Tissue Sections Research Articles

Electromagnetic Field-Assisted Frozen Tissue Planarization Enhances MALDI-MSI in Plant Spatial Omics.

Plant samples with irregular morphology are challenging for longitudinal tissue sectioning. This has restricted the ability to gain insight into some plants using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Herein, we develop a novel technique termed electromagnetic field-assisted frozen tissue planarization (EMFAFTP). This technique involves using a pair of adjustable electromagnets on both sides of a plant tissue. Under an optimized electromagnetic field strength, nondestructive planarization and regularization of the frozen tissue is induced, allowing the longitudinal tissue sectioning that favors subsequent molecular profiling by MALDI-MSI. As a proof of concept, flowers, leaves and roots with irregular morphology from six plant species are chosen to evaluate the performance of EMFAFTP for MALDI-MSI of secondary metabolites, amino acids, lipids, and proteins among others in the plant samples. The significantly enhanced MALDI-MSI capabilities of these endogenous molecules demonstrate the robustness of EMFAFTP and suggest it has the potential to become a standard technique for advancing MALDI-MSI into a new era of plant spatial omics.

Identification of cellular and noncellular components of mature intact human peripheral nerve.

The goal of this study was to define basic constituents of the adult peripheral nervous system (PNS) using intact human nerve tissues. We combined fluorescent and chromogenic immunostaining methods, myelin-selective fluorophores, and routine histological stains to identify common cellular and noncellular elements in aldehyde-fixed nerve tissue sections. We employed Schwann cell (SC)-specific markers, such as S100β, NGFR, Sox10, and myelin protein zero (MPZ), together with axonal, extracellular matrix (collagen IV, laminin, fibronectin), and fibroblast markers to assess the SC's relationship to myelin sheaths, axons, other cell types, and the acellular environment. Whereas S100β and Sox10 revealed mature SCs in the absence of other stains, discrimination between myelinating and non-myelinating (Remak) SCs required immunodetection of NGFR along with axonal and/or myelin markers. Surprisingly, our analysis of NGFR+ profiles uncovered the existence of at least 3 different novel populations of NGFR+/S100β- cells, herein referred to as nonglial cells, residing in the stroma and perivascular areas of all nerve compartments. An important proportion of the nerve's cellular content, including circa 30% of endoneurial cells, consisted of heterogenous S100β negative cells that were not associated with axons. Useful markers to identify the localization and diversity of nonglial cell types across different compartments were Thy1, CD34, SMA, and Glut1, a perineurial cell marker. Our optimized methods revealed additional detailed information to update our understanding of the complexity and spatial orientation of PNS-resident cell types in humans.

QMAP enabled microanatomical mapping of human skin aging.

Aging is a major driver of diseases in humans. Identifying features associated with aging is essential for designing robust intervention strategies and discovering novel biomarkers of aging. Extensive studies at both the molecular and organ/whole-body physiological scales have helped determined features associated with aging. However, the lack of meso-scale studies, particularly at the tissue level, limits the ability to translate findings made at molecular scale to impaired tissue functions associated with aging. In this work, we established a tissue image analysis workflow - quantitative micro-anatomical phenotyping (qMAP) - that leverages deep learning and machine vision to fully label tissue and cellular compartments in tissue sections. The fully mapped tissue images address the challenges of finding an interpretable feature set to quantitatively profile age-related microanatomic changes. We optimized qMAP for skin tissues and applied it to a cohort of 99 donors aged 14 to 92. We extracted 914 microanatomic features and found that a broad spectrum of these features, represented by 10 cores processes, are strongly associated with aging. Our analysis shows that microanatomical features of the skin can predict aging with a mean absolute error (MAE) of 7.7 years, comparable to state-of-the-art epigenetic clocks. Our study demonstrates that tissue-level architectural changes are strongly associated with aging and represent a novel category of aging biomarkers that complement molecular markers. Our results highlight the complex and underexplored multi-scale relationship between molecular and tissue microanatomic scales.

