Molecular Analysis Research Articles

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

Rediscovery and phylogenetic analysis of Agnostrup, a least known genus of Mecistocephalidae (Chilopoda, Geophilomorpha) in China

The genus Agnostrup Foddai, Bonato, Pereira & Minelli, 2003 is one of the least known genera within the family Mecistocephalidae Bollman, 1893. In this study, we rediscovered the genus Agnostrup in China and provided a comprehensive redescription of A. striatus (Takakuwa, 1949). Additionally, we conducted a molecular phylogenetic analysis using CO1, 16S, and 28S sequence data, revealing a low genetic distance between Agnostrup and Nannarrup Foddai, Bonato, Pereira & Minelli, 2003. After comparing the morphological characteristics of these two genera, we synonymized the genus Nannarrupsyn. nov. with Agnostrup. As a result, three species previously belonging to the genus Nannarrup were transferred to Agnostrup: A. hoffmani (Foddai et al., 2003), comb. nov., A. innuptus (Tsukamoto, 2022), comb. nov., and A. oyamensis (Tsukamoto, 2022), comb. nov.

Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling

The endogenous neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) modulates inflammatory and neuroinflammatory signaling through toll-like receptors (TLRs) in human and mouse macrophages, human blood cells and alcohol-preferring (P) rat brains. Although it is recognized that 3α,5α-THP inhibits TLR4 activation by blocking interactions with MD2 and MyD88, the comprehensive molecular mechanisms remain to be elucidated. This study explores additional TLR4 activation sites, including TIRAP binding to MyD88, which is pivotal for MyD88 myddosome formation, as well as LPS interactions with the TLR4:MD2 complex. Both male and female P rats (n = 8/group) received intraperitoneal administration of 3α,5α-THP (15 mg/kg; 30 min) or a vehicle control, and their hippocampi were analyzed using immunoprecipitation and immunoblotting techniques. 3α,5α-THP significantly reduces the levels of inflammatory mediators IL-1β and HMGB1, confirming its anti-inflammatory actions. We found that MyD88 binds to TLR4, IRAK4, IRAK1, and TIRAP. Notably, 3α,5α-THP significantly reduces MyD88-TIRAP binding (Males: −31 ± 9%, t-test, p < 0.005; Females: −53 ± 15%, t-test, p < 0.005), without altering MyD88 interactions with IRAK4 or IRAK1, or the baseline expression of these proteins. Additionally, molecular docking and molecular dynamic analysis revealed 3α,5α-THP binding sites on the TLR4:MD2 complex, targeting a hydrophobic pocket of MD2 usually occupied by Lipid A of LPS. Surface plasmon resonance (SPR) assays validated that 3α,5α-THP disrupts MD2 binding of Lipid A (Kd = 4.36 ± 5.7 μM) with an inhibition constant (Ki) of 4.5 ± 1.65 nM. These findings indicate that 3α,5α-THP inhibition of inflammatory mediator production involves blocking critical protein-lipid and protein-protein interactions at key sites of TLR4 activation, shedding light on its mechanisms of action and underscoring its therapeutic potential against TLR4-driven inflammation.

MitoQ enhances CYP19A1 expression to stimulate WNT/β-catenin signaling pathway for promoting hair growth in androgenetic alopecia.

Increased sensitivity to androgens and androgen receptors is the underlying cause of androgenetic alopecia (AGA), a hereditary disease. Our study investigated the preventive effects of MitoQ on dihydrotestosterone (DHT)-induced mitochondrial dysfunction and subsequent hair loss from three perspectives: in vivo, in vitro, and network pharmacology. A mouse model of AGA was used to assess the effectiveness of MitoQ intervention. Seventy-five drug targets and 367 disease targets were identified through network pharmacology analysis. Molecular docking analysis revealed that the androgen receptor (AR) and CYP19A1, which are key targets of MitoQ, may play a role in AGA treatment. CYP19A1 expression was downregulated in lesions from patients with AGA compared to healthy scalp tissue, while AR expression was upregulated. Cellular tests of human dermal papilla cells (DPCs) treated with MitoQ revealed that the mRNA and protein expression of AR remained unchanged, but the mRNA expression of CYP19A1 was upregulated. Our experiments also confirmed that CYP19A1 overexpression prevented DHT-induced apoptosis and upregulated the expression levels of WNT3A and β-catenin, whereas increased apoptosis levels and the downregulation of WNT3A and β-catenin due to CYP19A1 knockdown were reduced by MitoQ. We verified that MitoQ enhanced hair growth in DHT-induced hair loss model mice and reversed DHT-induced apoptosis by enhancing the expression of CYP19A1 in DPCs and that MitoQ may act by mediating the WNT/β-catenin pathway. These findings indicate that MitoQ could be a promising intervention for AGA and that CYP19A1 may serve as a valuable therapeutic target for AGA.

