Tissue Patterning Research Articles

HHLA2 deficiency inhibits pancreatic cancer progression and THP-1 macrophage M2 polarization via EGFR/MAPK/ERK and mTOR/AKT pathway

BackgroundHuman endogenous retrovirus subfamily H long terminal repeat associating protein 2, (HHLA2), a member of B7 family, exhibits heightened expression in various malignant tumors. However, the exact functions of HHLA2 in pancreatic cancer (PC) remain incompletely elucidated.MethodsWe initially conducted an analysis of the B7 family members’ expression pattern in pancreatic tumor samples and adjacent normal tissues using The Cancer Genome Atlas (TCGA) database. Subsequently, immunohistochemistry, RT-qPCR and western blot methods were used to assess HHLA2 expression levels in PC tissues and cell lines. Furthermore, after silencing HHLA2 in PC cell lines, cell migration and proliferation of PC cells were detected by wound healing and CCK-8 assays, and cell invasion of PC cells was detected by transwell assays. We also investigated the regulation of epithelial-mesenchymal transition (EMT) markers and levels of EGFR, MEK, ERK1/2, mTOR and AKT via western blot analysis. Finally, the correlation between HHLA2 expression and immune infiltration was further explored.ResultsSilencing of HHLA2 resulted in the inhibition of PC cell proliferation, migration and invasion, potentially through the suppression of the EGFR/MAPK/ERK and mTOR/AKT signaling pathway. Additionally, silencing HHLA2 led to the inhibition of M2-type polarization of tumor associated macrophages (TAMs).ConclusionThe knockdown of HHLA2 was observed to inhibit the migration and invasion of PC cells through the regulation of the EMT process and EGFR/MAPK/ERK and mTOR/AKT pathway. Furthermore, silencing HHLA2 was found to modulate M2 polarization of TAMs. These finding suggest that HHLA2 could be a promising therapeutic target for Pancreatic cancer.

Autonomous and Self-sustained Circadian Oscillators Displayed in Human Primary Parathyroid Cell Culture Drive Parathormone Secretion

Abstract Background Most cells in our body possess circadian oscillators controlling diurnal rhythmicity of body metabolism. The role of molecular clocks in regulating the function and disfunction of human parathyroid gland (PG), responsible for calcium homeostasis, has not been unraveled. Hyperparathyroidism is a common endocrine pathology, characterized by an unadapted PTH secretion to calcium levels. Aims Here we aimed at characterizing molecular makeup of circadian clocks in human primary parathyroid cell culture (HPPCC), and at establishing differential transcriptional patterns of normal and pathological PGs. Methods RNA extracted from normal, adenomatous, and hyperplastic PG tissues was subjected to RNA sequencing (RNAseq) analysis. The rim of normal PG tissue was dissected from parathyroid adenoma specimens based on the higher autofluorescence intensity assessed by the near infra-red imaging. HPPCCs were established from adenomatous and hyperplastic PG biopsies. Efficient circadian clock disruption was achieved in HPPCCs by small interfering RNA-mediated knockdown of CLOCK. The functionality of circadian clock machinery was assessed by continuous recording of circadian bioluminescence introduced via lentivectors and paralleled with around-the-clock measurement of PTH secretion using perfusion system. Results The RNAseq analyses revealed transcriptional signatures that distinguished between pathological and normal PGs, and among adenomatous and hyperplastic PGs. We report, for the first time, robust anti-phasic circadian rhythmicity of Per2-luciferase and Bmal1-luciferase reporters in HPPCCs synchronized in vitro. Strikingly, we observed circadian rhythmicity of PTH secretion by parathyroid adenoma cells. Moreover, clock disruption in PG adenoma cells has an impact on transcription of functional genes. Conclusion Our data indicate presence of cell-autonomous molecular clock in human PG cells paralleled with circadian rhythmic PTH secretion and provide large-scale transcriptional pattern of parathyroid adenoma and hyperplastic tissues.

Sequence and epigenetic variations of R2R3-MYB transcription factors determine the diversity of taproot skin and flesh colors in different cultivated types of radish (Raphanus sativus L.).

