Clonal hematopoiesis in lung cancer and its impact on liquid biopsy based on targeted deep sequencing.

e20532 Background: Blood-based liquid biopsy has proved its potential in enormous clinical applications, such as cancer screening, diagnosis, treatment guidance, disease tracking and monitoring. In certain scenarios (e.g., molecular residual disease), it requires the technique to be able to detect mutation with very low frequency (0.001% ̃ 1%). The major hurdle of ultra-sensitive circulating tumor DNA sequencing is the high background noise of plasma cell-free DNA (cfDNA) and clonal hematopoiesis (CH). Here in this study, we investigated the prevalence of CH in lung cancer patients and its interference with liquid biopsy. Methods: We retrospectively analyzed cfDNA and blood cell genomic DNA (gDNA) sequencing data sets (n = 1,261) from a group of Chinese lung cancer patients. Threshold (1%) and subthreshold (0.2%) for variant allele frequency were set and compared. We focused on 23 clonal hematopoiesis genes that were selected based on previous publications. Results: CH mutations were detected in 27.68% of all the patients at the threshold and 62.01% at the subthreshold, and the detection rate increased with age. DNMT3A was the most frequently mutated CH gene, accounting for more than half of the CH mutations. The CH mutations had a higher detection rate in smokers (72%) than non-smokers (59.4%) at subthreshold. VAFs of CH mutations in cell-free DNA strongly correlated with their VAFs in gDNA (Pearson’s R = 0.92, p &lt; 2.2x10-16), while tumor-derived somatic mutations didn’t have such correlation. Conclusions: Our study showed that clonal hematopoiesis is very common in lung cancer patients, especially when examining low-frequency mutations. Sequencing of gDNA at equivalent depth is very important to filter out CH mutation in cancer liquid biopsy.

The prevalence of HPV infection in Chinese solid tumor patients.

e22519 Background: Human papillomavirus (HPV) is a family of small double-stranded DNA viruses. It is associated with a series of cancer types, such as cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. In the world, nearly 70% of HPV-related cancer cases are HPV types 16 and 18. In this study, we investigated HPV infection prevalence among a cohort of Chinese solid tumor patients. Methods: The study enrolled thousands of Chinese patients with different types of cancers. We finally collected 4,606 tumor tissue DNA (ttDNA), 7,296 plasma cell-free DNA (cfDNA), and 7,509 peripheral blood mononuclear cell genomic DNA (gDNA) samples, respectively. Using a targeted capture technique that covering the whole reference genome of HPV16 and HPV18 and the next generation sequencing (NGS) technology, we calculated the percentage of HPV16 and HPV 18 reads in all sequencing reads for each sample. Finally, we explored the prevalence of HPV infection in each cancer type. Results: Based on different types of DNA samples, the HPV infection rate was 38.75%, 28.62%, and 10.43% in ttDNA, cfDNA, and gDNA samples, respectively. Besides, the magnitudes of the mean percentage of HPV reads in all the sequencing reads in ttDNA, cfDNA and gDNA samples were 10-5, 10-6 and 10-8, respectively, while in the HPV infection positive the percentages of ttDNA, cfDNA, and gDNA samples increased to 10-4, 10-5, and 10-7, respectively. The prevalence of HPV infection rate in different cancer types was shown in table. Conclusions: ttDNA and cfDNA are better material for HPV infective detection compared with gDNA, and NGS is capable of HPV detection.[Table: see text]

Changes in Higher-Order Chromosomal Structure of Klebsiella pneumoniae Under Simulated Microgravity.

Our previous work have shown that certain subpopulations of Klebsiella pneumoniae exhibit significant phenotypic changes under simulated microgravity (SMG), including enhanced biofilm formation and cellulose synthesis, which may be evoked by changes in gene expression patterns. It is well known that prokaryotic cells genomic DNA can be hierarchically organized into different higher-order three-dimensional structures, which can highly influence gene expression. It is remain elusive whether phenotypic changes induced by SMG in the subpopulations of K. pneumoniae are driven by genome higher-order structural changes. Here, we investigated the above-mentioned issue using the wild-type (WT) K. pneumoniae (WT was used as a control strain and continuously cultivated for 2 weeks under standard culture conditions of normal gravity) and two previous identified subpopulations (M1 and M2) obtained after 2 weeks of continuous incubation in a SMG device. By the combination of genome-wide chromosome conformation capture (Hi-C), RNA-seq and whole-genome methylation (WGS) analyses, we found that the along with the global chromosome interactions change, the compacting extent of M1, M2 subpopulations were much looser under SMG and even with an increase in active, open chromosome regions. In addition, transcriptome data showed that most differentially expressed genes (DEGs) were upregulated, whereas a few DEGs were downregulated in M1 and M2. The functions of both types DEGs were mainly associated with membrane fractions. Additionally, WGS analysis revealed that methylation levels were lower in M1 and M2. Using combined analysis of multi-omics data, we discovered that most upregulated DEGs were significantly enriched in the boundary regions of the variable chromosomal interaction domains (CIDs), in which genes regulating biofilm formation were mainly located. These results suggest that K. pneumoniae may regulate gene expression patterns through DNA methylation and changes in genome structure, thus resulting in new phenotypes in response to altered gravity.