A New Genus Neotricholomopsis Gen. Nov and Description of Neotricholomopsis globispora Sp. Nov. (Phyllotopsidaceae, Agaricales) from Northwestern China Based on Phylogeny, Morphology, and Divergence Time

While investigating macrofungi diversity in Gansu province, northwestern China, five fresh and fleshy specimens were collected, which are characterized by nearly white to buff hemispherical pileus with waved margins, a disc depressed with coral to brownish red fibrillose scales, adnate to sub-decurrent lamellae with four relatively regular rows of lamellulae, a stipe that is central, hollow, frequently straight to curved; basidiospores that are globose to subglobose, 5.0–6.0 × (3.5−) 4.0–5.0 (−5.5) μm, narrowly clavate cheilocystidia predominantly, pleurocystidia and caulocystidia not observed; and a cutis pileipellis, with hyphae slightly inflated in the KOH. The results of phylogeny analysis indicated that the species forms an independent lineage in Phyllotopsidaceae based on the ITS (ITS5/ITS4) and nLSU (LR0R/LR7) dataset. Molecular clock analyses suggested the common ancestor of Neotricholomopsis emerged later than upper Cretaceous with a mean crown age of 229.36 Mya (95% highest posterior density of 129.63–343.08 Mya). These five specimens were described as an unreported taxon based on the phylogeny analysis combined with morphological examination and ecological and geographical distribution. Detailed descriptions, illustrations, and phylogenetic trees to demonstrate the placement of this species and discussions with its related species are provided.

The Role of Polyploidy in the Genetic Structure and Expansion of Lepisorus clathratus in the Qinghai–Tibetan Plateau and Hengduan Mountains

Polyploidy plays a crucial role in plant evolution, particularly in shaping genetic diversity and geographic distribution. This study investigates the genetic diversity and distribution of Lepisorus clathratus (C. B. Clarke) Ching, a polyploid fern species endemic to the Qinghai–Tibetan Plateau and Hengduan Mountains. We sampled 586 individuals from 66 populations and identified three ploidy levels: diploid, tetraploid, and hexaploid. Flow cytometry and chloroplast DNA sequencing were used to assess ploidy variation and genetic structure. Tetraploid populations dominated the Hengduan Mountains and exhibited wider geographic ranges, while diploids were largely confined to the Qinghai–Tibetan Plateau. Molecular variance analysis revealed significant genetic differentiation among regions, with polyploid populations demonstrating higher cross-region migration rates compared with diploids, as evidenced by the historical gene flow analysis. Ecological niche modeling suggested that polyploids expanded more successfully in post-glacial periods, likely due to their greater ecological flexibility and capacity for long-distance colonization. These findings highlight the critical role of polyploidy in shaping genetic structure and species expansion, contributing to the understanding of plant adaptation in response to historical climatic changes.

Predictive Analysis of Yi-Gai-San's Multifaceted Mechanisms for Tremor-dominant Parkinson's Disease via Network Pharmacology and Molecular Docking Validation.