This study found that three paralogous R2R3-MYB transcription factors exhibit functional divergence among different subspecies and cultivated types in radish. Cultivated radish taproots exhibit a wide range of color variations due to unique anthocyanin accumulation patterns in various tissues. This study investigated the universal principles of taproot color regulation that developed during domestication of different subspecies and cultivated types. The key candidate genes RsMYB1 and RsMYB2, which control anthocyanin accumulation in radish taproots, were identified using bulked segregant analysis in two genetic populations. We introduced the RsMYB1-RsF3'H-RsMYB1Met genetic model to elucidate the complex and unstable genetic regulation of taproot flesh color in Xinlimei radish. Furthermore, we analyzed the expression patterns of three R2R3-MYB transcription factors in lines with different taproot colors and investigated the relationship between RsMYB haplotypes and anthocyanin accumulation in a natural population of 56 germplasms. The results revealed that three paralogous RsMYBs underwent functional divergence during radish domestication, with RsMYB1 regulating the red flesh of Xinlimei radish, and RsMYB2 and RsMYB3 regulating the red skin of East Asian big long radish (R. sativus var. hortensis) and European small radish (R. sativus var. sativus), respectively. Moreover, RsMYB1-H1, RsMYB2-H10, and RsMYB3-H6 were identified as the primary haplotypes exerting regulatory functions on anthocyanin synthesis. These findings provide an understanding of the genetic mechanisms regulating anthocyanin synthesis in radish and offer a potential strategy for early prediction of color variations in breeding programs.

SPINK4 modulates inhibition of glycolysis against colorectal cancer progression.

Dysregulation of glycolysis is frequently linked to aggressive tumor activity in colorectal cancer (CRC). Although serine peptidase inhibitor, Kazal type 4 (SPINK4) has been linked to CRC, its exact linkage to glycolytic processes and gene expression remains unclear. Differentially expressed genes (DEGs) were screened from two CRC-related datasets (GSE32323 and GSE141174), followed by expression and prognostic analysis of SPINK4. In vitro techniques such as flow cytometry, western blotting, transwell assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to assess SPINK4 expression in CRC cells. Its effects on apoptosis, glycolysis, and the cell cycle were also investigated. Finally, the impact of SPINK4 overexpression on tumor development was assessed using a xenograft model, while histological and immunohistochemical analyses characterized SPINK4 expression patterns in CRC tissues. SPINK4 expression was downregulated in CRC, correlating with poor patient prognosis. In vitro assays confirmed that overexpression of SPINK4 reduced CRC cell proliferation, invasion, and migration, while its knockdown promoted these processes and caused G1 arrest. SPINK4 also regulated apoptosis by altering caspase activation and Bcl-2 expression. Besides, SPINK4 overexpression altered glycolytic activity, reduced 2-Deoxy-D-glucose (2-DG) absorption, and controlled critical glycolytic enzymes, resulting in alterations in metabolic pathways, whereas SPINK4 knockdown reversed this effect. SPINK4 overexpression significantly reduced tumor volume in vivo, indicating its inhibitory role in carcinogenesis. Moreover, high expression of SPINK4, hexokinase 2 (HK2), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and pyruvate kinase M2 (PKM2) was observed in CRC tissues. As a key inhibitor of glycolytic metabolism in CRC, SPINK4 promises metabolic intervention in CRC therapy due to its impact on tumor growth and cell proliferation.

Histopathological and Molecular Characteristics of Malignant Breast Tumors in the Kurdish Population During 2019 - 2021

Background: Breast cancer, the most prevalent cancer in women and the second leading cause of cancer-related deaths worldwide, is a complex and heterogeneous disease characterized by multiple tumor types with distinct tissue patterns, biological characteristics, and clinical behaviors. Given the critical role of histopathological and molecular characteristics in the treatment and management of breast cancer. Objectives: This study was undertaken to investigate the histopathological characteristics of malignant breast masses in the city of Sanandaj from 2019 to 2021. Methods: In this descriptive-analytical study, out of 1,631 pathology reports from breast tissue samples collected in Sanandaj, a city in northwest Iran, between 2019 and 2021, 597 (36%) malignant cases were examined for their pathological type and molecular characteristics. Data analysis was performed using SPSS 26 software, employing chi-square and Fisher's exact tests. Results: The mean age of the patients at diagnosis was 49.7 ± 11.9 years. The most prevalent molecular subtype was luminal A (56.4%), followed by triple-negative (16.7%), human epidermal growth factor receptor 2 (HER2)-positive/enriched (14.1%), and luminal B (14%). Invasive ductal carcinoma was the predominant histological type, accounting for 82.7% of cases, followed by lobular invasive carcinoma (8.2%). Papillary carcinoma accounted for 5.9% of cases, metaplastic carcinoma for 1.8%, and mucinous carcinoma for 1.3%. Higher grades at the time of diagnosis were associated with the metaplastic subtype (P < 0.001). The only biomarker that showed a significant association with pathological types of breast cancer was Ki-67. The percentage of cases positive for Ki-67 was higher in three types of breast cancer: Infiltrating ductal carcinoma, metaplastic, and papillary (intraductal) carcinoma, compared to the other types (P < 0.001). Conclusions: According to our data, infiltrating ductal and lobular carcinoma are the most prevalent pathological types, and luminal A is the most common molecular subtype among Kurdish women in Iran. It is recommended that future studies examine patient survival based on the pathological and molecular types of tumors, including the evaluation of additional biomarkers. Furthermore, comparing these results with those from other ethnic groups could provide valuable insights into the impact of ethnicity on breast cancer characteristics and outcomes.