A Dual-Modal, Label-Free Raman Imaging Method for Rapid Virtual Staining of Large-Area Breast Cancer Tissue Sections.

As one of the most common cancers, accurate, rapid, and simple histopathological diagnosis is very important for breast cancer. Raman imaging is a powerful technique for label-free analysis of tissue composition and histopathology, but it suffers from slow speed when applied to large-area tissue sections. In this study, we propose a dual-modal Raman imaging method that combines Raman mapping data with microscopy bright-field images to achieve virtual staining of breast cancer tissue sections. We validate our method on various breast tissue sections with different morphologies and biomarker expressions and compare it with the golden standard of histopathological methods. The results demonstrate that our method can effectively distinguish various types and components of tissues, and provide staining images comparable to stained tissue sections. Moreover, our method can improve imaging speed by up to 65 times compared to general spontaneous Raman imaging methods. It is simple, fast, and suitable for clinical applications.

Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.

Generation of cardiomyocytes from stem cells cultured on nanofibrous scaffold: Experimental approach for attenuation of myocardial infarction

The current study was constructed to fabricate polyamide based nanofibrous scaffolds (NS) and to define the most promising one for the generation of cardiomyocytes from adipose tissue derived mesenchymal stem cells (ADMSCs). This purpose was extended to assess the potentiality of the generated cardiomyocytes in relieving myocardial infarction (MI) in rats. Production and characterization of NSs were carried out. ADMSCs were cultured on NS and induced to differentiate into cardiomyocytes by specific growth factors. Molecular analysis for myocyte-specific enhancer factor 2 C (MEF2C) and alpha sarcomeric actin (α-SCA) expression was done to confirm the differentiation of ADMSCs into cardiomyocytes for further transplantation into MI induced rats. Implantation of cells in MI afflicted rats boosted heart rate, ST height and PR interval and lessened P duration, RR, QTc and QRS intervals. Also, this type of medication minified serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) enzymes activity as well as serum and cardiac troponin T (Tn-T) levels and upraised serum and cardiac α-SCA and cardiac connexin 43 (CX 43) levels. Microscopic feature of cardiac tissue sections of rats in the treated groups revealed great renovation in the cardiac microarchitecture. Conclusively, this attempt gains insight into a realistic strategy for recovery of MI through systemic employment of in vitro generated cardiomyocytes.

Concomitant decrease of E- and A-FABP expression predicts worse survival in urothelial bladder cancer patients

Non-muscle invasive bladder cancers (NMIBC) pTa-pT1 are depicted by a high risk of recurrence and/or progression with an unpredictable clinical evolution. Our aim was to identify, from the original resection specimen, tumors that will progress to better manage patients. We previously showed that A-FABP (Adipocyte- Fatty Acid Binding Protein) loss predicted NMIBC progression. Here we determined by immunohistochemistry the prognostic value of E-FABP (Epidermal-Fatty Acid Binding Protein) expression in 210 tumors (80 pTa, 75 pT1, 55 pT2-T4). Thus, E-FABP low expression was correlated with a high grade/stage, the presence of metastatic lymph nodes, and visceral metastases (p < 0.001). Unlike A-FABP in NMIBC, E-FABP low expression was not associated with RFS or PFS in Kaplan–Meier analysis. But patients of the overall cohort with a high E-FABP expression had a longer mOS (53.8 months vs. 29.3 months, p = 0.029). The immunohistochemical analysis on the same NMIBC tissue sections revealed that when A-FABP is absent, a high E-FABP expression is detected. E-FABP could compensate A-FABP loss. Interestingly, patients, whose original tumor presents both low E-FABP and negative A-FABP, had the worse survival, those maintaining the expression of both markers had better survival. To conclude, the combined evaluation of A- and E-FABP expression allowed to stratify patients with urothelial carcinoma for optimizing treatment and follow-up.

Intrahepatic distribution of nerves in Suncus murinus by whole-mount immunohistological observation.