Based on comprehensive network-pharmacology and molecular docking analysis, this study was intended to unveil the multiple mechanisms of Yi- Gai-San (YGS) in treating the tremor-dominant subtype of Parkinson's disease (PD-DT). The compounds of YGS were meticulously analyzed, selected, and standardized with references to their pharmacological attributes. Its components included Gouteng (Uncaria rhynchophylla), Chaihu (Radix Bupleuri), Chuanxiong (Chuanxiong Rhizoma), Danggui (Angelicae sinensis radix), Fuling (Wolfiporia extensa), Baizhu (Atractylodis macrocephalae rhizoma), and Gancao (Licorice, Glycyrrhizae radix). We identified 75 active compounds within YGS. From these, we predicted 110 gene targets, which exhibited a direct association with PD-DT. PPI network results highlighted core target proteins, including TP53, SLC6A3, GAPDH, MAOB, AKT, BAX, IL6, BCL2, PKA, and CASP3. These proteins potentially alleviate PD-DT by targeting inflammation, modulating neuronal cell apoptosis, and regulating the dopamine system. Furthermore, GO and KEGG enrichment analyses emphasized that YGS might influence various mechanisms, such as the apoptotic process, mitochondrial autophagy, Age-Rage signaling, and dopaminergic and serotonergic synapses. The core proteins from the PPI analysis were selected for the docking experiment. The docking results demonstrated that the most stable ligand-receptor conformations were kaempferol with CASP3 (-9.5 kcal/mol), stigmasterol with SLC6A3 (-10.5 kcal/mol), shinpterocarpin with BCL2L1 (-9.6 kcal/mol), hirsutine with MAOB (-9.7 kcal/mol), hederagenin with PRKACA (-9.8 kcal/mol), and yatein with GAPDH (-9.8 kcal/mol). These results provide us with research objectives for future endeavors in extracting single compounds for drug manufacturing or in-depth studies on drug mechanisms. From these computational findings, we suggested that YGS might mitigate PD-DT via "multi-compounds, multi-targets, and multi-pathways."

Revalidation of Enteromius karkensis (Gilchrist and Thompson 1913) (Cypriniformes: Smiliogastrinae) from the southern temperate freshwater ecoregion, South Africa.

The chubbyhead barbs, a distinct group of fishes endemic to southern Africa, currently include eight valid species. Historically, Enteromius anoplus was the most widespread freshwater fish in South Africa due to synonymizations in the 1960s. It occurred in nearly every river system except coastal systems in the Cape Fold Ecoregion and the lower Orange River. However, a recent revision utilizing molecular and morphological analyses has led to significant taxonomic updates. Enteromius anoplus is restricted to the Gouritz River system, whereas Enteromius cernuus and Enteromius oraniensis have been revalidated and respectively confined to the Olifants and Orange River systems. Additionally, a new species Enteromius mandelai was described, with its range currently considered to encompass several river systems in the Eastern Cape. Despite these advances, the taxonomic status of fish previously assigned to E. anoplus from KwaZulu-Natal remained uncertain. In this study, an integrative approach, combining genetic analysis, morphological characteristics, and geographic data, was employed to resurrect Enteromius karkensis as a distinct species from E. anoplus. Enteromius karkensis is deeply genetically differentiated (3.99%-8.07% p-distance) from its congeners within the chubbyhead group. Morphologically, E. karkensis is easily distinguishable from its counterparts due to possession of a terminal mouth (vs. inferior in E. amatolicus), one pair of maxillary barbels (vs. two conspicuous pairs in E. motebensis, E. treurensis, and E. gurneyi), and a complete lateral line (vs. incomplete in E. anoplus and E. oraniensis). Furthermore, E. karkensis is differentiated from E. mandelai and E. cernuus by its long maxillary barbels that extend beyond the midpoint of the orbit. These findings highlight a consistent pattern from previous studies that show many freshwater fishes in South Africa have narrow geographic ranges. This emphasizes the importance of targeted conservation and management efforts, and our understanding of the biogeographic and evolutionary history of freshwater fishes in southern Africa.

Revealing the potential therapeutic mechanism of Lonicerae Japonicae Flos in Alzheimer’s disease: a computational biology approach

BackgroundAlzheimer’s disease (AD) is a degenerative brain disease without a cure. Lonicerae Japonicae Flos (LJF), a traditional Chinese herbal medicine, possesses a neuroprotective effect, but its mechanisms for AD are not well understood. This study aimed to investigate potential targets and constituents of LJF against AD.MethodsNetwork pharmacology and bioinformatics analyses were performed to screen potential compounds and targets. Gene Expression Omnibus (GEO) datasets related to AD patients were used to screen core targets of differential expression. Gene expression profiling interactive analysis (GEPIA) was used to validate the correlation between core target genes and major causative genes of AD. The receiver operating characteristic (ROC) analysis was used to evaluate the predictive efficacy of core targets based on GEO datasets. Molecular docking and dynamics simulation were conducted to analyze the binding affinities of effective compounds with core targets.ResultsNetwork pharmacology analysis showed that 112 intersection targets were identified. Bioinformatics analysis displayed that 32 putative core targets were identified from 112 intersection targets. Only eight core targets were differentially expressed based on GEO datasets. Finally, six core targets of MAPK8, CTNNB1, NFKB1, EGFR, BCL2, and NFE2L2 were related to AD progression and had good predictive ability based on correlation and ROC analyses. Molecular docking and dynamics simulation analyses elucidated that the component of lignan interacted with EGFR, the component of β-carotene interacted with CTNNB1 and BCL2, the component of β-sitosterol interacted with BCL2, the component of hederagenin interacted with NFKB1, the component of berberine interacted with EGFR and BCL2, and the component of baicalein interacted with NFKB1, EGFR and BCL2.ConclusionThrough a comprehensive analysis, this study revealed that six core targets (MAPK8, CTNNB1, NFKB1, EGFR, BCL2, and NFE2L2) and six practical components (lignan, β-carotene, β-sitosterol, hederagenin, berberine, and baicalein) were involved in the mechanism of action of LJF against AD. Our work demonstrated that LJF effectively treats AD through its multi-component and multi-target properties. The findings of this study will establish a theoretical basis for the expanded application of LJF in AD treatment and, hopefully, can guide more advanced experimental research in the future.

