Multiple Tissues Research Articles

Detect The Most Important Changes That Occur in The Stomach Tissue of Patients Infected With H. Pylori

The current study aimed to detect the most important changes that occur in the stomach tissue of patients infected with H. pylori. Samples were collected from the Endoscope Unit for the period from 12/14/2022 to 1/30/2024 at Azadi Teaching Hospital and Al-Watan Al-Ahli Hospital in Kirkuk Governorate, from both sexes for the age group (14-57) years. Stool samples were taken from the same patients those undergoing laparoscopy for the purpose of investigating the presence of Helicobacter pylori. Tissue biopsy samples were taken by the gastroenterologist using laparoscopic forceps, and (88) samples were obtained from these patients, with two tissue biopsies from each patient. The histological results of people infected with Helicobacter pylori bacteria showed many histological changes, including infiltration of inflammatory cells, which was accompanied by multiple tissue necrosis, and damage to some gastric glands as a result of degeneration of glandular epithelial cells with thickening of the basal membrane, in addition to necrosis and damage to most of the gastric glands. Narrowing of the lumen of some glands, sometimes their complete atrophy, metaplasia of the epithelial cells of the gastric glands and the epithelial tissue lining the antral area, and cell shedding occur. It is concluded from the current study that H. pylori has led to many tissue lesions in the stomach of patients, the most important of which is stomach ulceration.

Immunohistochemical analysis of the immune checkpoint molecule Galectin-9 in meningiomas

Purpose: Most meningiomas can be treated by surgical resection. However, depending on the location of the lesion, incomplete resection or high-grade meningiomas may have a poor prognosis. The new methods such as immunotherapy may improve our options for effective, patient-specific treatment of meningiomas. We aim to contribute to the development of new personalized treatment strategies by investigating the status of Gal-9 in meningiomas. Materials and Methods: Four hundred two cases diagnosed in our laboratory between 2007 and 2020 were used for the study. New blocks of multiple tissues were prepared for immunohistochemistry using the tissue microarray method. Immunohistochemical staining of Gal-9 antibody was evaluated using the H-score method. Results: Of the 402 cases studied, 289 were female and 113 were male. Two hundred and seventy-one (67.4%) cases were WHO grade 1; 121 (30.1%) were grade 2 and 10 (2.5%) were grade 3. A high H-score was observed in grade 1 and 2 tumors (H-score: 93.38 and 93.91) and a low H-score in grade 3 tumors (H-score: 59.40). There was no significant correlation between brain invasion and Gal-9 expression. No significant correlation was found between Gal-9 expression and minor criteria used in tumor grading. Conclusion: A statistically significant difference was found between Gal-9 H-score and tumor grade. Gal-9 had a lower H-score in high-grade meningiomas and its expression level decreased. Therefore, Gal-9 with different expression levels can be used as a prognostic and predictive biomarker as well as an important molecule for treatment.

An economically viable stable isotope-enhanced multiple reaction monitoring method for total fatty acid analysis in a mouse model of non-alcoholic fatty liver disease

The complex pathological mechanisms of non-alcoholic fatty liver disease (NAFLD) are closely related to dysregulated lipid metabolism, and the therapeutic effects of the traditional Chinese medicine Zexie-Baizhu Decoction (AA) on NAFLD have been gaining increasing attention. However, research into altered lipid metabolism, especially fatty acids, in NAFLD and the intervention of AA faces technical challenges, especially in the precise quantitative analysis of fatty acids in biological samples. The high complexity of biological matrices, particularly after drug intervention, greatly increases the difficulty of detection. Therefore, this study innovatively developed a simple and economical stable isotope derivatization technique by synthesizing d6N,N-dimethylethylenediamine (d6-DMED) in the laboratory, establishing a simple and economical method for fatty acid quantification. This method employs a chemical reaction under low-temperature conditions to ensure the efficient synthesis of d6-DMED. Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technique (UHPLC-MS/MS), combined with optimized chromatographic separation conditions and dynamic multiple reaction monitoring mode, the study established a highly sensitive detection method for 35 fatty acid derivatives. Methodological evaluation showed that the limits of quantification ranged from 0.002 to 0.060 μM, with high linearity of R² > 0.995. Additionally, the relative recovery rates were between 93.14% and 106.63%. To further demonstrate the feasibility of this method for fatty acid quantification, it was applied to measure fatty acids in multiple tissues in a mouse NAFLD model, as well as the effects of AA intervention on fatty acid metabolism. This rapid, simple, and cost-effective detection method not only enhances the understanding of NAFLD mechanisms but also provides a new strategy for evaluating the biological complex system after drug intervention.

