Similar Profiles Research Articles

DNA Methylation Profiles Are Stable in H3 K27M-Mutant Diffuse Midline Glioma Neurosphere Cell Lines.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but the degree to which such cultures retain the characteristics of the source tumors is unknown. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided a comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

Autistic and non-autistic transgender youth are similar in gender development and sexuality phenotypes.

Increasing rhetoric regarding the common intersection of autism and gender diversity has resulted in legislation banning autistic transgender youth from accessing standard of care supports, as well as legislative efforts banning all youth gender care in part justified by the proportional over-occurrence of autism. Yet, no study has investigated whether autistic and non-autistic transgender youth present fundamentally different gender-related phenotypes. To address this gap, we extensively characterized autism, gender diversity, and sexuality among autistic and non-autistic transgender binary youth (N = 66, Mage = 17.17, SDage = 2.12) in order to investigate similarities and/or differences in gender and sexuality phenotypes. Neither autism diagnostic status nor continuous autistic traits were significantly related to any gender or sexuality phenotypes. These findings suggest that the developmental and experiential features of gender diversity are very similar between autistic and non-autistic transgender adolescents. Future research is needed to determine whether the similarity in profiles is maintained over time into adulthood.

Clinical Characteristics and Compliance of Diabetic Macular Edema Patients to Intravitreal Anti-VEGF Injection Loading Dose at Cipto Mangunkusumo Hospital

Introduction & ObjectivesDiabetic retinopathy is the fifth most common cause of preventable blindness. In cases of diabeticmacular edema (DME), loading dose is a critical phase to achieve a stable concentration ofintravitreal vascular endothelial growth factor (VEGF) in order to prevent progressivity. This studyaims to determine the demographic and clinical characteristics, waiting time, compliance, as well asthe relationship between education, domicile and clinical outcomes to compliance during loadingdose in DME patients. MethodsIn this retrospective descriptive study, we reviewed medical records of DME patients who receivedanti-VEGF intravitreal injection between 1 January 2019 and 31 December 2020, while alsocomparing records in 2019 (pre-Covid-19) and 2020 (during Covid-19). ResultsThere were 160 patients (207 eyeballs) in 2019 and 83 patients (114 eyeballs) in 2020 who receivedloading dose with similar profiles of sex, age, education, initial visual acuity and central macularthickness (CMT). Median waiting times in 2019 and 2020 were 19 days and 23.5 days, respectively(p=0.016). Only 25 (30.5%) and 15 (35.7%) patients were punctual in 2019 and 2020, respectively.There was no significant relationship between education and domicile with compliance (p= 0.45, p=0.51). Furthermore, there were visual acuity improvements of -0.18 logMAR and -0.0 logMAR in thepunctual and unpunctual group, respectively, as well as CMT decrease of -181 ?m and -156 ?m,respectively. ConclusionMost patients who received loading dose were unpunctual. There was no significant relationshipbetween education and domicile with compliance. Furthermore, there were more significantimprovements in clinical outcomes among the punctual patients.

Single-cell ‘omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Single-cell 'omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations.

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Developing defined starter culture for reproducible profile of flavour compound in Chinese xiaoqu baijiu fermentation

Defined starter cultures, containing selected microbes could reduce the complexity of natural starter, are beneficial for controllable food fermentations. However, there are challenges in identifying key microbiota and constructing synthetic microbiota for traditional food fermentations. Here, we aimed to develop a defined starter culture for reproducible profile of flavour compounds, using Chinese Xiaoqu Baijiu fermentation as a case. We classified all microbes into 4 modules using weighted correlation network analysis. Module 3 presented significant correlations with flavour compounds (P < 0.05) and the highest gene abundance related with flavour compound production. 13 dominant species in module 3 were selected for mixed culture fermentation, and each species was individually deleted to analyse the effect on flavour compound production. Ten species, presenting significant effects (P < 0.05) on flavour compound production, were selected for developing the starter culture, including Rhizopus oryzae, Rhizopus microsporus, Saccharomyces cerevisiae, Pichia kudriavzevii, Wickerhamomyces anomalus, Lactobacillus acetotolerans, Levilactobacillus brevis, Weissella paramesenteroides, Pediococcus acidilactici, and Leuconostoc pseudomesenteroides. After optimising the structure of the starter culture, the profile similarity of flavour compounds produced by the starter culture reached 81.88% with that by the natural starter. This work indicated feasibility of reproducible profile of flavour compounds with defined starter culture for food fermentations.