Machine learning interatomic potential with DFT accuracy for general grain boundaries in α-Fe

AbstractTo advance the development of high-strength polycrystalline metallic materials towards achieving carbon neutrality, it is essential to design materials in which the atomic level control of general grain boundaries (GGBs), which govern the material properties, is achieved. However, owing to the complex and diverse structures of GGBs, there have been no reports on interatomic potentials capable of reproducing them. This accuracy is essential for conducting molecular dynamics analyses to derive material design guidelines. In this study, we constructed a machine learning interatomic potential (MLIP) with density functional theory (DFT) accuracy to model the energy, atomic structure, and dynamics of arbitrary grain boundaries (GBs), including GGBs, in α-Fe. Specifically, we employed a training dataset comprising diverse atomic structures generated based on crystal space groups. The GGB accuracy was evaluated by directly comparing with DFT calculations performed on cells cut near GBs from nano-polycrystals, and extrapolation grades of the local atomic environment based on active learning methods for the entire nano-polycrystal. Furthermore, we analyzed the GB energy and atomic structure in α-Fe polycrystals through large-scale molecular dynamics analysis using the constructed MLIP. The average GB energy of α-Fe polycrystals calculated by the constructed MLIP is 1.57 J/m2, exhibiting good agreement with experimental predictions. Our findings demonstrate the methodology for constructing an MLIP capable of representing GGBs with high accuracy, thereby paving the way for materials design based on computational materials science for polycrystalline materials.

Extra-Axial Poorly Differentiated Chordoma Initially Misdiagnosed as Epithelioid Sarcoma.

Poorly differentiated chordoma is an exceedingly rare, aggressive subtype of chordoma. These tumors typically arise in the axial skeleton of young patients, most commonly the skull base, followed by the cervical spine. Herein, we present a 60-year-old patient with longstanding knee pain and nondiagnostic imaging, initially thought to be due to osteoarthritis. No discrete mass-forming lesion was identified by radiology. Synovial histology at the time of arthroplasty revealed a multinodular proliferation of epithelioid-to-histiocytoid cells with a moderate amount of eosinophilic-to-clear, vacuolated cytoplasm. Scattered cells with high-grade nuclear atypia were present. A diagnosis of metastatic carcinoma was considered due to immunohistochemical positivity for keratin and GATA3. However, a diagnosis of epithelioid sarcoma was rendered based on clinical context, morphology, and loss of immunohistochemical expression for SMARCB1 (INI1). However, upon re-review of the tumor, brachyury was retrospectively added to the immunohistochemistry panel and showed strong positivity, thus prompting amendment of the initial diagnosis of epithelioid sarcoma to extra-axial poorly differentiated chordoma. Given the rarity of this diagnosis, molecular testing was performed which revealed a unique SMARCB1 molecular profile with a single-nucleotide variant in addition to the commonly reported loss of chromosome 22q. This report of an ultra-rare sarcoma in an uncommon anatomic site highlights multiple potential pitfalls in the diagnosis of poorly differentiated chordoma, emphasizes the importance of brachyury immunohistochemistry in rendering a correct interpretation, and underscores an opportunity for further molecular analysis to better define the molecular profile of this entity.