Detecting thyroid nodules along with surrounding tissues and tracking nodules using motion prior in ultrasound videos

Ultrasound examination plays a crucial role in the clinical diagnosis of thyroid nodules. Although deep learning technology has been applied to thyroid nodule examinations, the existing methods all overlook the prior knowledge of nodules moving along a straight line in the video. We propose a new detection model, DiffusionVID-Line, and design a novel tracking algorithm, ByteTrack-Line, both of which fully leverage the prior knowledge of linear motion of nodules in thyroid ultrasound videos. Among them, ByteTrack-Line groups detected nodules, further reducing the workload of doctors and significantly improving their diagnostic speed and accuracy. In DiffusionVID-Line, we propose two new modules: Freq-FPN and Attn-Line. Freq-FPN module is used to extract frequency features, taking advantage of these features to reduce the impact of image blur in ultrasound videos. Based on the standard practice of segmented scanning by doctors, Attn-Line module enhances the attention on targets moving along a straight line, thus improving the accuracy of detection. In ByteTrack-Line, considering the characteristic of linear motion of nodules, we propose the Match-Line association module, which reduces the number of nodule ID switches. In the testing of the detection and tracking datasets, DiffusionVID-Line achieved a mean Average Precision (mAP50) of 74.2 for multiple tissues and 85.6 for nodules, while ByteTrack-Line achieved a Multiple Object Tracking Accuracy (MOTA) of 83.4. Both nodule detection and tracking have achieved state-of-the-art performance.

Identification of conserved and tissue-restricted transcriptional profiles for lipid associated macrophages (LAMs).

Macrophages are essential immune cells present in all tissues, and are vital for maintaining tissue homeostasis, immune surveillance, and immune responses. Considerable efforts have identified shared and tissue-specific gene programs for macrophages across organs during homeostasis. This information has dramatically enhanced our understanding of tissue-restricted macrophage programming and function. However, few studies have addressed the overlapping and tissue-specific responses of macrophage subsets following inflammatory responses. One subset of macrophages that has been observed across several studies, lipid-associated macrophages (LAMs), have gained interest due to their unique role in lipid metabolism and potential as a therapeutic target. LAMs have been associated with regulating disease outcomes in metabolically related disorders including atherosclerosis, obesity, and nonalcoholic fatty liver disease (NAFLD). In this study, we utilized single-cell RNA sequencing (scRNAseq) data to profile LAMs across multiple tissues and sterile inflammatory conditions in mice and humans. Integration of data from various disease models revealed that LAMs share a set of conserved transcriptional profiles, including Trem2 and Lpl, but also identified key sets of tissue-specific LAM gene programs. Importantly, the shared LAM markers were highly conserved with human LAM populations that also emerge in chronic inflammatory settings. Overall, this analysis provides a detailed transcriptional landscape of tissue-restricted and shared LAM gene programs and offers insights into their roles in metabolic and chronic inflammatory diseases. These data may help instruct appropriate targets for broad or tissue-restricted therapeutic interventions to modulate LAM populations in disease.