Draft genome sequence and tissue expression panel of Pacific saury (Cololabis saira).

Pacific saury (Cololabis saira) is an important fish in several countries. Notably, the catch of this fish has markedly decreased recently, which might be due to environmental changes, including feeding habitat changes. However, no clear correlation has been observed. Therefore, the physiological basis of Pacific saury in relation to possible environmental factors must be understood. We sequenced the genome of Pacific saury and extracted RNA from nine tissues (brain, eye, gill, anterior/posterior guts, kidney, liver, muscle, and ovary). In 1.09 Gb assembled genome sequences, a total of 26,775 protein-coding genes were predicted, of which 26,241 genes were similar to known genes in a public database. Transcriptome analysis revealed that 24,254 genes were expressed in at least one of the nine tissues, and 7,495 were highly expressed in specific tissues. Based on the similarity of the expression profiles to those of model organisms, the transcriptome obtained was validated to reflect the characteristics of each tissue. Thus, the present genomic and transcriptomic data serve as useful resources for molecular studies on Pacific saury. In particular, we emphasize that the gene expression data, which serve as the tissue expression panel of this species, can be employed in comparative transcriptomics on marine environmental responses.

A Cone Beam CT Bronchoscopy Study of the Ultrathin Cryoprobe for Biopsy of Peripheral Lung Lesions.

Compared with the standard cryoprobe, the novel ultrathin 1.1mm cryoprobe (UTCP) has improved ergonomics, shape memory, and flexibility. The performance of UTCP has demonstrated promising results in several small trials. In this single-center, retrospective review, we examine 200 (N=200) consecutive patients referred for cone beam CT bronchoscopic biopsy of peripheral lung lesions. We utilized an extended multimodality approach, including transbronchial needle aspirate, brush, traditional forces biopsies, UTCP biopsies, and BAL. We analyzed tool in lesion, tool touch lesion, center strike rates, and diagnostic yield. We assessed for molecular adequacy and analyzed safety. A total of 222 lesions were biopsied. We achieved a tool in lesion or tool touch lesion confirmation for all biopsy attempts (100%) and a center strike rate of 68%. AQuIRE diagnostic yield was 90%, with 60% malignant, 30% benign lung nodules, and 10% nondiagnostic. UTCP was diagnostic in 3.6 % of peripheral lung lesions biopsies when all other modalities were nondiagnostic; thus, raising our overall diagnostic yield from 86.4% to 90.1%. Our analysis demonstrates superior adequacy for molecular analysis for histologic samples (TBBX or UTCP) versus cytologic samples (FNA) ( P <0.001). Three patients (1.5%) had a pneumothorax, and 1 patient (0.5%) had moderate bleeding. UTCP was diagnostic in 3.6% of peripheral lung lesions when all other modalities were nondiagnostic. In the setting of CBCT guidance, UTCP has a similar safety profile to standard biopsy tools. Future trials are warranted to assess UTCP and its impact on peripheral lung lesion biopsies.

High Tidal Volume, High Positive End Expiratory Pressure and Apneic Breath Hold Strategies (Lung Navigation Ventilation Protocol) With Cone Beam Computed Tomography Bronchoscopic Biopsy of Peripheral Lung Lesions: Results in 100 Patients.

A dedicated anesthesia protocol for bronchoscopic lung biopsy-lung navigation ventilation protocol (LNVP)-specifically designed to mitigate atelectasis and reduce unnecessary respiratory motion, has been recently described. LNVP demonstrated significantly reduced dependent ground glass, sublobar/lobar atelectasis, and atelectasis obscuring target lesions compared with conventional ventilation. In this retrospective, single-center study, we examine the impact of LNVP on 100 consecutive patients during peripheral lung lesion biopsy. We report the incidence of atelectasis using cone beam computed tomography imaging, observed ventilatory findings, anesthesia medications, and outcomes, including diagnostic yield, radiation exposure, and complications. Atelectasis was observed in a minority of subjects: ground glass opacity atelectasis was seen in 30 patients by reader 1 (28%) and in 18 patients by reader 2 (17%), with good agreement between readers (κ = 0.78). Sublobar/lobar atelectasis was observed in 23 patients by reader 1 and 26 patients by reader 2, also demonstrating good agreement (κ = 0.67). Atelectasis obscured target lesions in very few cases: 0 patients (0%, reader 1) and 3 patients (3%, reader 2). Diagnostic yield was 85.9% based on the AQuIRE definition. Pathology demonstrated 57 of 106 lesions (54%) were malignant, 34 lesions (32%) were benign, and 15 lesions (14%) were nondiagnostic. Cone beam computed tomography images confirmed low rates of atelectasis, high tool-in-lesion confirmation rate, and high diagnostic yield. LNVP has a similar safety profile to conventional bronchoscopy. Most patients will require intravenous fluid and vasopressor support. Further study of LNVP and other ventilation protocols are necessary to understand the impact of ventilation protocols on bronchoscopic peripheral lung biopsy.