First records and extension of the geographic distribution of Cynomops kuizha (Chiroptera: Molossidae) in the Colombian Caribbean

Abstract The insectivorous bat genus Cynomops belongs to the Neotropical fraction of the family Molossidae, which includes Free-tailed Bats or Mastiff Bats. Currently, nine species are recognized within Cynomops. In this study, we present the first records of Cynomops kuizha for the Colombian Caribbean Region. The two female specimens were recovered in the campus of the Puerta Roja of the University of Sucre, Sucre and represent a significant extension in the geographical and ecological distribution of the species. With these new records, the known distribution of this species is extended approximately 520 km northeast of the nearest previous record in the department of Valle del Cauca, Colombia. Identification of both specimens was carried out through morphological comparisons and analysis of the mitochondrial gene cytochrome-b (Cyt-b). Molecular analyses revealed that the Cyt-b sequences from the Sucre specimens are >99 % similar to those documented for C. kuizha in southwestern Colombia. With these new records, the known distribution of this species is extended approximately 520 km northeast of the nearest previous record in the department of Valle del Cauca, Colombia. Additionally, we discuss some morphological peculiarities observed in our Sucre specimens and their relationship with closely related species present in the area: Cynomops abrasus, Cynomops greenhalli, and Cynomops mastivus.

Abstract PR010: High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer

Abstract Background: Metastatic prostate cancer is an androgen receptor (AR) driven disease for which multiple AR targeted therapies are FDA approved, leading to improvements in patient outcomes. However, development of treatment resistance remains universal, driven by AR alterations as well as lineage state transitions that bypass AR signaling and culminate in neuroendocrine prostate cancer (NEPC) with poor prognosis. Liquid biopsies could enable longitudinal molecular analysis to identify such lineage transitions, but bulk RNA sequencing of circulating tumor cells (CTCs) has been limited by the challenge of isolating sufficiently pure samples for global signaling pathway analysis. We developed a novel approach to CTC purification that results in purity comparable to tissue biopsies and performed the first large-scale CTC RNA-seq study in a multi-institutional cohort of patients with metastatic prostate cancer. Using this approach, we identified multiple prostate cancer lineage states associated with prognosis. Methods: 273 blood samples were collected from 117 patients with histologically confirmed metastatic prostate cancer treated at the University of Wisconsin Carbone Cancer Center, William S. Middleton Memorial Veterans Hospital, UC San Diego Moores Cancer Center and Dana Farber Cancer Institute. CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA sequencing. A modification of the ESTIMATE algorithm was used to infer sample tumor purity, and CIBERSORTx was used to infer immune content. Overall survival (OS) was defined as date of death or last contact relative to first CTC sample collection. Results: 146 samples from 70 patients met our >50% purity threshold for gene expression pathway analysis. Single sample pathway analysis identified four CTC transcriptional phenotypes: luminal A (high AR signaling, intermediate proliferation), luminal B (high AR signaling, high proliferation), low proliferation, and neuroendocrine. Compared to patients with low CTC burden/low tumor purity (median OS NR, n=63), patients with luminal A (n=21) and low proliferation (n=12) phenotypes had similar survival, while patients with luminal B (n=18) and NE (n=3) had markedly shorter survival (LumB: median OS 6 months, HR 9.1, log rank p<0.0001, NE: median OS 3.7 months, HR 11.8, log rank p=0.0019). Conclusion: We report the largest CTC RNA sequencing cohort of patients with metastatic prostate cancer and demonstrate that CTC RNA-seq identifies prostate cancer lineage states mirroring those described in tissue profiling. In addition to an NE phenotype concordant with tissue histology and associated with markedly inferior overall survival, we identified a more common luminal B CTC subtype defined by persistent AR signaling and high proliferation and associated with poor prognosis comparable to NEPC. Citation Format: Marina N Sharifi, Jamie M Sperger, Amy K Taylor, Katharine E Tippins, Shannon R Reese, Viridiana Carreno, Katherine R Kaufmann, Alex H Chang, Luke A Nunamaker, Charlotte Linebarger, Kyle T Helzer, Matthew Bootsma, Grace C Blitzer, John Floberg, David Kosoff, Rana R McKay, Xiao X Wei, Shuang G Zhao, Joshua M Lang. High purity CTC RNA sequencing identifies poor prognosis lineage states in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR010.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