First Report of Root-Knot Nematode Meloidogyne enterolobii onCockscomb (Celosia argentea var. cristata) in Taiwan.

Cockscomb (Celosia argentea) is commonly found in subtropical and temperate zones of Africa, South America and South East Asia, and is a popular ornamental plant in the family Amaranthaceae. Cockscomb has been known to contain antiviral proteins, betalains, and anthocyanin, which can be applied in beneficial ways (2). In September 2020, a cockscomb plant (Celosia argentea var. cristata) showing typical galling root symptoms likely infected by root-knot nematodes (Meloidogyne sp.) was collected from a garden in Taichung, Taiwan, and a quick exam of several individuals using MK7F/R primers (7) indicating they were M. enterolobii. Nematode population was established from a single egg mass and was later used for species identification and pathogenicity tests. Five perineal patterns of mature females from the single female population show round to oval shapes with weak lateral lines. Dorsal arches are moderate to high, almost squared, with the smooth ventral striae. Second-stage juveniles are vermiform and have a slender tail, tapering to rounded tip with distinct hyaline region at the tail terminus. Morphological measurements of 28 J2s revealed body length = 457.2 ± 20.6 (416.1-506.9) μm, body width = 16.0 ± 2.0 (13.4-20.3) μm, stylet length = 14.7 ± 0.5 (13.9-15.9) μm, dorsal gland orifice to the stylet base = 4.0 ± 0.5 (2.0-4.8) μm, and tail length = 56.0 ± 3.8 (47.4-60.3) μm. Female perineal patterns and morphometric data are similar to the original description of Meloidogyne enterolobii (9). DNA purified from approximately 1500 juveniles using GeneMark Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan) was used to amplify 18S rDNA fragment, D2-D3 expansion segments of 28S rDNA, and a COII region on mtDNA with primer sets 1A/MelR, D2A/D3B, and C2F3/1108, respectively (4,5,6). The 18S rDNA sequence (OK076893) of this study shares 99.94% nucleotide identity with those of M. enterolobii isolated from the United States (KP901058) and China (MN832688). D2D3 sequence of haplotype 1 (OK076898) shows 100% identity to those of M. enterolobii from China (MT193450) and Taiwan (KP411230). Sequence of haplotype 2 (OK076899) shows 99.86% identity to those of M. enterolobii from the United States (MN809527) and China (MN269945). Sequence of the COII region (OK086042) show 99.86% identity to that of M. enterolobii from China (MN269945). Phylogenetic trees of the three gene sequences were plotted following Ye et al.(10), revealing that the newly described root-knot nematode on Cockscomb is grouped with other M. enterolobii isolates. DNA fragment amplified by primer sets Me-F/R(3) and MK7F/R specifically targeting of M. enterolobii yielded 236 bp and 520 bp, respectively. Pathogenicity tests were assayed, from July to September 2021, on three-week-old nematode-free cockscomb plant directly germinated from seeds of SkyStar® (ASUSA SPIKE SEEDS, Taipei, Taiwan) planted in a 10.5 cm diameter pot filled with 600 ml sterilized peat moss: sand (1:1, v/v) soil in a 28℃walk-in chamber. Nematode eggs were extracted using 0.05% NaoCl as described by Vrain(8), and cockscomb plants (n=3) were inoculated by adding 6000 eggs (10 eggs/ cm3). Cockscomb plants treated with water were used as mock controls. Rf value of the inoculated plants were determined by the method of Belair and Benoit (1) 45 days after inoculation, and the average was 4.13. No galls were observed on the roots of control plants. The results confirmed that cockscomb is the new host of M. enterolobii. To the best of our knowledge, this is the first report of M. enterolobii on Celosia argentea var. cristata in Taiwan.