Adaptive-to-maladaptive gradient of emotion regulation tendencies are embedded in the functional-structural hybrid connectome.

Emotion regulation tendencies are well-known transdiagnostic markers of psychopathology, but their neurobiological foundations have mostly been examined within the theoretical framework of cortical-subcortical interactions. We explored the connectome-wide neural correlates of emotion regulation tendencies using functional and diffusion magnetic resonance images of healthy young adults (N = 99; age 20-30; 28 females). We first tested the importance of considering both the functional and structural connectome through intersubject representational similarity analyses. Then, we employed a canonical correlation analysis between the functional-structural hybrid connectome and 23 emotion regulation strategies. Lastly, we sought to externally validate the results on a transdiagnostic adolescent sample (N = 93; age 11-19; 34 females). First, interindividual similarity of emotion regulation profiles was significantly correlated with interindividual similarity of the functional-structural hybrid connectome, more so than either the functional or structural connectome. Canonical correlation analysis revealed that an adaptive-to-maladaptive gradient of emotion regulation tendencies mapped onto a specific configuration of covariance within the functional-structural hybrid connectome, which primarily involved functional connections in the motor network and the visual networks as well as structural connections in the default mode network and the subcortical-cerebellar network. In the transdiagnostic adolescent dataset, stronger functional signatures of the found network were associated with higher general positive affect through more frequent use of adaptive coping strategies. Taken together, our study illustrates a gradient of emotion regulation tendencies that is best captured when simultaneously considering the functional and structural connections across the whole brain.

Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor

The manipulation of cell identity by reprograming holds immense potential in regenerative medicine, but is often limited by the inefficient acquisition of fully functional cells. This problem can potentially be resolved by better understanding the reprogramming process using in vivo genetic models, which are currently scarce. Here we report that both enterocytes (ECs) and enteroendocrine cells (EEs) in adult Drosophila midgut show a surprising degree of cell plasticity. Depleting the transcription factor Tramtrack in the differentiated ECs can initiate Prospero-mediated cell transdifferentiation, leading to EE-like cells. On the other hand, depletion of Prospero in the differentiated EEs can lead to the loss of EE-specific transcription programs and the gain of intestinal progenitor cell identity, allowing cell cycle re-entry or differentiation into ECs. We find that intestinal progenitor cells, ECs, and EEs have a similar chromatin accessibility profile, supporting the concept that cell plasticity is enabled by pre-existing chromatin accessibility with switchable transcription programs. Further genetic analysis with this system reveals that the NuRD chromatin remodeling complex, cell lineage confliction, and age act as barriers to EC-to-EE transdifferentiation. The establishment of this genetically tractable in vivo model should facilitate mechanistic investigation of cell plasticity at the molecular and genetic level.

A simple analytical model for turbulent kinetic energy dissipation for self-similar round turbulent jets

This work presents a simple analytical model for the streamwise and radial variations of turbulent kinetic energy dissipation in an incompressible round turbulent jet. The key assumptions in the model are: similarity in the axial velocity profile with a Gaussian shape, axisymmetric flow and the dominance of radial derivatives of the mean velocity over axial direction derivatives (similar to boundary layer theory). Initially, a simplified eddy-viscosity relation for turbulent stresses is derived using the algebraic stress model by Gatski &amp; Speziale (J. Fluid Mech., vol. 254, 1993, pp. 59–78). Subsequently, with this eddy-viscosity relation, the relation for variations of turbulent kinetic energy dissipation is formulated using the conservation of turbulent kinetic energy. To extract the necessary constants of the model, experimental velocity statistics for round jets are obtained through particle image velocimetry measurements. The experimental results of the mean entrainment coefficient for turbulent jets are also analysed. When comparing the radial variation of turbulent kinetic energy dissipation from the model with experimental results at Reynolds number $1.4\times 10^5$ and numerical results at Reynolds number $1200$ from the available literature, we observe a maximum error of $10\,\%$ and $15\,\%$ , respectively. Finally, using the validated model, we analyse the impact of mean velocity evolution parameters on the behaviour of turbulent kinetic energy dissipation and discuss its potential significance in future studies.