Dynamic microvilli sculpt bristles at nanometric scale

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.

Disruption of microRNA pathway core genes inhibits molting and reproduction of the cigarette beetle, Lasioderma serricorne.

MicroRNA (miRNA) pathway genes have been widely reported to participate in several physiological events in insect lifecycles. The cigarette beetle Lasioderma serricorne is an economically important storage pest worldwide. However, the functions of miRNA pathway genes in L. serricorne remain to be clarified. Herein, we investigated the function of molting and reproduction of the miRNA pathway in L. serricorne. LsDicer-1, LsArgonaute-1, LsLoquacious and LsExportin-5 were universally expressed in adults, whereas LsPasha and LsDrosha were mainly expressed in the pupae. The genes presented different patterns in various tissues. Silencing of LsDicer-1, LsArgonaute-1, LsDrosha and LsExportin-5 resulted in a high proportion of wing deformities and molting defects. Silencing of LsDicer-1, LsArgonaute-1, LsPasha and LsLoquacious affected the development of the ovary and the maturation of oocytes, resulting in a significant decrease in fecundity. Further investigation revealed that the decreases in LsDicer-1 and LsArgonaute-1 expression destroyed follicular epithelia and delayed vitellogenesis and oocyte development. In addition, the expression levels of several miRNAs (let-7, let-7-5p, miR-8-3p, miR-8-5p, miR-9c-5p, miR-71, miR-252-5p, miR-277-3p, miR-263b and Novel-miR-50) were decreased significantly after knockdown of these miRNA pathway core genes, indicating that they played important roles in regulating miRNA-mediated gene expression. The results indicate that miRNA pathway genes play important roles in the molting, ovarian development and female fecundity of L. serricorne, and thus are potentially suitable target genes for developing an RNAi strategy against a major pest of stored products. © 2024 Society of Chemical Industry.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of ‘open’ chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

WhARIO: whole-slide-image-based survival analysis for patients treated with immunotherapy.

Immune checkpoint inhibitors (ICIs) are now one of the standards of care for patients with lung cancer and have greatly improved both progression-free and overall survival, although of the patients respond to the treatment, and some face acute adverse events. Although a few predictive biomarkers have integrated the clinical workflow, they require additional modalities on top of whole-slide images and lack efficiency or robustness. In this work, we propose a biomarker of immunotherapy outcome derived solely from the analysis of histology slides. We develop a three-step framework, combining contrastive learning and nonparametric clustering to distinguish tissue patterns within the slides, before exploiting the adjacencies of previously defined regions to derive features and train a proportional hazards model for survival analysis. We test our approach on an in-house dataset of 193 patients from 5 medical centers and compare it with the gold standard tumor proportion score (TPS) biomarker. On a fivefold cross-validation (CV) of the entire dataset, the whole-slide image-based survival analysis for patients treated with immunotherapy (WhARIO) features are able to separate a low- and a high-risk group of patients with a hazard ratio (HR) of 2.29 ( to 3.56), whereas the TPS 1% reference threshold only reaches a HR of 1.81 ( to 2.69). Combining the two yields a higher HR of 2.60 ( to 3.94). Additional experiments on the same dataset, where one out of five centers is excluded from the CV and used as a test set, confirm these trends. Our uniquely designed WhARIO features are an efficient predictor of survival for lung cancer patients who received ICI treatment. We achieve similar performance to the current gold standard biomarker, without the need to access other imaging modalities, and show that both can be used together to reach even better results.