Isolation and identification of hybrid snakehead rhabdovirus (HSHRV) and its immune response in the hybrid Snakehead((male Channa argus × female Channa maculata)

Hybrid snakehead is an emerging aquaculture species obtained from the mating of Channa argus (♂) and Channa maculate (♀). It has the advantages of fast growth and strong disease resistance. Viral diseases caused by hybrid snakehead rhabdovirus (HSHRV) critically affect the hybrid snakehead industry. We isolated and identified a highly virulent strain of HSHRV from a naturally occurring hybrid snakehead, namely HSHRV-GZ22. It showed clinical signs of sinking, superficial blackening, spinning, acute internal congestion, and hemorrhage, along with blackening and enlargement of the liver, spleen, and kidneys. Histopathological analysis showed multiple tissue lesions in the liver, spleen, and kidneys, characterized mainly by massive inflammatory cell infiltration, interstitial hemorrhage, and partial cell necrosis. Pathogen analysis identified the virus as HSHRV. Immunofluorescence analysis (IFA) with HSHRV-specific antibodies confirmed the virus and electron microscopic observation showed that the bullet-like virus particles had a size of approximately 150 nm. The replication efficiency of HSHRV was 107.33 TCID50/mL. The glycoproteins of the isolates were cloned and sequenced, and a phylogenetic tree was constructed. The HSHRV-GZ22 isolates clustered into a single branch with the reported HSHRV-C1207, and it had a high degree of homology with Siniperca chuatsi rhabdovirus (SCRV). HSHRV-GZ22 was regressively infected, clinical and pathological symptoms were similar to naturally occurring fish, with a fatality rate of about 85 %. qRT-PCR was performed to determine the viral replication in different tissues of hybrid snakehead, and the viral copies were found to be highly expressed in the liver, spleen, kidney, and intestine. HSHRV-GZ22 activated the antiviral immune pathway in hybrid snakeheads during infection, and the expressions of IgM, IRF7, ISG12, and IFNγ were significantly altered. In this study, we isolated a strong virulent strain of HSHRV and characterized it; in addition, it provided insights into the pathogenesis of HSHRV and immune response in hybrid snakehead, while also advancing the methods for diagnosing and preventing diseases caused by HSHRV.

MAX inactivation deregulates the MYC network and induces neuroendocrine neoplasia in multiple tissues.

MAX inactivation deregulates multiple transcription factors to induce neuroendocrine cancers.

Elavl1 is dispensable for appendicular skeletal development.

Elavl1/HuR is a RNA binding protein implicated in multiple developmental processes with pleiotropic roles in RNA life cycle. Loss of Elavl1 is incompatible with life with early embryonic loss of Elavl1 in epiblast cells being lethal with defects in placental branching and embryonic tissue growth. Postnatal global deletion of Elavl1/HuR results in lethality with atrophy in multiple tissues mainly due to loss of progenitor cells. However, roles of Elavl1 specifically during embryonic limb development is not well understood. Here we report that deletion of Elavl1 in limb bud mesenchyme in mouse did not reveal any abnormalities during embryonic development with normal development in pre- and postnatal limb skeleton. Analyses of skeletal patterning, morphogenesis and skeletal maturation including skeletal elements in stylopod, zeugopod and autopod during development did not reveal any significant differences between long bones from control and Elavl1 conditional knockout animals. Our study indicates differential dependency and susceptibility to loss of Elavl1 in different stem cell lineages with its functions being dispensable during limb development.