Abstract 2853: Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions

Abstract Introduction High grade hemispheric gliomas in infancy (IHG) are characterized by a more favorable outcome compared to high grade gliomas in older children. We have shown in previous studies that these tumors are characterized by a stable genome. The occurrence of tyrosine kinase gene fusions in high-grade hemispheric gliomas of infancy may represent therapeutic targets. Methods: 33 high-grade hemispheric gliomas occurring in children younger than three years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, University of Bonn. DNA and RNA were extracted from FFPE tumor samples. Methylation-based classification was done by hybridization to Illumina methylation arrays and subsequent use of the Heidelberg brain tumor classifier versions v11b4, v12.5 and v12.8. In addition UMAP plots were used to visualize similarities of methylation profiles. Gene fusions were identified on the DNA level by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET and Molecular Inversion Probe (MIP) methodology. On the RNA level, fusion transcripts were detected by targeted RNA sequencing as well as Nanostring assay with fusion-specific probes. Results: 32 IHG occurred in the first year of life, one at the age of two years. 23/33 IHG were classified as such by methylation arrays; 6 further cases could be annotated to the IHG group by UMAP plots. 18/33 cases (54.5 %) showed fusions of ALK to different partners; all occurred in the first year of life. Fusions of ROS1 were found in 4, MET in 2, NTRK1 and -3 in 7 cases. 2 cases showed no fusions. The different methods for fusion detection led to comparable results. Only recurrent fusions with known fusion partners were detectable with Nanostring probes designed for the fusion sequence, and library construction for targeted RNA sequencing was not successful in a fraction of cases. Break-apart FISH led to reliable results on the next day, and MIP technology represented the most sensitive method for analysis of FFPE samples. Conclusions: Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1 and -3 occurred in 93 % of high grade hemispheric gliomas of infancy; the most frequent were ALK fusions occurring in children younger than 1 year. DNA-based MIP technology represented the most robust and sensitive assay. A combination of RNA- and DNA-based methods to detect these fusions with high reliability is recommended. Citation Format: Torsten Pietsch, Gerrit Gielen, Andreas Waha, Evelyn Dörner, Verena Dreschmann, Christof Kramm, Christian Vokuhl. Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions [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 2853.

Abstract 619: Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses

Abstract Dysregulation of the Mitogen-Activated Protein Kinase (MAPK) pathway is a frequent event in many cancers. The RAF family of kinases, which includes ARAF, BRAF, and CRAF (RAF1), plays a key role in MAPK signaling. Of the three isoforms, only BRAF is currently targeted by approved RAF inhibitors. These inhibitors were designed to target BRAF-mutant variants that act as oncogenic monomers. However, BRAF-monomer inhibitors can induce activation of wild-type RAF by the formation of dimers, resulting in hyperproliferative lesions of BRAF wild-type cells. Additionally, BRAF-monomer inhibitors do not target RAF-dimers, although this is a potential vulnerability of many MAPK-driven cancers. Novel inhibitors are in development to circumvent these limitations. Further insights into the precise targeting and mechanism of action of RAF inhibitors can help in determining the most promising avenues for drug development. We performed extensive cellular profiling experiments with three approved and five clinical stage inhibitors of RAF to gain more insight into the differences and cellular targeting of these agents. The inhibitors were tested in cell viability assays on a panel of 130 human cancer cell lines from diverse tissue origins. Cell lines were exposed to dose ranges of each inhibitor spanning four logarithmic orders, and viability was determined by measurement of intracellular ATP. Effects of compound on cell viability were related to genomic, transcriptomic, and proteomic status using bioinformatics, with the aim to identify predictive biomarkers for drug response, and to determine selective targeting of MAPK-driven cell lines. Additionally, the primary mechanism of cellular inhibitor activity was determined by relating cell line responses to gene dependency data from large CRISPR knockout screens. Our results showed striking similarity in the overall inhibitory profile of BRAF-monomer inhibitors and the vemurafenib-derivative and paradox breaker plixorafenib, which all selectively targeted BRAF-mutant cell lines. However, whereas vemurafenib increased cell viability in RAS-mutant cell lines at high drug concentrations, this effect was greatly reduced for plixorafenib. Western blot analysis of phosphorylated MEK and ERK levels in RAS-mutant cell lines treated with BRAF-monomer inhibitors confirmed MAPK pathway activation as cause of the increased cell viability. In addition, RAF-dimer inhibitors potently targeted BRAF-mutant cell lines, as well as several KRAS- and NRAS-mutant cell lines. Lastly, correlation analyses of cell line profiling data with CRISPR knockout data revealed the preferential targeting of RAF1-dependent cell lines by the dual RAF/MEK inhibitor avutometinib. The results of our analyses shed light on the intricacies of cellular targeting by RAF inhibitors and provide insights to guide the development of new RAF inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Sjoerd B. van der Leeuw, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Nicole Willemsen-Seegers, Guido J. Zaman. Comprehensive cell line profiling of monomer- and dimer-selective small molecule RAF inhibitors uncovers determinants of cellular responses [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 619.