In Situ X-ray and Infrared Spectroscopic Studies of Electrochemical Systems

Abstract Despite a wealth of data and intense research, the phenomena occurring during electrocatalysis are still a major obstacle in many chemical processes. Molecular analysis of the electrode/electrolyte interface is needed to correctly describe the reaction through identifying the species involved, their interaction with the environment and kinetics in situ, i.e. while the reaction is taking place. That can be done by coupling the electrochemical system with complementary non-electrochemical techniques. In addition, in situ x-ray spectroscopic techniques analyze the electrode itself, providing the information on the changes in the catalyst during the reaction. The synergy of the traditional electrochemical techniques with the complementary spectroscopic methodologies offer understanding of the electrode/electrolyte interface above and beyond traditional experimental mainframe. Here we demonstrate how in situ X-ray absorption spectroscopy, in situ infrared reflection/absorption spectroscopy, and traditional voltammetric studies can reveal electrochemical processes during oxidation of ethanol, enhance the structure and selectivity of the catalyst to complete oxidation pathway, and provide understanding of the parameters that enhance its oxidation for future designing catalysts for alcohol oxidation fuel cells.

Species Identification and Genotyping of Cutaneous Leishmaniasis in Clinical Samples Based on ITS1-PCR- Sequencing in Southeast Iran

Background: Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases in many regions of Iran. It has a major role in deprived societies. We aimed to identify Leishmania species based on molecular as ITS1-rDNA-PCR internal transcribed spacer 1 (ITS1) region, microscopy, and culture techniques in diagnosing cutaneous leishmaniasis. Methods: From April 2018 to May 2020, we conducted a cross-sectional study involving 32 patients with suspected CL lesions in Sistan and Baluchistan Province, located in southeast Iran. Samples were subjected to microscopic examination, culture, and PCR amplification targeting the internal transcribed spacer 1 (ITS1) region. DNA sequencing was performed on PCR-positive samples for species identification and phylogenetic analysis. Results: PCR demonstrated superior sensitivity (93.75%, 30/32) compared to culture (56.25%, 18/32) and microscopic examination (53.1%, 17/32). Molecular analysis revealed that L. major was the predominant causative agent of CL in the study area, with L. tropica occurring less frequently. Sequencing and phylogenetic analysis of the ITS1 region showed high intraspecies similarity among L. tropica isolates, while L. major isolates exhibited greater genetic diversity. Conclusion: This study shows the co-existence of L. major and L. tropica in Mirjaveh, southeast Iran, with L. major as the primary cause. While L. tropica isolates displayed high genetic similarity, L. major samples were more diverse, indicating different epidemiological patterns. These findings highlight the importance of molecular methods for accurately identifying Leishmania species and understanding CL epidemiology in the region

Mouse models for pancreatic ductal adenocarcinoma are affected by the cre-driver used to promote KRASG12D activation.

The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically engineered mouse models that allow temporal and spatial activation of oncogenic KRAS (KRASG12D). One of the most commonly used models involves targeted insertion of a cre-recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRASG12D). This study aims to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRASG12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three genetically engineered mouse models that express a tamoxifen-inducible cre-recombinase to activate KrasG12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a or Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Histological and RNA-seq analyses were used to delineate differences between the models. Up to two months after tamoxifen induction of KRASG12D, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in widespread PanIN lesions in Ptf1acreERT pancreata within seven days and maintained for at least five weeks post-injury, which was not seen in the models with two functional Ptf1a alleles. RNA-seq analysis prior to injury induction suggested Ptf1acreERT and Mist1creERT mice have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the models. These findings suggest Ptf1a haploinsufficiency in Ptf1acreERT mouse models promotes KRASG12D priming of genes for promotion of PDAC.

Investigation of Antibiotic Resistance of E. coli Associated with Farm Animal Feces with Participation of Citizen Scientists

This paper presents the findings of a large-scale study on antibiotic resistance in bacteria found in farm animal feces across Russia. The study included 6578 samples of farm animal manure from 13 regions in Russia, with the help of citizen scientists. Molecular and microbiological methods were used to analyze 1111 samples of E. coli. The microbiological analysis focused on culturing the microorganisms present in the fecal samples on selective media for E. coli and evaluating the sensitivity of the bacteria to different antibiotics, including ampicillin, tetracycline, chloramphenicol, cefotaxime, and ciprofloxacin. The molecular analysis involved isolating the genomic DNA of the bacteria and conducting PCR assays to detect the vanA, vanB, and mcr-1 antibiotic resistance genes. The results demonstrated significant differences in antibiotic sensitivity of the samples that are morphologically identical to E. coli from different regions. For example, 98.0% and 82.5% of E. coli and other fecal bacterial isolates from the Omsk and Vologda regions lacked antibiotic resistance genes, while 97.7% of samples from the Voronezh region possessed three resistance genes simultaneously. The phenotypic antibiotic sensitivity test also revealed regional differences. For instance, 98.1% of fecal bacterial samples from cattle in the Udmurt Republic were sensitive to all five antibiotics tested, whereas 92.8% of samples from the Voronezh region showed resistance to all five antibiotics. The high level of antibiotic resistance observed may be attributed to their use in farming practices. The distinctive feature of our research is that comprehensive geographical coverage was achieved by using a citizen science platform. Citizen scientists, specifically students from colleges and universities, were responsible for the collection and initial analysis of samples. The project attracted 3096 student participants, enabling the collection and analysis of a significant number of samples from various locations in Russia.