In recent years, elucidating the actual state of the liver nervous system has attracted attention, owing to clinical needs, such as liver transplantation. Conventional methods for studying the intrahepatic nerve distribution mostly use liver tissue sections, specific markers for immunohistological studies, or anterograde/retrograde tracing in animals. However, knowledge of the three-dimensional structure of intrahepatic innervation is vague or speculative. In this study, Suncus murinus (Suncus) (n = 10) were perfused and fixed, the livers were excised, and the liver parenchyma was carefully removed, leaving only the intrahepatic vasculature. Specimens were prepared to study the three-dimensional structure of Suncus intrahepatic and hilar innervation by whole-mount immunohistochemical staining using a neurofilament protein antibody. After the nerves running along the intrahepatic arterial system entered the liver parenchyma from the hepatic hilum, they maintained a relatively rich distribution along the interlobular arteries until the distal end. The innervation of the portal system began to decrease after entering the liver parenchyma and decreased significantly after reaching the deep parts. By the time it reached the end of the interlobular vein, there was very little left. The number of nerves running along the intrahepatic bile duct system was significantly reduced after entering the porta hepatis, and innervation was difficult to observe after completely entering the liver parenchyma. Whole-mount immunohistochemical analyses with an anti-NFP antibody showed that intrahepatic innervation mainly accompanied the hepatic interlobular arteries and extended to their terminal ends. Neuronal regulation is very important in the functional regulation of intrahepatic nutritional vessels. However, there were very few NFP-immunoreactive nerves accompanying the intrahepatic bile duct system, possibly suggesting that the functional regulation of the intrahepatic biliary system mainly relies on hormones and neuropeptides.

Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

P-335 Integrative analysis of endometriosis and adenomyosis through single-cell transcriptomics

Abstract Study question What are the shared and unique cell type -specific molecular pathways in endometriosis and adenomyosis? Summary answer Our analysis focuses on hormonal pathways and those regulating cell proliferation and apoptosis, which are crucial for the persistence of the lesions. What is known already Estrogen-driven proliferation and progesterone unresponsiveness are key mechanisms in the pathogenesis of endometriosis and adenomyosis. It has been recently observed that core pathways governing proliferation and apoptosis, such as the Wnt/beta-catenin and KRAS pathways, are involved in the etiology. The precise mechanisms of how estrogen and progesterone signaling interact with the Wnt/beta-catenin and KRAS pathways, which contribute to lesion growth maintenance, are not fully understood, but preliminary evidence suggests some level of cross-talk in both conditions. Study design, size, duration Although endometriosis and adenomyosis share similar mechanisms, they have mostly been studied in isolation. We propose a comprehensive study using an integrated approach to gain a deeper understanding of their pathophysiology. We will combine recent single-cell study data from both disorders, and examine the shared and unique cell-type-specific molecular pathways diverging from the eutopic endometrium. Participants/materials, setting, methods We will employ publicly available single-cell transcriptomic data from cases of endometriosis, adenomyosis, and controls with eutopic endometrium. Tissue sections from an extensive collection of patient samples will be examined for validation. Main results and the role of chance We will integrate single-cell transcriptomic data for endometriosis, adenomyosis, and eutopic endometrium controls. For each cell type, we will conduct comprehensive differential expression analysis between lesions and endometrium, study cell-to-cell communication among major cell types, and construct gene regulatory networks that are either shared or specific to endometriosis and adenomyosis. We will also consider GWAS lead genes and investigate their cell type-specific expression, focusing on hormonally regulated genes. Tissue sections of patient samples, apoptosis measurements, and clinical parameters including pain data will be utilized for validation. Limitations, reasons for caution The public single-cell transcriptomic data for endometriosis and adenomyosis have limitations in sample size and menstrual cycle distribution. The utilized single-cell transcriptomics data and the in-house tissue collections for validations studies are not matched. Wider implications of the findings This project aims to identify molecular pathways in endometriosis and adenomyosis, potentially revealing common disease development mechanisms and unique molecular signatures. Both endometriosis and adenomyosis are significant health issues affecting women, and their study contributes to the broader understanding of women’s health. Trial registration number not applicable