Abstract 4920: A nanopore sequencing approach characterizes cell-free DNA methylation-fragmentomics profiles indicative of breast cancer in a large cohort

Abstract The detection of breast cancer from cell-free DNA (cfDNA) offers a new approach with the potential to benefit individual screening and early detection. However, identifying breast cancer from cfDNA remains a major challenge when one uses conventional short-read sequencing. Methylation and DNA fragmentation characteristics provide highly informative biomarkers for detecting and monitoring cancer. To characterize these genomic features from cfDNA, we developed an approach using single molecule Nanopore sequencing. Single cfDNA molecules are sequenced intact with the methylation and fragment size directly extrapolated from Nanopore data. Moreover, our approach requires small amounts of cfDNA to discern specific genomic features from individual molecules. Thus, the use of Nanopore sequencing provides both methylation and fragment size as directly measured from native cfDNA without the use of any chemical processing or other molecular manipulation. We studied an extended breast cancer cohort with in total 1080 of samples, consisting of 440 untreated breast cancer patients and 640 healthy controls. Nanopore sequencing analysis of these cfDNA samples identified a range of 1 to 4 million CpG sites from 1 ml of plasma. Leveraging the cohort, we developed an ensemble classifier for breast cancer detection based on cfDNA methylation and fragmentation. The classifier leveraged a leave-one-out cross-validation (LOOCV) to generate predicted probability. We created nucleosome-specific methylation scores as the input features to the classifier, which scores quantified the similarity of each sample's methylation profile to a training set on a selected set of CpG sites associated with cancers. Furthermore, we also identified DNA fragmentation features, revealing distinct mono-nucleosome and di-nucleosome patterns that distinguish cancers from healthy controls. Aggregating the DNA nucleosome-specific scores and fragmentation features, we built an ensemble LOOCV random forest model. We applied the model to the breast cancer cohort and achieved a detection specificity of 74%, a sensitivity of 71%, and an overall accuracy of approximately 73%. The area under the receiver operating characteristic curve (AUROC) reached 80%. We validated the classifier with two external independent cohorts. Validation cohort 1 demonstrated as a specificity of 85%, a sensitivity of 80%, and overall accuracy of 83% with AUROC of 90%. Validation cohort 2 without controls demonstrated that the classifier achieved a sensitivity of 94%. Both validation sets confirm that our model can achieve excellent performance for early breast cancer detection. In summary, our findings demonstrate that nanopore sequencing provides cfDNA methylation and fragment profiling that enables breast cancer detection. This approach holds the potential to improve cancer diagnosis and treatment outcomes. Citation Format: Xiangqi Bai, Billy T. Lau, Alison Almada, Sue Grimes, Tianqi Chen, Hojoon Lee, Hanlee P. Ji. A nanopore sequencing approach characterizes cell-free DNA methylation-fragmentomics profiles indicative of breast cancer in a large cohort [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 4920.