Cytogenetic and molecular identification of novel wheat-Elymus sibiricus addition lines with resistance to leaf rust and the presence of leaf pubescence trait

IntroductionEmerging new races of leaf rust (Puccinia triticina Eriks) are threatening global wheat (Triticum aestivum L.) production. Identifying additional resistance genes from all available gene pools is crucial to expanding wheat resistance to these virulent leaf rust races. Siberian wild rye (Elymus sibiricus L.) possesses numerous beneficial traits that can be valuable in wheat improvement. Three new wheat-E. sibiricus addition lines, O27-2 (BC8), O27-3 (BC12) and O193-3 (BC12), were developed through a backcrossing scheme in this study, using leaf rust field evaluations, molecular marker assays and cytogenetic analysis. MethodsThese three lines were derived from progeny of the bread wheat cultivar ‘Obriy’ (2n = 6x = 42, AABBDD) and partial octoploid amphiploid wheat-E. sibiricus (2n = 8x = 56, AABBDDStSt). Results and discussionThe lines (O27-2, O27-3 and O193-3) demonstrated strong specific leaf pubescence (hairiness) and resistance at the adult stage to a local population of leaf rust races. The response to leaf rust in these three lines significantly differed from that of the Lr24 gene, providing evidence for a distinct resistance mechanism associated with the 3St chromosome. This study is the first to report the transfer of an E. sibiricus chromosome into wheat that confers leaf rust resistance. Molecular marker analysis and genomic in situ hybridization confirmed that lines O27-2, O27-3 and O193-3 each possess one pair of E. sibiricus 3St chromosomes. The resistance gene was determined to be on the additional alien chromosome in these lines. Molecular markers (Xwmc221, Lr29F18, Sr24/Lr24) confirmed that the lines O27-2, O27-3, and O193-3 each contain a pair of E. sibiricus 3St chromosomes carrying leaf rust resistance genes. These findings demonstrate that the E. sibiricus 3St chromosome carries the leaf rust resistance gene and that the O27-2, O27-3, and O193-3 lines can serve as novel germplasm sources for introducing this resistance into wheat breeding programs. This study contributes to broadening the genetic diversity of resistance genes available for combating leaf rust in wheat.

Sequencing the Parasitic Nematode Contracaecum spp. in Edible Fish (Planiliza Abu)

Contracaecum rudolphii Hartwich,1 964 (Nematoda: Ascaridoidea, Anisakidae) is a typical anisakid recorded globally. Consumption of undercooked seafood, raw or infected, contains the nematode larvae that cause human anisakidosis . Fish (Planiliza abu) specimens were obtained from a local market in Karbala, Iraq. This type of fish came from the Razzaza Lake. The prevalence of Contracaecum spp. parasites was done over eight months from June 2022 until January 2023, using ITS1, morphological and molecular analysis of the nematodes Contracaecum spp from fish. Out of 395 fish, 124(31.3%) had visceral infections caused by Contracaecum spp. larval type (L3). The morphological and genetic identification of Contracaecum spp. was validated in the parasitology laboratory of the veterinary medicine college at Kerbala University. The result showed the infection rate in January 2023 was (46.6%) and it dropped in 2022, September with a percentage of (20%). With the use of ITS1 gene, the molecular analysis for Contracaecum spp. was to investigate Contracaecum spp. and to confirm it. However, the nematode count, number of infected fish, and length were all substantially different at the P≤0.05. This study detected the isolate=(a1) at the locus=OP787071 and sequencing the parasites. The isolates were confirmed as Contracaecum rudolphii, isolate a1 internal transcribed spacer 1, partial sequence. In conclusion, molecular genotyping might be a useful technique for identifying the Contracaecum L3 larval species., life-cycle biology, transmission methods, and types of intermediate hosts.