Use of Sodium-glucose cotransporter 2 (SGLT 2) inhibitor is associated with reduced emergency room visits and hospitalizations in patients with Chronic obstructive pulmonary disease (COPD) and type 2 Diabetes Mellitus

BackgroundThe Sodium-Glucose Cotransporter 2 inhibitors (SGLT2i) are a class of anti-diabetic medications that confer cardio-renal-metabolic (CRM) benefits. Emerging evidence also suggests that these agents provide better benefits for chronic pulmonary conditions, especially chronic obstructive pulmonary disease (COPD). Research questionWe aimed to assess the association between SGLT2i use and outcomes in patients with COPD and concomitant Type 2 Diabetes Mellitus (T2DM). Study design and methodsWe conducted a retrospective cohort study on adults with T2DM and COPD in a primary care clinic from January 01, 2019 to 01/01//2023. Patients were categorized into two groups based on SGLT2i use. We collected demographic information and outcomes such as emergency room (ER) visits, hospitalizations secondary to COPD exacerbation over the period of four years and time to hospitalization and ER visits. Chi-square analysis was used for categorical variables, whereas an unpaired t-test was used for continuous variables. Cox regression was performed to identify significant prognostic factors of hospitalization and ER visits. A Kaplan-Meir analysis was used to visualize the probability of non-hospitalization and the probability of not visiting the ER. Statistical significance was set at p-value <0.05. ResultsOf the 220 patients screened, 94 met the inclusion criteria, of which 20 patients (21.3 %) had SGLT2i use at admission, and 74 (78.7 %) did not. Baseline demographic information were well-matched between the two groups. SGLT2i use was associated with a significant reduction in ER visits (70 % vs. 97.3 %, p-0.001) and the number of hospitalizations (55 % vs 87.8 %, p-0.001). Further multivariate analysis showed lower hazards of hospitalization (adjusted HR-0.156; CI:0.073 to 0.331) and ER visits (HR)-0.232; CI:0.118 to 0.453) in patients on SGLT2i. InterpretationIn patients with T2DM with COPD, SGLT2i use was associated with reduced ER visits and hospitalizations related to COPD. This protective effect of SGLT2i could be explained by reduced systemic proinflammatory markers and increased anti-inflammatory markers via inhibition of Node like receptor protein 3(NLRP3) inflammasome activation in multiple tissues, including the lungs.

Sex-specific decline in prefrontal cortex mitochondrial bioenergetics in aging baboons correlates with walking speed.

Mitochondria play a crucial role in brain aging due to their involvement in bioenergetics, neuroinflammation and brain steroid synthesis. Mitochondrial dysfunction is linked to age-related neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. We investigated changes in the activities of the electron transport chain (ETC) complexes in normally aging baboon brains and determined how these changes relate to donor sex, morning cortisol levels, and walking speed. Using a novel approach, we assessed mitochondrial bioenergetics from frozen prefrontal cortex (PFC) tissues from a large cohort (60 individuals) of well-characterized aging baboons (6.6-22.8 years, approximately equivalent to 26.4-91.2 human years). Aging was associated with a decline in mitochondrial ETC complexes in the PFC, which was more pronounced when activities were normalized for citrate synthase activity, suggesting that the decline in respiration is predominantly driven by changes in the specific activity of individual complexes rather than changes in mitochondrial number. Moreover, when donor sex was used as a covariate, we found that mitochondrial respiration was preserved with age in females, whereas males showed significant loss of ETC activity with age. Males had higher activities of each individual ETC complex and greater lactate dehydrogenase activity relative to females. Circulating cortisol levels correlated only with complex II-linked respiration in males. We also observed a robust positive predictive relationship between walking speed and respiration linked to complexes I, III, and IV in males but not in females. This data reveals a previously unknown link between aging and bioenergetics across multiple tissues linking frailty and bioenergetic function. This study highlights a potential molecular mechanism for sexual dimorphism in brain resilience and suggests that in males changes in PFC bioenergetics contribute to reduced motor function with age.

An initial game-theoretic assessment of enhanced tissue preparation and imaging protocols for improved deep learning inference of spatial transcriptomics from tissue morphology.

The application of deep learning to spatial transcriptomics (ST) can reveal relationships between gene expression and tissue architecture. Prior work has demonstrated that inferring gene expression from tissue histomorphology can discern these spatial molecular markers to enable population scale studies, reducing the fiscal barriers associated with large-scale spatial profiling. However, while most improvements in algorithmic performance have focused on improving model architectures, little is known about how the quality of tissue preparation and imaging can affect deep learning model training for spatial inference from morphology and its potential for widespread clinical adoption. Prior studies for ST inference from histology typically utilize manually stained frozen sections with imaging on non-clinical grade scanners. Training such models on ST cohorts is also costly. We hypothesize that adopting tissue processing and imaging practices that mirror standards for clinical implementation (permanent sections, automated tissue staining, and clinical grade scanning) can significantly improve model performance. An enhanced specimen processing and imaging protocol was developed for deep learning-based ST inference from morphology. This protocol featured the Visium CytAssist assay to permit automated hematoxylin and eosin staining (e.g. Leica Bond), 40×-resolution imaging, and joining of multiple patients' tissue sections per capture area prior to ST profiling. Using a cohort of 13 pathologic T Stage-III stage colorectal cancer patients, we compared the performance of models trained on slide prepared using enhanced versus traditional (i.e. manual staining and low-resolution imaging) protocols. Leveraging Inceptionv3 neural networks, we predicted gene expression across serial, histologically-matched tissue sections using whole slide images (WSI) from both protocols. The data Shapley was used to quantify and compare marginal performance gains on a patient-by-patient basis attributed to using the enhanced protocol versus the actual costs of spatial profiling. Findings indicate that training and validating on WSI acquired through the enhanced protocol as opposed to the traditional method resulted in improved performance at lower fiscal cost. In the realm of ST, the enhancement of deep learning architectures frequently captures the spotlight; however, the significance of specimen processing and imaging is often understated. This research, informed through a game-theoretic lens, underscores the substantial impact that specimen preparation/imaging can have on spatial transcriptomic inference from morphology. It is essential to integrate such optimized processing protocols to facilitate the identification of prognostic markers at a larger scale.

BayeSMART: Bayesian clustering of multi-sample spatially resolved transcriptomics data.

The field of spatially resolved transcriptomics (SRT) has greatly advanced our understanding of cellular microenvironments by integrating spatial information with molecular data collected from multiple tissue sections or individuals. However, methods for multi-sample spatial clustering are lacking, and existing methods primarily rely on molecular information alone. This paper introduces BayeSMART, a Bayesian statistical method designed to identify spatial domains across multiple samples. BayeSMART leverages artificial intelligence (AI)-reconstructed single-cell level information from the paired histology images of multi-sample SRT datasets while simultaneously considering the spatial context of gene expression. The AI integration enables BayeSMART to effectively interpret the spatial domains. We conducted case studies using four datasets from various tissue types and SRT platforms, and compared BayeSMART with alternative multi-sample spatial clustering approaches and a number of state-of-the-art methods for single-sample SRT analysis, demonstrating that it surpasses existing methods in terms of clustering accuracy, interpretability, and computational efficiency. BayeSMART offers new insights into the spatial organization of cells in multi-sample SRT data.

Mass mortality associated with carpione rhabdovirus in golden pompano (Trachinotus ovatus) in China: First report

From August to November 2023, a severe epizootic outbreak occurred in an offshore cultured golden pompano (Trachinotus ovatus) population in Zhanjiang City, Guangdong Province, China, resulting in a cumulative mortality rate of 65–82 %. The diseased fish showed clinical signs of lethargy, anorexia, whirling movements, and hemorrhage at the base of the fins and in the upper and lower jaws before mass mortality. A rhabdovirus strain, designated as CARPV2023, was successfully isolated using fathead minnow (FHM) cells. The cytopathic effects of the tissue filtrate from the diseased golden pompano on FHM and epithelioma papulosum cyprini (EPC) cell monolayers were characterized by rounded cells, grape-like cluster formation, detachment, and lysis. Histopathology revealed congestion, hemorrhage, and necrosis in the spleen, liver, trunk kidney, and intestinal tissues of naturally and experimentally infected golden pompanos. Electron microscopy showed that bullet-shaped viral particles, measuring 183.5–217.6 nm in length and 57.3–82.7 nm in width, budded from the cell membrane or aggregated around the infected FHM cells. The virus remained stable for 7 d at 4–33 °C and replicated optimally at 28 °C. Whole-genome sequencing and subsequent phylogenetic analysis further indicated that the carpione rhabdovirus (CARPV)2023 strain is a new carpione rhabdovirus, and is related to novirhabdoviruses, however, is thus far unclassified. A specific nested reverse transcription-polymerase chain reaction assay confirmed the presence of CARPV in the affected golden pompano. Significantly higher mortality was observed in challenged golden pompanos than that in the controls through intraperitoneal injection and immersion infection. Fluorescence in situ hybridization showed showed positive reactions for RNA in multiple tissues (liver, spleen, trunk kidney, brain, heart, gill, and muscle) from infected fish. The highest expression level was confirmed in the spleen through qPCR. This is the first study to report natural CARPV infections in marine fish.

Alkyl Pyridinol Compounds Exhibit Antimicrobial Effects against Gram-Positive Bacteria.

Background/Objectives. The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen, causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit the growth or impair the survival of bacterial pathogens. Given their large structural and chemical diversity, as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against Gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin-resistant S. aureus (MRSA) and the multi-drug-resistant community-acquired strain USA300LAC. Methods. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Results/Conclusions. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 μg/mL), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane-associated mechanism of action.

Expression patterns of housekeeping genes and tissue-specific genes in black goats across multiple tissues

Black goats are a significant meat breed in southern China. To investigate the expression patterns and biological functions of genes in various tissues of black goats, we analyzed housekeeping genes (HKGs), tissue-specific genes (TSGs), and hub genes (HUBGs) across 23 tissues. Additionally, we analyzed HKGs in 13 tissues under different feeding conditions. We identified 2968 HKGs, including six important ones. Interestingly, HKGs in grazing black goats demonstrated higher and more stable expression levels. We discovered a total of 9912 TSGs, including 134 newly identified ones. The number of TSGs for mRNA and lncRNA were nearly equal, with 127 mRNA TSGs expressed solely in one tissue. Additionally, the predicted functions of tissue-specific long non-coding RNAs (lncRNAs) targeting mRNAs corresponded with the physiological functions of the tissues.Weighted gene co-expression network analysis (WGCNA) constructed 30 modules, where the dark grey module consists almost entirely of HKGs, and TSGs are located in modules most correlated with their respective tissues. Additionally, we identified 289 HUBGs, which are involved in regulating the physiological functions of their respective tissues. Overall, these identified HKGs, TSGs, and HUBGs provide a foundation for studying the molecular mechanisms affecting the genetic and biological processes of complex traits in black goats.

Transcriptomic Analysis Reveals Diverse Expression of Scorpion Toxin Genes in Mesobuthus martensii.

Scorpions, an ancient group of venomous invertebrates, have existed for over 430 million years. Their toxins, important for predation and defense, exhibit a variety of biological and pharmacological activities. Research on scorpion toxins has spanned decades. Notably, the toxin genes of Mesobuthus martensii (Scorpiones: Buthidae), a well-known Chinese herbal medicine, have been described at genomic and proteomic levels. However, previous studies primarily focused on the toxin genes expressed in the venom glands, overlooking their expression in multiple tissues. This study analyzed transcriptomes from 14 tissues of M. martensii. Gene annotation revealed 83 toxin and toxin-like genes, including those affecting sodium, potassium, calcium, and chloride ion channels. Approximately 70% of toxin genes were highly expressed in the vesicle; additionally, some exhibited low or no expression in the vesicle while showing high expression in other tissues. Beyond the vesicle, high expression levels of toxin genes were observed in metasoma segments II-V, blood, lateral eyes, chelicerae, legs, pedipalp chelae, femurs, and patellae. This expression pattern suggests that toxin genes are recruited from multiple tissues and may help prevent intraspecific harm during courtship and competition for prey. These findings inspire further research into the evolutionary recruitment process of scorpion toxins.

Progress on three-dimensional visualizing skin architecture with multiple immunofluorescence staining and tissue-clearing approaches.

The skin forms the external covering of the body and is its largest organ, comprising many different cell types. Although the diversity of these cells has been widely studied with various histological methods, our understanding of skin architecture is mainly established on thin tissue sections, which restricted the information available to two dimensions. The development of innovative techniques to induce optical transparency ("clearing") in biological tissues has enabled researchers to visualize the three-dimensional reconstruction of intact organs and thick tissue sections at a cellular resolution. With the aid of tissue-clearing treatment, the labeled cutaneous nerve fibers and blood vessels can be followed for a longer distance on the thicker skin section or the whole mount skin under a fluorescence microscopy or a confocal microscopy. It is beneficial for demonstrating the morphological characteristics of nerve fibers and blood vessels themselves, as well as their spatial interconnection. In this review, we provide a brief summary of the literature on the use of tissue optical clearing methods and describe our experience of multiple fluorescent staining and tissue clearing approaches on thicker skin sections and whole-mount skin in our laboratory. Given the existing conventional methods, we expected to provide a more effective approach to comprehensively study skin architecture.

Abstract C055: Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element

Abstract Genome-wide association studies (GWAS) have identified independent signals at 5p15.33 across numerous cancers, with protective alleles for one cancer often conferring risk for another. Many of these associations are thought to act via allele-specific alterations in the cis- regulation of target genes. Transcriptomic analyses in multiple tissue types implicate two plausible target genes: TERT and CLPTM1L. However, the mechanisms linking cancer susceptibility alleles to their presumed target genes, along with the observed antagonistic pleiotropy between different cancers remain elusive. With this in mind, we employed a comprehensive approach, integrating statistical fine-mapping of GWAS summary statistics with massively parallel reporter assays (MPRA) and a densely tiled CRISPRi screen performed across eight cell lines, representative of four distinct cancers associated with 5p15.33 alleles – pancreatic, melanoma, lung, and bladder cancer. Integrative screening identified up to 30 variants across multiple signals in the locus with putative allele-specific transcriptional activity. Furthermore, a variable number tandem repeat (VNTR) in intron 9 of CLPTM1L emerged as a potent transcriptional enhancer in CRISPRi screening. Leveraging PacBio sequencing, VNTR alleles of European ancestry samples from the 1000 Genomes Project were genotyped and found to be highly polymorphic, with longer VNTRs linked to the haplotype tagged by rs31490-A (risk increasing for pancreatic cancer). Subsequent imputation into a pancreatic cancer GWAS of 9,040 cases and 12,496 controls revealed an association between longer VNTRs and risk of pancreatic cancer (OR for VNTR in the 75th percentile = 1.17, 95% CI = 1.13-1.22, P = 2.21x10-12). The significance of the association for the lead SNP rs31490 after conditioning on the VNTR genotype dropped to P= 2.20x10-5, suggesting that VNTR alleles merit consideration as part of the credible variant set for pancreatic cancer. Targeted CRISPRi assays suggested VNTR-mediated cis-regulation of both TERT and CLPTM1L in PANC-1 and MIA PaCa-2 pancreatic cancer cell lines, while dual luciferase reporter assays in the same cell lines revealed that VNTR enhancer activity is mediated by Hippo pathway transcription factors. In ongoing experiments, we aim to expand on luciferase reporter assays and DNA-protein binding assays in relevant cell lines with the aim of not only deciphering how longer VNTRs differentially exert their transcriptional effects compared to shorter alleles but also characterizing the divergent directions of effect between different cancer types. These findings underscore the proposition that pancreatic cancer susceptibility at the 5p15.33 locus may be mediated by both SNPs and an uncharted class of variants, yet to be characterized in the post-GWAS landscape. Citation Format: Aidan O'Brien, Hyunkyung Kong, Minal Patel, Katelyn E Connelly, Mai Xu, Irene Collins, Jun Zhong, Jason Hoskins, Michelle Ho, Brenen Papenburg, Mark M Iles, Matthew H Law, Maria-Teresa Landi, Rachael Stolzenber- Solomon, Brian M Wolpin, Alison P Klein, Nick Orr, Stephen J Chanock, Sara Lindström, Marc-Henri Stern, Ludmila Prokunina-Olsson, Jieyon Choi, Kevin M Brown, Laufey T Amundadottir. Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C055.

Immune and microbiome modulatory effects of Limosilactobacillus fermentum NCDC 400 in an immunocompromised mouse model

The present study was aimed to assess and validate the safety and functional efficacy of an indigenous probiotic strain Limosilactobacillus fermentum NCDC 400 (hereafter, LFN400) in an immunocompromised murine model. The study included four groups; a normal control (NC) group without immune suppression; an experimental model control (MC) with immune suppression induced via intraperitoneal cyclophosphamide (Cy) administration; and two MC groups orally administered with either low dose (LD) or high dose (HD) of LFN400 at dose 108 and 1010 CFU/mouse/day, respectively, for 15-days. Both control groups received normal saline as placebo control. LFN400 improved specific experimental characteristics including hematological and serum biochemical markers. Compared to MC group, LFN400-fed groups showed markedly (P < 0.05) decreased arrays of detrimental caecal enzymes. We did not observe instances of bacterial translocation of LFN400 from gut to bloodstream or extra-intestinal organs. LFN400 intake significantly (P < 0.05) enhanced spleen cell differentiation, immune and oxidative stress markers, and restored Cy-induced histopathological changes in multiple tissues, including the spleen. There was no genotoxic effect of LFN400 on bone marrow cells. Although not statistically significant, LFN400 feeding moderately increased gut microbiome diversity, supporting the growth of beneficial saccharolytic microorganisms and reducing the presence of pathobionts. The findings demonstrate that the probiotic strain LFN400 possesses in vivo safety and immunomodulatory potency and thus should be considered a potential candidate for future human clinical studies.

Switch-like Gene Expression Modulates Disease Susceptibility.

A fundamental challenge in biomedicine is understanding the mechanisms predisposing individuals to disease. While previous research has suggested that switch-like gene expression is crucial in driving biological variation and disease susceptibility, a systematic analysis across multiple tissues is still lacking. By analyzing transcriptomes from 943 individuals across 27 tissues, we identified 1,013 switch-like genes. We found that only 31 (3.1%) of these genes exhibit switch-like behavior across all tissues. These universally switch-like genes appear to be genetically driven, with large exonic genomic structural variants explaining five (~18%) of them. The remaining switch-like genes exhibit tissue-specific expression patterns. Notably, tissue-specific switch-like genes tend to be switched on or off in unison within individuals, likely under the influence of tissue-specific master regulators, including hormonal signals. Among our most significant findings, we identified hundreds of concordantly switched-off genes in the stomach and vagina that are linked to gastric cancer (41-fold, p<10-4) and vaginal atrophy (44-fold, p<10-4), respectively. Experimental analysis of vaginal tissues revealed that low systemic levels of estrogen lead to a significant reduction in both the epithelial thickness and the expression of the switch-like gene ALOX12. We propose a model wherein the switching off of driver genes in basal and parabasal epithelium suppresses cell proliferation therein, leading to epithelial thinning and, therefore, vaginal atrophy. Our findings underscore the significant biomedical implications of switch-like gene expression and lay the groundwork for potential diagnostic and therapeutic applications.