Abstract 6335: Armoring CAR-T therapy with PD-1 blockade: a powerful strategy for enhancing anti-tumor effects in pancreatic cancer

Abstract Chimeric antigen receptor-modified T cells (CAR-T) have been widely applied in the treatment of hematological malignancies and are currently under investigation for their potential effectiveness against solid tumors. Nevertheless, the effectiveness of CAR-T in treating solid tumors remains unsatisfactory due to a range of factors, including CAR-T cell infiltration and heterogeneity. The efficacy of CAR-T therapy is substantially influenced by the tumor microenvironment. This is particularly evident when PD-L1 is activated through CAR-T, leading to the inhibition of CAR-T cell activity via the PD-1 pathway. In this study, we engineered a CAR-T therapy that targets mesothelin and concurrently expresses a membrane-bound PD-1 receptor. This design blocks the PD-1 on CAR-T cells, effectively counteracting the suppression of CAR-T function mediated by PD-L1. We observed a substantial upregulation of PD-L1 on AsPC-1 tumor cells after 4 hours of IFN-γ stimulation in vitro. CAR-T cells engineered to co-express PD-1 antibody (aMSLN-28BBZ-mPD-1) exhibited similar short-term cytotoxicity and cytokine secretion profiles to those of the traditional third-generation CAR-T (aMSLN-28BBZ). Notably, aMSLN-28BBZ-mPD-1 displayed significantly improved long-term target cell killing. Furthermore, in an in vivo model of pancreatic cancer, aMSLN-28BBZ-mPD-1 demonstrated enhanced anti-tumor activity when compared to the traditional aMSLN-28BBZ, as supported by RNAscope analysis and immunofluorescence assays of tumor tissues. These analyses revealed a marked upregulation of PD-L1 expression and enhanced CAR-T cell infiltration in the aMSLN-28BBZ-mPD-1 treatment group. In summary, our study demonstrates that co-expression of a membrane-bound PD-1 antibody overcomes PD-L1 mediated CAR-T suppression, as observed in both in vitro and in vivo. This innovative strategy underscores its therapeutic potential and value in treating solid tumors. Keywords: CAR-T, PD-1, Tumor Microenvironment, Pancreatic Cancer Citation Format: Zhixiang Zhang, Jingwei Huang, Zhifeng Zhao, Xiangyi Wang, Cnhui he, Sen Yang, Qingyang Gu. Armoring CAR-T therapy with PD-1 blockade: a powerful strategy for enhancing anti-tumor effects in pancreatic 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 6335.

Mother adversity and co-residence time impact mother–child similarity in genome-wide and gene-specific methylation profiles

BackgroundThe effects of adverse life events on physical and psychological health, with DNA methylation (DNAm) as a critical underlying mechanism, have been extensively studied. However, the epigenetic resemblance between mother and child in the context of neglectful caregiving, and whether it may be shaped by the emotional impact of maternal stressful events and the duration of co-residence (indexed by child age), remains unknown. The present study examined mother–child similarity in methylation profiles, considering the potential effect of mother adversity, mother empathy, neglect-control group, child age (an index of years of mother–child co-residence), and mother age. Using Illumina Epic arrays, we quantified DNAm in 115 mother–child saliva samples. We obtained a methylation similarity index by computing correlation coefficients between methylation profiles within dyads, for the entire epigenome, and five specific genes related to stress and empathy: NR3C1, FKPB5, OXTR, SCL6A4, and BDNF.ResultsThe methylation profiles of the mother–child familial pairs significantly correlated as compared to mother–child random pairs for the entire epigenome and NR3C1, FKBP5, OXTR and BDNF genes. Next, multiple linear regression models observed associations of mother adversity, child age, and neglect-control group on mother–child methylation similarity, only significant in mother–child familial pairs, after correcting for multiple comparisons. Higher mother adversity was associated with lower mother–child methylation similarity for the epigenome-wide analysis, for the BDNF gene, and in the neglect-control group for the OXTR gene. In turn, being an older child (longer co-residence) was associated with higher mother–child methylation similarity.ConclusionsMother adversity and co-residence time are modulating factors in the intergenerational methylation process that offer a window into development-dependent adaptations that can be affected by both hereditary and environmental factors, significantly observed only in biological dyads. A twofold implication for child well-being emerges, one is positive in that children of mothers exposed to life adversity or neglect did not necessarily inherit their methylation patterns. The other is concerning due to the influence of time spent living together, which affects similarity with the mother and potentially increases the risk of inheriting an epigenetic profile associated with future dysfunctional parenting patterns. This underscores the importance of the 'the earlier, the better' recommendation by the Child Protection System, which is not always followed.