Genome-wide identification of SAMS gene family in Cucurbitaceae and the role of ClSAMS1 in watermelon tolerance to abiotic stress

S-Adenosyl-L-methionine (SAM) is widely involved in plant growth, development, and abiotic stress response. SAM synthetase (SAMS) is the key enzyme that catalyzes the synthesis of SAM from methionine and ATP. However, the SAMS gene family has not been identified and their functions have not been characterized in most Cucurbitaceae plants. Here, a total of 30 SAMS genes were identified in nine Cucurbitaceae species and they were categorized into 3 subfamilies. Physicochemical properties and gene structure analysis showed that the SAMS protein members are tightly conserved. Further analysis of the cis-regulatory elements (CREs) of SAMS genes' promoter implied their potential roles in stress tolerance. To further understand the molecular functions of SAMS genes, watermelon SAMSs (ClSAMSs) were chosen to analyze the expression patterns in different tissues and under various abiotic stress and hormone responses. Among the investigated genes, ClSAMS1 expression was observed in all tissues and found to be up-regulated by abiotic stresses including salt, cold and drought treatments as well as exogenous hormone treatments including ETH, SA, MeJA and ABA. Furthermore, knockdown of ClSAMS1 via virus-induced gene silencing (VIGS) decreased SAM contents in watermelon seedings. The pTRSV2-ClSAMS1 plants showed reduced susceptibility to drought, cold and NaCl stress, indicating a positive role of ClSAMS1 in abiotic stresses tolerance. Those results provided candidate SAMS genes to regulate plant resistance against abiotic stresses in Cucurbitaceae plants.

Characterization and expression profiling of buffalo IFN-lambda family

Interferon lambda (IFN-λ) is an important type III interferon triggered mainly by viral infection. IFN-λ binds to their heterodimeric receptors and signals through JAK-STAT pathways similar to type I IFN. In this study, we deduced the buffalo IFN-λ sequences through the polymerase chain reaction, and then studied IFN-λ’s expression patterns in different tissues, and post induction with poly I:C and live MRSA using RT-qPCR. The full-length sequences of buffalo IFN-λ3, IFN-λ receptors, and a transcript variant of IFN-λ4 were determined. IFN-λ1 is identified as a pseudogene. Virus response elements and a recombination hotspot factor was observed in the regulatory region of IFN-λ. The IFN-λ3 expressed highest in lungs and monocytes but IFN-λ4 did not. The expression of Interferon Lambda Receptor 1 was tissue specific, while Interleukin 10 Receptor subunit beta was ubiquitous. Following poly I:C induction, IFN-λ3 expression was primarily observed in epithelial cells as opposed to fibroblasts, displaying cell type-dependent expression. The cytosolic RNA sensors were expressed highest in endometrial epithelial cells, whereas the endosomal receptor was higher in fibroblasts. 2’,5’-oligoadenylate synthetase expressed higher in fibroblasts, myxoma resistance protein 1 and IFN-stimulated gene 56 in epithelial cells, displaying cell-specific antiviral response of the interferon stimulated genes (ISGs). The endometrial epithelial cells expressed IFN-λ3 after live S. aureus infection indicating its importance in bacterial infection. The induction of IFN-λ3 was S. aureus isolate specific at the same multiplicity of infection (MOI). This study elucidates the IFN-λ sequences, diverse expression patterns revealing tissue specificity, and specificity in response to poly I:C and bacterial stimuli, emphasising its crucial role in innate immune response modulation.

Transforming 'Junk' DNA into Cancer Warriors: The Role of Pseudogenes in Hepatocellular Carcinoma.

In the dynamic landscape of hepatocellular carcinoma (HCC) or the liver cancer research, pseudogenes have emerged from the shadows of genetic obscurity to become central figures, significantly influencing the disease molecular development and clinical trajectory. This review explores a transformative shift in perspective, recognizing pseudogenes not as genetic remnants without function, but as critical regulators in the molecular underpinnings of HCC. Engaging in complex interactions such as microRNA sponging, gene expression modulation, and signaling pathway disruptions, pseudogenes orchestrate a part of the molecular complexity driving tumor genesis, progression, and drug resistance in the liver cancer. Their unique expression patterns in hepatoma tissues herald new opportunities for early HCC detection, offering insights into patient prognosis, and identifying novel targets for therapeutic intervention of this disease. Such advancements underscore the importance of pseudogenes in enriching our understanding and management of HCC, paving the way for more effective diagnostic strategies and targeted therapies in the ongoing battle against this challenging malignancy.

Abstract PO5-07-06: Impact of patient-specific factors on quantitative breast parenchymal texture features

Abstract Breast parenchymal texture characterizes the spatial and structural patterns of breast tissue and is an independent predictor of breast cancer risk. Although it has conceptual similarities with another breast composition measure, breast density, parenchymal texture is more complex and is represented by multiple quantitative measures. The extent to which the epidemiology and biological basis of parenchymal texture and breast density differ has not been well described. In this cross-sectional study, we evaluated quantitative measures of parenchymal texture among 308 postmenopausal women aged ≥ 45 years who participated in a routine breast cancer screening at an academic medical center between 2020 and 2022. After providing informed consent, participants completed a short interview-based questionnaire. Additional demographic and breast cancer risk factor information was obtained from the medical record. Parenchymal texture features were measured from craniocaudal views of full-field digital mammograms. Measured texture values were standardized and averaged across left and right breasts. For this analysis, we evaluated associations between features previously validated as being associated with breast cancer risk (fractal dimension, grey-level mean, co-occurrence entropy and inverse difference moment) and patient personal and clinical characteristics reported at the time of screening. Associations between texture features and participant characteristics were evaluated using Pearson correlations (continuous variables) or the Kruskal-Wallis test (categorical variables). Generalized linear models were used to adjust for technical factors associated with image acquisition, including mammography machine, compression force, and software version. P-values less than 0.05 were considered statistically significant. Among the 308 participants, 70% were White, 24% were Black, 1% were Asian, 1% were American Indian, and 4% were another race that was not specified. 4% of participants reported Hispanic ethnicity. The mean age was 64.7 years (range 45 – 84 years). Fewer than half of the women were reported by a radiologist to have dense breasts (heterogeneously dense – 24%, extremely dense – 5%, vs. almost entirely fatty – 21%, scattered fibroglandular densities – 50%). The mean body mass index (BMI) was 29.4 kg/m2 (range 19.4 to 53.1 kg/m2) and 77% of the women were parous. BMI was strongly correlated with fractal dimension (r=0.62, P< 0.01) and grey-level mean (r=-0.48, P< 0.01), and had a weaker correlation with co-occurrence entropy (r=-0.19, P< 0.01). Age was weakly correlated with co-occurrence inverse difference moment (r=-0.12, P=0.03). BI-RADS breast density was inversely associated with fractal dimension, grey-level mean, and co-occurrence entropy (all P< 0.01). Adjustment for technical factors attenuated some associations, but BMI associations with fractal dimension (P< 0.01) and gray-level mean (P< 0.01), and breast density associations with fractal dimension, grey-level mean, and co-occurrence entropy (all P< 0.01) all remained statistically significant. Parity was not associated with any of the features evaluated in unadjusted or adjusted analyses (all P >0.05). These data suggest that, in postmenopausal women, risk-associated parenchymal texture features are associated with measures affected by body size and adiposity, but not with measures related to age or reproductive status. Longitudinal studies are needed to understand the temporality of these associations. Additionally, future studies will address the joint effects of texture features and the impact of endogenous estrogen levels on the features, which will provide insight into the biological mechanisms that influence texture feature associations with personal characteristics and breast cancer risk. Citation Format: Sarah Nyante, Yukie Kajita, Walter Mankowski, Ley Killeya, Despina Kontos, Xianming Tan, Eric Cohen, Cherie Kuzmiak. Impact of patient-specific factors on quantitative breast parenchymal texture features [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-07-06.

Abstract PO3-13-08: D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease

Abstract Background: Obesity is a recognized risk factor for the development of metabolic disease and breast cancer possibly due to chronic inflammation mediated by cytokines secreted by adipose tissue. Given this, our group examined differential expression of several markers of chronic inflammation in adipose tissue of both benign and malignant breast tissue from patients. The aryl hydrocarbon receptor (AhR) is an important cytosolic, ligand-dependent transcription factor found within hepatocytes and adipocytes that is upregulated in many types of cancer. AhR promotes the initiation, progression, and metastasis of cancer cells allowing certain cancers to evade immune recognition (Xue et al. Front. Immunology 2018). Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that generates kynurenine (KYN) from tryptophan (TRP), which acts as an AhR agonist. We sought to determine expression of IDO1 via immunohistochemistry (IHC) in mammary adipose tissue associated with benign or malignant breast. We hypothesized that adipose tissue adjacent to malignant breast tissue may have heightened expression of IDO1 in addition to Cyp1b1, and SPP1; both of which are also markers of increased aryl-hydrocarbon transcriptional activity, compared to adipose tissue noncontiguous to malignant lesions. KYN affects gene expression for insulin resistance and levels are elevated in plasma of older obese women compared to lean women; however, the relationship to breast malignancy is not known. Methods: IDO1 (Cell Signaling, 1:200), Cyp1b1 (Thermo Fisher, 1:300), and SPP1 (Thermo Fisher, 1:200) IHC was performed according to the manufacturers' protocols. Adipose tissue immediately adjacent to, and >2 mm away from, tumors (three DCIS, 11 invasive carcinomas) was evaluated in 14 patients. Adipose tissue in separate tissue blocks from benign breast tissue (three ipsilateral, 11 contralateral) in the same patients was also evaluated. Human plasma KYN levels were assayed by HPLC in the Dartmouth Clinical Pharmacology Shared Resource as described previously (Rojas et al. Obesity 2021). Results: In this preliminary pilot study, there was no difference in staining pattern for IDO1 in adipose tissue present in malignant and benign samples. Two patients showed increased Cyp1b1 and SPP1 staining immediately adjacent to the invasive carcinoma (Figure 1). Patients with benign breast disease did not show increased Cyp1b1 or SPP1 staining. Results will be confirmed using qPCR for mRNA. Plasma samples from patients with benign disease showed a lower level of KYN expression on average (0.370 ug/mL, N = 8) compared to plasma from patients with malignant disease (0.424, N = 10). Additionally, the ratio of KYN:TRP was lower in the benign group (0.065) compared to the cohort with malignancy (0.080). Univariate linear regression will be used to further assess relationships between continuous variables in the study population (e.g., patient age, BMI, KYN plasma levels, benign vs malignant breast disease). Regression lines and the associated slope estimates (b-value) and p-values for regression models will be provided on scatter plots. Conclusions: While there was no difference in IDO1 staining patterns in adipose tissue of malignant and benign samples in this group, tissue samples from patients with invasive carcinoma demonstrated increased Cyp1b1 and SPP1 staining adjacent to the malignant tumor; while benign tissue samples did not show a positive stain. This suggests increased aryl-hydrocarbon transcriptional activity in invasive breast carcinoma—further linking AhR signaling with adipose tissue immune dysregulation and invasive malignancy. Additionally, KYN plasma levels were higher in patients with malignant disease indicating a possible relationship between insulin resistance and breast malignancy. Additional research may offer new insights relating to this important pathway and ultimately treatments aimed at blunting its nefarious effects. Citation Format: Adam Ephraim, Jonathan Pirruccello, Scott Palisoul, Jonathan Marotti, Mary Chamberlin. D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-08.

Overexpression of phosphatidylserine synthetase IbPSS1 enhances salt tolerance by stimulating ethylene signaling-dependent lignin synthesis in sweetpotato roots

Phosphatidylserine (PS) is an important lipid signaling required for plant growth regulation and salt stress adaptation. However, how PS positively regulate plant salt tolerance is still largely unknown. In this study, IbPSS1-overexpressed sweetpotato plants that exhibited overproduction of PS was employed to explore the mechanisms underlying the PS stimulation of plant salt tolerance. The results revealed that the IbPSS1-overexpressed sweetpotato accumulated less Na+ in the stem and leaf tissues compared with the wild type plants. Proteomic profile of roots showed that lignin synthesis-related proteins over-accumulated in IbPSS1-overexpressed sweetpotato. Correspondingly, the lignin content was enhanced but the influx of Na+into the stele was significantly blocked in IbPSS1-overexpressed sweetpotato. The results further revealed that ethylene synthesis and signaling related genes were upregulated in IbPSS1-overexpressed sweetpotato. Ethylene imaging experiment revealed the enhancement of ethylene mainly localized in the root stele. Inhibition of ethylene synthesis completely reversed the PS-overproduction induced lignin synthesis and Na+ influx pattern in stele tissues. Taken together, our findings demonstrate a mechanism by which PS regulates ethylene signaling and lignin synthesis in the root stele, thus helping sweetpotato plants to block the loading of Na+ into the xylem and to minimize the accumulation of Na+ in the shoots.

Two FT genes synergistically regulate the reproductive transition of loquat

Unlike annual flowering plants that exhibit a single reproductive cycle encompassing vegetative growth, flowering, and senescence within a single year, perennial woody trees undergo a transition between vegetative and reproductive growth every year after a long juvenile phase. Most woody fruit trees bloom when it turns warm in spring, and the fruit ripens in summer or autumn. However, as a member of the Maloideae subfamily, the loquat tree blooms in the cold autumn and winter, and the fruit matures in the spring. To explore the regulatory mechanism of loquat flowering, we characterized two FLOWERING LOCUS T homologous genes in loquat, EjFT1 and EjFT2. qRT-PCR results revealed that EjFT1 and EjFT2 exhibited nearly opposite expression patterns in various tissues. EjFT1 was mainly expressed in young tissues; EjFT2 was mainly expressed in mature leaves, open flowers, and fruits. EjFT2 exhibited an obvious circadian rhythm and was regulated by EjCO. After the trees were exposed to short-day conditions or sprayed with exogenous GA3, the expression of EjFT2 was strongly inhibited, and the loquat tree did not produce floral buds. Furthermore, the yeast two-hybrid, bimolecular fluorescence complementation, and Dual-luciferase assay experiments revealed that both EjFT1 and EjFT2 interacted with EjFD, with the EjFT2-EjFD protein complex enhancing the activity of EjAP1-1 and EjAP1-2 promoters, while EjFT1-EjFD inhibited the activity of the EjAP1-1 promoter. Protein structural analysis of EjFT1 and EjFT2 suggested that differences in amino acid residues at Val123/Leu123, Ser157/Ala157, and Val158/Ala158 may be the reason for their functional differences. Our results showed that EjFT1 and EjFT2 may cooperatively regulate the floral bud differentiation of loquat by competitively binding with EjFD. EjFT2 regulates the onset of loquat floral bud differentiation by responding to photoperiod and gibberellin signals, while EjFT1 is involved in the vegetative growth of loquat. These findings provide important clues for the investigation of the regulatory mechanism of loquat flowering and advance the exploration into the multiple roles of FT homologous genes in regulating the reproductive transformation and vegetative growth of flowering plants.

Genome-Wide Characterization of the MADS-Box Gene Family and Expression Pattern in Different Tissues and Stresses in Zanthoxylum armatum

Genome-Wide Characterization of the MADS-Box Gene Family and Expression Pattern in Different Tissues and Stresses in Zanthoxylum armatum

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress

BackgroundWhite clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored.ResultsIn this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana.ConclusionThese findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Thermal Effects of 445 nm Laser Irradiation on the Bovine Tongue Tissues Ex Vivo

The purpose of this study was to employ the use of thermographic techniques to compare temperature changes and thermal heat transfer patterns in soft tissues when using the 445nm-diode laser with initiated and non-initiated tips. Methods: Bovine tongue slices measuring 5 mm in thickness were placed in between two microscopy glass slides at a distance of 11 cm from a thermographic camera. Fifteen 2 cm long incisions were made along the surface of the soft tissue parallel to the glass slides and the camera capture field. Incisions were performed using a 445nm diode laser (continuous wave at 2 watts) with 320μm-thick glass initiated and non-initiated fiber tips for a total 30-second irradiation period. The maximum temperature changes in oC (ΔT) in the soft tissues, as well as the vertical and lateral heat transfer (in mm), were recorded at 10-second intervals for a duration of 30 seconds, using the thermographic images captured using the infrared camera and a special image analysis software. Descriptive statistical analysis occurred to evaluate the temperature changes over the critical temperature threshold. Results: The maximum ΔT in oC for the initiated lasers was 17.48 ± 9.05 and for non-initiated lasers was 10.72 ± 2.29. The heat transfer in the two groups for vertical/lateral was 7.50 ± 1.4/18.30 ± 4.01 for the initiated lasers and 7.10 ± 1.82/19.24 ± 3.86 for the non-initiated lasers. Conclusion: Initiated tips and non-initiated tips of 445nm laser present deep penetration depths and high temperatures in bovine homogenous soft tissues. The irradiation period and clinical indication must be strictly followed to avoid complications from overheating in soft tissues.