Effect of Plasma Electric Field Assistance on the Freezing Process and Ice Crystal Formation of Bactrian Camel Meat

Background: The longissimus dorsi muscle of the camel was chosen as the experimental material in order to investigate the impact of plasma electric field assisted freezing on the quality and freezing process of Bactrian camel meat. Methods: The effect of the auxiliary plasma electric field of different field strengths (1 kV/m, 3 kV/m and 5 kV/m) on the freezing velocity of the camel meat and the distribution of ice crystals as well as the diameter and roundness of ice crystal in the frozen meat were studied and analyzing them with the help of the thermodynamic theory. Result: The findings demonstrated that the meat samples in the auxiliary 5 kV/m plasma field environment took 60 min and 30 min, respectively, at -20°C and -35°C, to pass the maximal ice crystal production zone. The tissue sections revealed that at -20°C and -35°C assisted 5 kV/m plasma field freezing, the smallest ice crystal area and grain size were formed, the roundness of the ice crystals was highest. Using Boltzmann’s entropic equation to calculate the system’s entropy, the results revealed that the meat samples frozen by the aided 5 kV/m plasma electric field had the lowest entropy at -20°C and -35°C. The electric field assisted freezing requires less energy to improve the subcooling degree and accelerate the freezing rate of meat samples, proving that the auxiliary 5 kV/m plasma electric field can significantly improve the system frozen meat moisture vitrification degree and can make the crystallization process more controllable.

P-105 Gender affirming hormone therapy affects the spermatogonial cell compartment in transwomen

Abstract Study question What are the specific changes under gender affirming hormone therapy (GAHT) in the spermatogonial cell compartment in testicular tissues of transwomen? Summary answer GAHT has a negative effect on the most undifferentiated PIWIL4+ spermatogonia potentially leading to reduced chances for fertility preservation in transwomen. What is known already Transwomen take GAHT to achieve transition between the gender assigned at birth and their gender identity. GAHT leads to a reduction of the most advanced germ cells and a reduction of MAGEA4 positive spermatogonia (pan spermatogonial marker). Single-cell RNA sequencing studies identified spermatogonial subpopulations based on the expression of the marker genes PIWIL4, FGFR3, NANOS3, GFRA1, KIT, UTF1. PIWIL4+ cells are considered the origin of germ cell differentiation (state 0). State 1 and 2 spermatogonia are characterized by expression of GFRA1 and KIT, respectively. Study design, size, duration On the day of gender affirming surgery (GAS) testicular tissues and blood samples were collected of 25 age matched trans women (mean 28.1 yr), who underwent comparable GAHT regimens (10 or 12.5 mg cyproterone acetate and estrogens) between 2013 and 2018. 8 adult cis men (mean 34.5 yr) with complete spermatogenesis served as controls. Age, start and type of GAHT were assessed using a questionnaire. This study was approved by the local ethics committee. Participants/materials, setting, methods We performed immunohistochemical stainings to evaluate the expression of spermatogonial markers (MAGEA4, UTF1, PIWIL4, FGFR3, NANOS3, GFRA1) in Bouin’s-fixed testicular tissue sections. For each marker the number of positive cells per round tubular cross-section was determined in 25 round tubules. The spermatogonial proliferation rate was evaluated using immunofluorescence co-stainings for GFRA1/MIKI67/MAGEA4 and KIT/MKI67/MAGEA4. We scored 400 MAGEA4+ cells per individual and determined the proportion of GFRA1+/KIT+. MKI67+ cells among the GFRA1+/KIT+ were evaluated. Main results and the role of chance The number of MAGEA4+ spermatogonia and the proportion of PIWIL4+ spermatogonia within the MAGEA4+ spermatogonia were reduced in transwomen compared to controls (p &amp;lt; 0.001). In contrast, the relative proportion of UTF1+, FGFR3+ and NANOS3+ spermatogonia was comparable. The number of spermatogonia per tubule correlated negatively with the age at start of GAHT (p &amp;lt; 0.01) and the age on the day of GAS (p &amp;lt; 0.001). The treatment duration correlated negatively with the proportion of PIWIL4+ (p &amp;lt; 0.01) and NANOS3+ spermatogonia (p &amp;lt; 0.05). The immunofluorescence stainings in transwomen revealed a similar ratio of proliferating KI67+ spermatogonia, both within the GFRA1+ and KIT+ cells, compared to controls. These results show the negative effects of the GAHT itself on the population of spermatogonia as well as the individual susceptibility depending on age-related factors. These results have the potential to better define the chances for fertility preservation under GAHT. Limitations, reasons for caution Transwomen are a very heterogeneous patient group varying in age, different GAHT, different treatment durations, intake of co-medications and possible pre-existing medical conditions. Hence, testicular size and histological appearance of transwomen’s testes are heterogeneous. Wider implications of the findings Transwomen, who wish to have own biological children, have to be counseled with regard to fertility preservation, best before initiation of GAHT, in order to avoid adverse effects on spermatogonia. Individual counselling has to take place with regard to fertility preservation. Trial registration number 2012-555-f-S

Functional Study of the Role of the Methyl Farnesoate Epoxidase Gene in the Ovarian Development of Macrobrachium nipponense.

Methyl farnesoate epoxidase (MFE) is a gene encoding an enzyme related to the last step of juvenile hormone biosynthesis. Mn-MFE cDNA has a total length of 1695 bp and an open reading frame (ORF) length of 1482 bp, encoding 493 amino acids. Sequence analysis showed that its amino acid sequence has a PPGP hinge, an FGCG structural domain, and other structural domains specific to the P450 family of enzymes. Mn-MFE was most highly expressed in the hepatopancreas, followed by the ovary and gill, weakly expressed in heart and muscle tissue, and barely expressed in the eyestalk and cranial ganglion. Mn-MFE expression remained stable during the larval period, during which it mainly played a critical role in gonadal differentiation. Expression in the ovary was positively correlated and expression in the hepatopancreas was negatively correlated with ovarian development. In situ hybridization (ISH) showed that the signal was expressed in the oocyte, nucleus, cell membrane and follicular cells, and the intensity of expression was strongest at stage O-IV. The knockdown of Mn-MFE resulted in a significantly lower gonadosomatic index and percentage of ovaries past stage O-III compared to the control group. However, no differences were found in the cumulative frequency of molting between the experimental and control groups. Moreover, the analysis of ovarian tissue sections at the end of the experiment showed differences between groups in development speed but not in subcellular structure. These results demonstrate that Mn-MFE promotes the ovarian development of Macrobrachium nipponense adults but has no effect on molting.

Highly Sensitive Spatial Glycomics at Near-Cellular Resolution by On-Slide Derivatization and Mass Spectrometry Imaging.

Glycans on proteins and lipids play important roles in maturation and cellular interactions, contributing to a variety of biological processes. Aberrant glycosylation has been associated with various human diseases including cancer; however, elucidating the distribution and heterogeneity of glycans in complex tissue samples remains a major challenge. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is routinely used to analyze the spatial distribution of a variety of molecules including N-glycans directly from tissue surfaces. Sialic acids are nine carbon acidic sugars that often exist as the terminal sugars of glycans and are inherently difficult to analyze using MALDI-MSI due to their instability prone to in- and postsource decay. Here, we report on a rapid and robust method for stabilizing sialic acid on N-glycans in FFPE tissue sections. The established method derivatizes and identifies the spatial distribution of α2,3- and α2,6-linked sialic acids through complete methylamidation using methylamine and PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate). Our in situ approach increases the glycans detected and enhances the coverage of sialylated species. Using this streamlined, sensitive, and robust workflow, we rapidly characterize and spatially localize N-glycans in human tumor tissue sections. Additionally, we demonstrate this method's applicability in imaging mammalian cell suspensions directly on slides, achieving cellular resolution with minimal sample processing and cell numbers. This workflow reveals the cellular locations of distinct N-glycan species, shedding light on the biological and clinical significance of these biomolecules in human diseases.

Enhancing Spatial Resolution in Tandem Mass Spectrometry Ion/Ion Reaction Imaging Experiments through Image Fusion.

We have recently developed a charge inversion ion/ion reaction to selectively derivatize phosphatidylserine lipids via gas-phase Schiff base formation. This tandem mass spectrometry (MS/MS) workflow enables the separation and detection of isobaric lipids in imaging mass spectrometry, but the images acquired using this workflow are limited to relatively poor spatial resolutions due to the current time and limit of detection requirements for these ion/ion reaction imaging mass spectrometry experiments. This trade-off between chemical specificity and spatial resolution can be overcome by using computational image fusion, which combines complementary information from multiple images. Herein, we demonstrate a proof-of-concept workflow that fuses a low spatial resolution (i.e., 125 μm) ion/ion reaction product ion image with higher spatial resolution (i.e., 25 μm) ion images from a full scan experiment performed using the same tissue section, which results in a predicted ion/ion reaction product ion image with a 5-fold improvement in spatial resolution. Linear regression, random forest regression, and two-dimensional convolutional neural network (2-D CNN) predictive models were tested for this workflow. Linear regression and 2D CNN models proved optimal for predicted ion/ion images of PS 40:6 and SHexCer d38:1, respectively.

First Report of Fruit Scab Caused by Alternaria alternata on Actinidia chinensis in Korea.

Kiwi (Actinidia chinesis) is an economically important fruit in Korea, with 1,300 ha cultivated and a production of approximately 25,000 tons per year (Kim and Koh, 2018; Kim and Choi, 2023). In late June 2020, fruit scab symptoms were observed on A. chinensis var. rufopulpa in an orchard in Suncheon, Korea. The incidence of scab symptoms among 20-year-old trees was over 75%, primarily superficial, but rendered the fruit less marketable. In the initial stages of the disease, small, light-brown, circular, and oval spots were formed. As the superficial spots expanded, they became cracked scabs measuring 1 to 7 cm with light edges at the later stages. To isolate the causal pathogen, two lesions were cut from two sections of symptomatic tissue, from each of seven fruits from seven trees. Lesions were surface-sterilized with 70% ethanol for 1 min and washed three times with sterilized distilled water (SDW). The sterilized pieces were placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for one week. After subculturing on PDA, single-spore isolation produced 14 isolates: SYP-410 to 423). All 14 colonies appeared greyish-green and cottony on PDA after 7 d. Conidia were pale brown, ellipsoid to obclavate, with ornamented walls, 1 to 6 transverse and 0 to 3 vertical septa, and length × width of 21.5 to 53.4 × 7.3 to 19.2 μm (avg. 33.0 × 12.0 μm, n = 100). Their morphological characteristics were consistent with Alternaria spp. (van der Waals et al. 2011; Woudenberg et al. 2015). We randomly selected three isolates from the morphologically similar cultures and named them SYP-412 to 414 for further investigation. The ITS (GenBank accession nos.: OR901850 to 52), gapdh (OR924309 to 11), tef1 (OR924312 to 14), rpb2 (OR924315 to 17), Alt a1 (OR924318 to 20), endoPG (OR924321 to 23), and OPA10-2 (OR924324 to 26) sequences from SYP-412 to 414 had a 100% (515 bp/515 bp), 100% (578/578), 100% (240/240), 100% (724/724), 95.55% (451/472), 99.33% (445/448), and 100% (634/634) identity with that of type strain A. alternata CBS 918.96 (AF347032, AY278809, KC584693, KC584435, AY563302, KP124026, and KP124633), respectively. Results from the maximum likelihood phylogenetic analysis, based on the seven concatenated gene sequences, placed the representative isolates in a clade with A. alternata. Pathogenicity of SYP-412 was tested using 12 surface-sterilized two-month-old kiwifruits on a 20-year-old trees. Six kiwifruits were spray-inoculated with 5 mL of a conidial suspension (1 × 106 conidia/ml) generated after culturing in PDA medium for 7 d, with or without wounding. Another six control fruits were inoculated with SDW with and without wounding. The inoculated kiwifruits were enclosed in plastic bags to maintain high humidity for one day. Scab symptoms were observed in both wounded and unwounded fruits six weeks after inoculation, but not in the control. The pathogenicity test was performed on a total of three separate trees twice. To satisfy Koch's postulates, A. alternata was re-isolated from all the symptomatic tissues and confirmed by analyzing the ITS and rpb2 genes. Although scab disease caused by A. tenuissima (now A. alternata) has been previously reported in kiwifruit of A. chinensis var. rufopulpa in China (Woudenberg et al. 2015; Ma et al., 2019), this is the first report of its occurrence on kiwifruit in Korea and will help in future detection and control.

Disturbance in communication between mitochondrial redox processes and the AMPK/PGC-1/SIRT-1 axis influences diverse organ symptoms in lupus-affected mice

BackgroundMechanisms behind multiple organ involvement in lupus, is still an enigma for researchers. Mitochondrial dysfunction and oxidative stress are known to be important aspects in lupus etiology however, their role in lupus organ manifestation is yet to be understood. The present study is based on the understanding of interplay between AMPK/PGC-1/SIRT-1 axis, mitochondrial complexes, and anti-oxidants levels, which might be involved in lupus organ pathology. MethodologyPristane-induced Balb/c mice lupus model (PIL) was utilised and evaluation of anti-oxidants, mitochondrial complexes, pro-inflammatory cytokines levels, biochemical parameters were performed by standard procedures. Tissues were studied by haematoxylin and eosin staining followed by immunohistochemistry. The AMPK/PGC-1/SIRT-1 expression was analysed by using qPCR and flowcytometry. Analysis of reactive oxygen species (ROS) among WBCs was performed by using various dyes (DCFDA, Mitosox, JC-1) on flowcytometry. ResultSignificant presence of immune complexes (Tissue sections), ANA (Serum), and pro-inflammatory cytokines (plasma), diminished anti-oxidants and altered biochemical parameters depict the altered pathology in PIL which was accompanied by dysregulated mitochondrial complex activity. Differential expression of the AMPK/PGC-1/SIRT-1 axis was detected in tissue and correlation with mitochondrial and antioxidant activity emerged as negative in PIL group while positive in controls. Close association was observed between ROS, mitochondrial membrane potential, and AMPK/PGC-1/SIRT-1 axis in WBCs. ConclusionThis study concludes that mitochondria play a dual role in lupus organ pathology, contributing to organ damage while also potentially protecting against damage through the regulation of interactions between antioxidants and the AMPK axis expression.

Multiplexed in situ RNA imaging by combFISH.

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity.

FTO promotes the progression of bladder cancer via demethylating m6A modifications in PTPN6 mRNA

Bladder cancer (BC), a highly prevalent malignancy of the urinary system, necessitates further investigation into its progression mechanisms. N6-methyladenosine (m6A) RNA methylation, a prevalent modification in cellular RNA, has been implicated in the tumorigenesis and metastasis of various cancers. In this study, the upregulation of FTO in human BC samples and its association with poor prognosis were demonstrated using immunohistochemistry (IHC) on tissue sections collected from BC patients. The functional role of FTO in promoting the proliferation and metastasis abilities of BC cells was determined using a combination of in vitro and in vivo assays. In vitro, we conducted cell proliferation assays, such as the Cell Counting Kit-8 (CCK-8) assay, and metastasis assays, including the wound healing assay and transwell invasion assay. In vivo, we employed xenograft models to assess tumor growth and metastasis. Furthermore, our investigation into potential FTO targets in BC cells revealed that FTO modifies PTPN6 mRNA, leading to increased stability and expression of PTPN6, thereby enhancing proliferation and metastasis abilities. In conclusion, our findings indicate that FTO serves as an oncogenic factor in BC, suggesting its potential utility as a diagnostic or prognostic biomarker for bladder cancer.