Spectrum and antibiotic resistance in community- and hospital-acquired urinary tract infections among adults: Experience from a large tertiary care center in a developing country.

It is important to note that the causative agents and patterns of antibiotic resistance vary between urinary tract infections (UTIs) acquired in the community and those acquired in a hospital setting. Therefore, the aim of this study was to compare the types of organisms and patterns of antibiotic resistance in adult patients with community-acquired urinary tract infections (CA-UTIs) and hospital-acquired urinary tract infections (HA-UTIs). Retrospectively, we collected urine samples from patients at An-Najah National University Hospital who experienced nonrecurring urinary tract infections (UTIs) between January 2019 and December 2020. The data were subsequently analyzed using IBM-SPSS® 21.0 software to determine the distribution of microorganisms and evaluate the rates of antibiotic resistance. A total of 798 nonrepetitive UTI patients were included in our study, in which more than half of the UTIs occurred in female patients (472; 59.1%), and most of the UTIs were of community origin (611; 76.6%). Both community-acquired urinary tract infections (CA-UTIs) and hospital-acquired urinary tract infections (HA-UTIs) were more common in female patients (45.6% and 13.5%, respectively). Escherichia coli was the most commonly isolated urinary pathogen in both the CA-UTIs and HA-UTIs. The five most common isolated urinary pathogens were E. coli, Klebsiella pneumoniae, Enterococcus faecalis, E. faecium, and Pseudomonas aeruginosa. Among the isolated bacteria, 28.2% were extended-spectrum beta-lactamase (ESBL)-producing bacteria, 4.1% were carbapenem-resistant Enterobacterales (CRE), and 6.5% were vancomycin-resistant Enterococci. The most commonly isolated urinary pathogens from HA-UTIs showed higher resistance rates against all the tested antibiotics except for E. faecium, which showed greater resistance to tetracycline (42.5%) and quinupristin/dalfopristin (17.5%) in CA-UTIs. There are similarities in the etiological profiles of CA-UTI and HA-UTI, but the resistance rates are high, and urine culture is essential for the correct treatment of individual cases, even in primary care.

Uncovering a hidden functional role of the XRE-cupin protein PsdR as a novel quorum-sensing regulator in Pseudomonas aeruginosa.

XRE-cupin family proteins containing an DNA-binding domain and a cupin signal-sensing domain are widely distributed in bacteria. In Pseudomonas aeruginosa, XRE-cupin transcription factors have long been recognized as regulators exclusively controlling cellular metabolism pathways. However, their potential functional roles beyond metabolism regulation remain unknown. PsdR, a typical XRE-cupin transcriptional regulator, was previously characterized as a local repressor involved solely in dipeptide metabolism. Here, by measuring quorum-sensing (QS) activities and QS-controlled metabolites, we uncover that PsdR is a new QS regulator in P. aeruginosa. Our RNA-seq analysis showed that rather than a local regulator, PsdR controls a large regulon, including genes associated with both the QS circuit and non-QS pathways. To unveil the underlying mechanism of PsdR in modulating QS, we developed a comparative transcriptome approach named "transcriptome profile similarity analysis" (TPSA). Using this TPSA method, we revealed that PsdR expression causes a QS-null-like transcriptome profile, resulting in QS-inactive phenotypes. Based on the results of TPSA, we further demonstrate that PsdR directly binds to the promoter for the gene encoding the QS master transcription factor LasR, thereby negatively regulating its expression and influencing QS activation. Moreover, our results showed that PsdR functions as a negative virulence regulator, as inactivation of PsdR enhanced bacterial cytotoxicity on host cells. In conclusion, we report on a new QS regulation role for PsdR, providing insights into its role in manipulating QS-controlled virulence. Most importantly, our findings open the door for a further discovery of untapped functions for other XRE-Cupin family proteins.

The actinomycete Kitasatospora sp. SeTe27, subjected to adaptive laboratory evolution (ALE) in the presence of selenite, varies its cellular morphology, redox stability, and tolerance to the toxic oxyanion

The effects of oxyanions selenite (SeO32−) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized.