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Background: Breast cancer (BC) is the most common cancer in women. Likewise, canine mammary tumors (CMT) represent the most common cancer in intact female dogs and develop in the majority spontaneously. Similarities exist in clinical presentation, histopathology, biomarkers, and treatment. However, CMT subtype-specific genomic background is less investigated. Here, we assess the genetic etiology of two histomorphological (HM) subtypes with BC counterparts, the CMT invasive ductal simple solid carcinoma (SC) and comedocarcinoma (CC), and compare the results with BC data. Methods: Groups of 11–13 transformed ductal luminal epithelial cells were laser-capture microdissected from snap-frozen invasive mammary SC and CC subtypes of one intact female dog. HM unaffected lobular luminal epithelial cells were controls. Single-cell whole genome libraries were generated using PicoPLEX and sequenced to compare the subtypes' somatic coding variant landscapes with each other and with BC data available in COSMIC-CGC and KEGG. Furthermore, HM and immunohistochemical (IHC) subtype characteristics were compared with the genomic results. Results: The CC had six times more variants than the SC. The SC showed variants in adherens junction genes and genes of the MAPK, mTOR and NF-kappa-B signaling pathways. In the CC, the extracellular matrix (ECM) receptor interaction, cell adhesion, PI3K-Akt and cGMP-PKG pathways were enriched, reflecting the higher cellular malignancy. Affected pathways in both CMT subtypes overlapped with BC pathways in KEGG. Additionally, we identified ATP6V1C2, GLYATL3, CARMIL3, GATAD2B, OBSCN, SIX2, CPEB3 and ZNF521 as potential new subtype-distinct driver genes. Furthermore, our results revealed biomarker alterations in IHC in the basal/myoepithelial cell layer without respective genetic mutations, suggesting changes to their complex signaling pathways, disturbed regulative feedback loops or other silencing mechanisms. Conclusions: This study contributes to understanding the subtype-specific molecular mechanisms in the canine mammary invasive ductal simple SC and CC, and revealed subtype-specific molecular complexity for phenotypically similar characteristics. Several affected genes and signaling pathways overlapped with BC indicating the potential use of CMT as model for BC. Our findings emphasize the need for thorough characterization of cancer specimens with respect to translational cancer research, but also how insight into tumor heterogeneity will be crucial for the development of targeted prognostics and therapeutic interventions. [ABSTRACT FROM AUTHOR]

This study aims to elucidate if the regulation of plant aquaporins by the arbuscular mycorrhizal (AM) symbiosis occurs only in roots or cells colonized by the fungus or at whole root system. Maize plants were cultivated in a split‐root system, with half of the root system inoculated with the AM fungus and the other half uninoculated. Plant growth and hydraulic parameters were measured and aquaporin gene expression was determined in each root fraction and in microdissected cells. Under well‐watered conditions, the non‐colonized root fractions of AM plants grew more than the colonized root fraction. Total osmotic and hydrostatic root hydraulic conductivities (Lo and Lpr) were higher in AM plants than in non‐mycorrhizal plants. The expression of most maize aquaporin genes analysed was different in the mycorrhizal root fraction than in the non‐mycorrhizal root fraction of AM plants. At the cellular level, differential aquaporin expression in AM‐colonized cells and in uncolonized cells was also observed. Results indicate the existence of both, local and systemic regulation of plant aquaporins by the AM symbiosis and suggest that such regulation is related to the availability of water taken up by fungal hyphae in each root fraction and to the plant need of water mobilization. Summary statement: A differential aquaporin gene regulation by the arbuscular mycorrhizal (AM) symbiosis was observed in maize roots or cells colonized by the AM fungus as compared to uncolonized roots or cells. Results suggest that such regulation is related to the availability or not of water taken up by fungal hyphae and to the plant's need for water mobilization. [ABSTRACT FROM AUTHOR]

Objective: In schizophrenia, impaired working memory is associated with transcriptome alterations in layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC). Distinct subtypes of L3PNs that send axonal projections to the DLPFC in the opposite hemisphere (callosal projection [CP] neurons) or the parietal cortex in the same hemisphere (ipsilateral projection [IP] neurons) play critical roles in working memory. However, how the transcriptomes of these L3PN subtypes might shift during late postnatal development when working memory impairments emerge in individuals later diagnosed with schizophrenia is not known. The aim of this study was to characterize and compare the transcriptome profiles of CP and IP L3PNs across developmental transitions from prepuberty to adulthood in macaque monkeys. Methods: The authors used retrograde labeling to identify CP and IP L3PNs in the DLPFC of prepubertal, postpubertal, and adult macaque monkeys, and used laser microdissection to capture these neurons for RNA sequencing. Results: At all three ages, CP and IP L3PNs had distinct transcriptomes, with the number of genes differentially expressed between neuronal subtypes increasing with age. For IP L3PNs, age-related shifts in gene expression were most prominent between prepubertal and postpubertal animals, whereas for CP L3PNs such shifts were most prominent between postpubertal and adult animals. Conclusions: These findings demonstrate the presence of cell type–specific profiles and developmental trajectories of the transcriptomes of PPC-projecting IP and DLPFC-projecting CP L3PNs in monkey DLPFC. The evidence that IP L3PNs reach a mature transcriptome earlier than CP L3PNs suggests that these two subtypes differentially contribute to the maturation of working memory performance across late postnatal development and that they may be differentially vulnerable to the disease process of schizophrenia at specific stages of postnatal development. [ABSTRACT FROM AUTHOR]

[Objective] To study the effect of different fixatives and section methods on RNA quality of flower primordia of Chrysanthemum buds collected by microdissection. [Method] Chrysanthemum buds was fixed by acetone or ethanol acetic acid and then paraffin section/frozen section was prepared for laser microdissection respectively. Outermost two whorls of flower primordia were identified and collected by laser microdissection. The quantity and quality of isolated RNA were assessed. [Result] Both tissues embedded in paraffin for paraffin section or OCT for cryosection preserves well the anatomical structure and morphology of the buds. For paraffin-section, ethanol acetic acid was a better choice being a fixative than that of acetone, as that RNA extracted from the microdissection samples was less degraded. There was no significant difference in RNA integrity of cryosection between these two fixatives. The tissues conducted cryosection could get higher quality of RNA compared with that of paraffin-section. Furthermore, when microdissection was conducted, containing the extraction buffer from RNA isolation kit in the cap of PCR tube could preserved RNA integrity of the samples. Genes expression of flowering locus T in flower primordia was different in species of chrysanthemum showed that samples captured by microdissection can be used for subsequent research. [Conclusion] Plant tissues fixed by ethanol-acetic acid and protected by gradient sucrose before cryosection could collect specific cell-type populations by laser microdissection. From those populations we could obtain enough RNA with high integrity. [ABSTRACT FROM AUTHOR]

Abstract Background With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed. Results We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach. Conclusions We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Background: With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed. Results: We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach. Conclusions: We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues. [ABSTRACT FROM AUTHOR]

Abstract Objective Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important grain produced in the world. Interest for cultivating sorghum is increasing all over the world in the context of climate change, due to its low input and water requirements. Like other cultivated cereals, sorghum has significant nutritional value thanks to its protein, carbohydrate and dietary fiber content, these latter mainly consisting of cell wall polysaccharides. This work describes for the first time a transcriptomic analysis dedicated to identify the genes involved in the biosynthesis and remodelling of cell walls both in the endosperm and outer layers of sorghum grain during its development. Further analysis of these transcriptomic data will improve our understanding of cell wall assembly, which is a key component of grain quality. Data description This research delineates the steps of our analysis, starting with the cultivation conditions and the grain harvest at different stages of development, followed by the laser microdissection applied to separate the endosperm from the outer layers. It also describes the procedures implemented to generate RNA libraries and to obtain a normalized and filtered table of transcript counts, and finally determine the number of putative cell wall-related genes already listed in literature.

Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but requires clear understanding of the tradeoffs between analytical throughput and precision. Recent advances in mass spectrometry acquisition techniques, including updated instrumentation and sample preparation, have improved the quality of peptide signals obtained from single cell data. However, much of the proteome remains uncharacterized, and higher throughput techniques often come at the expense of reduced sensitivity and coverage, which diminish the ability to measure proteoform heterogeneity, including splice variants and post-translational modifications, in single cell data analysis. Here, we assess the growing body of ultrasensitive single-cell approaches and their tradeoffs as researchers try to balance throughput and precision in their experiments. [ABSTRACT FROM AUTHOR]

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions.We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme. [ABSTRACT FROM AUTHOR]

Nutritropism is a positive tropism toward nutrients in plant roots. An NH4+ gradient is a nutritropic stimulus in rice (Oryza sativa L.). When rice roots are exposed to an NH4+ gradient generated around nutrient sources, root tips bend toward and coil around the sources. The molecular mechanisms are largely unknown. Here, we analyzed the transcriptomes of the inside and outside of bending root tips exhibiting nutritropism to reveal nutritropic signal transduction. Tissues facing the nutrient sources (inside) and away (outside) were separately collected by laser microdissection. Principal component analysis revealed distinct transcriptome patterns between the two tissues. Annotations of 153 differentially expressed genes implied that auxin, gibberellin and ethylene signaling were activated differentially between the sides of the root tips under nutritropism. Exogenous application of transport and/or biosynthesis inhibitors of these phytohormones largely inhibited the nutritropism. Thus, signaling and de novo biosynthesis of the three phytohormones are necessary for nutritropism. Expression patterns of IAA genes implied that auxins accumulated more in the inside tissues, meaning that ammonium stimulus is transduced to auxin signaling in nutritropism similar to gravity stimulus in gravitropism. SAUR and expansin genes, which are known to control cell wall modification and to promote cell elongation in shoot gravitropism, were highly expressed in the inside tissues rather than the outside tissues, and our transcriptome data are unexplainable for differential elongation in root nutritropism. [ABSTRACT FROM AUTHOR]

Background: Solid-predominant lung adenocarcinoma (SPA), which is one of the high-risk subtypes with poor prognosis and unsatisfactory response to chemotherapy and targeted therapy in lung adenocarcinoma, remains molecular profile unclarified. Weighted correlation network analysis (WGCNA) was used for data mining, especially for studying biological networks based on pairwise correlations between variables. This study aimed to identify diseaserelated protein co-expression networks associated with early-stage SPA. Methods: We assessed cancerous cells laser-microdissected from formalin-fixed paraffin-embedded (FFPE) tissues of a SPA group (n = 5), referencing a low-risk subtype, a lepidic predominant subtype group (LPA) (n = 4), and another high-risk subtype, micropapillary predominant subtype (MPA) group (n = 3) and performed mass spectrometry-based proteomic analysis. Disease-related co-expression networks associated with the SPA subtype were identified by WGCNA and their upstream regulators and causal networks were predicted by Ingenuity Pathway Analysis. Results: Among the forty WGCNA network modules identified, two network modules were found to be associated significantly with the SPA subtype. Canonical enriched pathways were highly associated with cellular growth, proliferation, and immune response. Upregulated HLA class I molecules HLA-G and HLA-B implicated high mutation burden and T cell activation in the SPA subtype. Upstream analysis implicated the involvement of highly activated oncogenic regulators, MYC, MLXIPL, MYCN, the redox master regulator NFE2L2, and the highly inhibited LARP1, leading to oncogenic IRES-dependent translation, and also regulators of the adaptive immune response, including highly activated IFNG, TCRD, CD3-TCR, CD8A, CD8B, CD3, CD80/CD86, and highly inhibited LILRB2. Interestingly, the immune checkpoint molecule HLA-G, which is the counterpart of LILRB2, was highly expressed characteristically in the SPA subtype and might be associated with antitumor immunity. Conclusion: Our findings provide a disease molecular profile based on protein co-expression networks identified for the high-risk solid predominant adenocarcinoma, which will help develop future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies. [ABSTRACT FROM AUTHOR]

[Display omitted] • Meronts and megalomeronts of five co-infecting Haemoproteus spp. were described. • In situ hybridization combined with laser microdissection are effective in merogony research. • Lineage-specific tools revealed different tissue tropism in co-infecting parasites. • New pathologies during haemoproteosis were reported. • Haemoproteus magnus was molecularly characterized. Avian haemosporidians of the genera Plasmodium , Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs , a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6 , Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait. [ABSTRACT FROM AUTHOR]

Summary: Soybeans (Glycine max) develop newly differentiated aerenchymatous phellem (AP) in response to waterlogging stress. AP is formed in the hypocotyl and root, thus contributing to internal aeration and adaptation to waterlogging for several legumes. Extensive accumulation of triterpenoids – lupeol and betulinic acid – has been identified in AP. However, their physiological roles in plants remain unclarified. Lupeol is converted from 2,3‐oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Notably, soybeans have two LUS genes (GmLUS1 and GmLUS2).Functional analysis was performed to reveal the biological and physiological functions of triterpenoids in AP using lus mutants.The AP cells of lus1 mutant lacked triterpenoid accumulation and epicuticular wax. Lupeol and betulinic acid were the major components of epicuticular wax and contributed to tissue hydrophobicity and oxygen transport to the roots. Tissue porosity in AP was lower in the lus1 mutant than in the wild‐type, which resulted in reduced oxygen transport to the roots via AP. This reduction in oxygen transport resulted in shallow root systems under waterlogged conditions.Triterpenoid accumulation in AP contributes to effective internal aeration and root development for adaptation to waterlogging, suggesting the significance of triterpenoids in improving waterlogging tolerance. [ABSTRACT FROM AUTHOR]

Abstract Background Early diagnosis and typing are crucial for improving the prognosis of patients with renal amyloidosis. Currently, Untargeted proteomics based precise diagnosis and typing of amyloid deposits are crucial for guiding patient management. Although untargeted proteomics achieve ultra-high-throughput by selecting the most abundant eluting cationic peptide precursors in series for tandem MS events, it lacks in sensitivity and reproducibility, which may not be suitable for early-stage renal amyloidosis with minor damages. Here, we aimed to develop parallel reaction monitoring (PRM)-based targeted proteomics to achieve high sensitivity and specificity by determining absolute abundances and codetecting all transitions of highly repeatable peptides of preselected amyloid signature and typing proteins in identifying early-stage renal immunoglobulin-derived amyloidosis. Methods and results In 10 discovery cohort cases, Congo red-stained FFPE slices were micro-dissected and analyzed by data-dependent acquisition-based untargeted proteomics for preselection of typing specific proteins and peptides. Further, a list of proteolytic peptides from amyloidogenic proteins and internal standard proteins were quantified by PRM-based targeted proteomics to validate performance for diagnosis and typing in 26 validation cohort cases. The diagnosis and typing effectiveness of PRM-based targeted proteomics in 10 early-stage renal amyloid cases was assessed via a comparison with untargeted proteomics. A peptide panel of amyloid signature proteins, immunoglobulin light chain and heave chain in PRM-based targeted proteomics showed significantly distinguishing ability and amyloid typing performance in patients. The diagnostic algorithm of targeted proteomics with a low amount of amyloid deposits in early-stage renal immunoglobulin-derived amyloidosis showed better performance than untargeted proteomics in amyloidosis typing. Conclusions This study demonstrates that the utility of these prioritized peptides in PRM-based targeted proteomics ensure high sensitivity and reliability for identifying early-stage renal amyloidosis. Owing to the development and clinical application of this method, rapid acceleration of the early diagnosis, and typing of renal amyloidosis is expected.

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl- concentration and cell volume of neurons and/or glia. The Cl- extruder KCC2 is expressed at higher levels than the Cl- transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl- concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis. [ABSTRACT FROM AUTHOR]

Auxin distribution is essential for determining root developmental patterns. The formation of lateral roots and constitutive aerenchyma, which is a gas space developed through cell death, is regulated by auxin in rice (Oryza sativa). However, it is unclear whether the involvement of auxin in constitutive aerenchyma formation is conserved in other species. In this study, we found that constitutive aerenchyma formation was regulated by auxin in the nodal roots of Zea nicaraguensis, a wild relative of maize (Zea mays ssp. mays) grown naturally on frequently flooded coastal plains. Subsequent gravistimulation (root rotation) experiments showed opposite patterns of aerenchyma and lateral root formation. Lateral root formation on the convex side of rotated roots is known to be stimulated by a transient increase in auxin level in the pericycle. We found that aerenchyma formation was accelerated in the cortex on the concave side of the rotated nodal roots of Z. nicaraguensis. A cortex-specific expression analysis of auxin-responsive genes suggested that the auxin level was higher on the concave side than on the convex side. These results suggest that asymmetric auxin distribution underlies the regulation of aerenchyma and lateral root formation in the nodal roots of Z. nicaraguensis. As aerenchyma reduces the respiratory cost of the roots, constitutive aerenchyma on the concave side of the nodal root may balance resource allocation, thereby contributing to the uptake of water and nutrients by newly formed lateral roots. Our study provides insights into auxin-dependent asymmetric root patterning such as that of gravistimulation and hydropatterning response. [ABSTRACT FROM AUTHOR]

Abstract Background Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-β (Aβ) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation. Methods In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia. Results First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain. Conclusions Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aβ pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aβ-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aβ oligomer detection and in Aβ-plaque initiation, with increased contribution as the disease progresses. Graphical Abstract

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl− concentration and cell volume of neurons and/or glia. The Cl− extruder KCC2 is expressed at higher levels than the Cl− transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl− concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis.

Aim: Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. Methods: Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)‐based qPCR and enhanced computed tomography. LMD‐based RNA‐sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. Results: Liver fibrosis was mainly induced in the periphery of the liver and co‐localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD‐based RNA‐sequencing revealed the upregulation of extracellular matrix/collagen fibril‐, wound healing‐, angiogenesis‐, morphogenesis‐, and cell motility‐related signaling pathways in periphery of liver compared with liver center. Conclusions: Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis and typing are crucial for improving the prognosis of patients with renal amyloidosis. Currently, Untargeted proteomics based precise diagnosis and typing of amyloid deposits are crucial for guiding patient management. Although untargeted proteomics achieve ultra-high-throughput by selecting the most abundant eluting cationic peptide precursors in series for tandem MS events, it lacks in sensitivity and reproducibility, which may not be suitable for early-stage renal amyloidosis with minor damages. Here, we aimed to develop parallel reaction monitoring (PRM)-based targeted proteomics to achieve high sensitivity and specificity by determining absolute abundances and codetecting all transitions of highly repeatable peptides of preselected amyloid signature and typing proteins in identifying early-stage renal immunoglobulin-derived amyloidosis. Methods and results: In 10 discovery cohort cases, Congo red-stained FFPE slices were micro-dissected and analyzed by data-dependent acquisition-based untargeted proteomics for preselection of typing specific proteins and peptides. Further, a list of proteolytic peptides from amyloidogenic proteins and internal standard proteins were quantified by PRM-based targeted proteomics to validate performance for diagnosis and typing in 26 validation cohort cases. The diagnosis and typing effectiveness of PRM-based targeted proteomics in 10 early-stage renal amyloid cases was assessed via a comparison with untargeted proteomics. A peptide panel of amyloid signature proteins, immunoglobulin light chain and heave chain in PRM-based targeted proteomics showed significantly distinguishing ability and amyloid typing performance in patients. The diagnostic algorithm of targeted proteomics with a low amount of amyloid deposits in early-stage renal immunoglobulin-derived amyloidosis showed better performance than untargeted proteomics in amyloidosis typing. Conclusions: This study demonstrates that the utility of these prioritized peptides in PRM-based targeted proteomics ensure high sensitivity and reliability for identifying early-stage renal amyloidosis. Owing to the development and clinical application of this method, rapid acceleration of the early diagnosis, and typing of renal amyloidosis is expected. [ABSTRACT FROM AUTHOR]

An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation. [ABSTRACT FROM AUTHOR]

Simple Summary: Supernumerary marker chromosomes, as they are known in the human population, are usually small chromosomes that differ morphologically and structurally from the standard ones and in many cases are formed by genetically inert heterochromatin. Similar features were observed for a supernumerary chromosome discovered in two Asian elephants, a mother and her male offspring. In this study, we present its detailed analysis using several molecular cytogenetic techniques including laser microdissection and fluorescence in situ hybridization that allowed identification of this marker chromosome. Based on our findings, we propose the most possible mechanism for the origin of the marker studied. We extended our investigation and showed that the distribution of nucleolar organizer regions on the chromosomes of Asian and savanna elephants may be related to the distribution of heterochromatin. Supernumerary chromosomes or, in other words, additional or extra chromosomes added to typical human or animal karyotypes, have recently gained the attention of scientists as model systems for the study of chromosome evolution, which may include the chromosome marker described here. We identified a small, supernumerary marker chromosome (sSMC) in two phenotypically normal Asian elephants (Elephas maximus): a female (2n = 57,XX,+mar) and her male offspring (2n = 57,XY,+mar). sSMCs are defined as structurally abnormal chromosomes that cannot be identified by conventional banding analysis since they are usually small and often lack distinct banding patterns. Although current molecular techniques can reveal their origin, the mechanism of their formation is not yet fully understood. We determined the origin of the marker using a suite of conventional and molecular cytogenetic approaches that included (a) G- and C-banding, (b) AgNOR staining, (c) preparation of a DNA clone using laser microdissection of the marker chromosome, (d) FISH with commercially available human painting and telomeric probes, and (e) FISH with centromeric DNA derived from the centromeric regions of a marker-free Asian elephant. Moreover, we present new information on the location and number of NORs in Asian and savanna elephants. We show that the metacentric marker was composed of heterochromatin with NORs at the terminal ends, originating most likely from the heterochromatic region of chromosome 27. In this context, we discuss the possible mechanism of marker formation. We also discuss the similarities between sSMCs and B chromosomes and whether the marker chromosome presented here could evolve into a B chromosome in the future. [ABSTRACT FROM AUTHOR]

Lipid metabolic disturbances are associated with several diseases, such as type 2 diabetes or malignancy. In the last two decades, high-performance mass spectrometry-based lipidomics has emerged as a valuable tool in various fields of biology. However, the evaluation of macroscopic tissue homogenates leaves often undiscovered the differences arising from micron-scale heterogeneity. Therefore, in this work, we developed a novel laser microdissection-coupled shotgun lipidomic platform, which combines quantitative and broad-range lipidome analysis with reasonable spatial resolution. The multistep approach involves the preparation of successive cryosections from tissue samples, cross-referencing of native and stained images, laser microdissection of regions of interest, in situ lipid extraction, and quantitative shotgun lipidomics. We used mouse liver and kidney as well as a 2D cell culture model to validate the novel workflow in terms of extraction efficiency, reproducibility, and linearity of quantification. We established that the limit of dissectible sample area corresponds to about ten cells while maintaining good lipidome coverage. We demonstrate the performance of the method in recognizing tissue heterogeneity on the example of a mouse hippocampus. By providing topological mapping of lipid metabolism, the novel platform might help to uncover region-specific lipidomic alterations in complex samples, including tumors. [ABSTRACT FROM AUTHOR]

Aims: Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins. Methods: Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification. The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation. Results: More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones. Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA). Conclusions: In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillo-genesis is tightly regulated by the BAG3-associated protein quality system. [ABSTRACT FROM AUTHOR]

Moutan Cortex, is the root bark of Paeonia suffruticosa Andrews, which is classified into three specifications according to whether or not it is peeled and cored: Liandanpi, Guadanpi and whole root. In this study, the cork layer, cortex, phloem and xylem of P. suffruticosa fresh root were precisely separated by laser microdissection technique. UPLC-Q-Orbitrap-MS and UPLC-QQQ-MS techniques were used to analyse the differences in the chemical composition of different tissue parts of P. suffruticosa fresh root and Liandanpi, and to determine the optimal processing method of P. suffruticosa root. As a result, a total of 90 compounds were characterised, among which the cork layer had more types and higher contents of chemical constituents, and the xylem had fewer types and lower contents of chemical constituents. The proportion of xylem is larger, while the type and content of active ingredients is smaller. Therefore, the processing method of removing the wood core and retaining the cork bark can be used in the processing of Moutan Cortex. In this study, laser microdissection and ultra performance liquid chromatography-mass spectrometry were used to provide a theoretical basis for optimising the processing method of Moutan Cortex to enhance its pharmacological effects. • Accurate separation of different parts of P. suffruticosa root by LMD. • Characterization of different parts of P. suffruticosa root using UPLC-Q-Orbitrap-MS. • A total of 90 compounds were identified. • Quantitative analysis of four components in P. suffruticosa root using UPLC-QQQ-MS. • Determined the optimal processing method for P. suffruticosa root. [ABSTRACT FROM AUTHOR]

Samples for histological analyses are often formalin-fixed paraffin-embedded (FFPE) and slide-mounted, which complicates RNA extraction for many downstream molecular applications. Furthermore, when the region of interest is extremely small due to isolation with laser microdissection (LMD), extracting RNA of adequate quality and quantity is difficult. We describe an optimized protocol for maximizing RNA output from FFPE tissue devised to identify and analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars resulting from prior cesarean deliveries. Gomori trichrome staining allowed for region identification for LMD. Successful RNA isolation, reverse transcription and, importantly, quantitative real-time PCR (qRT-PCR) were performed. This report provides an optimized step-by-step protocol yielding sufficient RNA for qRT-PCR analyses from challenging tissue/LMD-FFPE samples.

Abstract Backgrounds Systemic amyloidosis is classified according to the deposited amyloid fibril protein (AFP), which determines its best therapeutic scheme. The most common type of AFP found are immunoglobulin light chains. The laser microdissection combined with mass spectrometry (LMD-MS) technique is a promising approach for precise typing of amyloidosis, however, the major difficulty in interpreting the MS data is how to accurately identify the precipitated AFP from background. Objectives The objective of the present study is to establish a complete data interpretation procedure for LMD-MS based amyloidosis typing. Methods Formalin-fixed paraffin-embedded specimens from patients with renal amyloidosis and non-amyloid nephropathies (including diabetic nephropathy, fibrillary glomerulonephritis, IgA nephropathy, lupus nephritis, membranous nephropathy, and normal tissue adjacent to tumors) were analyzed by LMD-MS. Forty-two specimens were used to train the data interpretation procedure, which was validated by another 50 validation specimens. Area under receiver operating curve (AUROC) analysis of amyloid accompanying proteins (AAPs, including apolipoprotein A-IV, apolipoprotein E and serum amyloid P-component) for discriminating amyloidosis from non-amyloid nephropathies was performed. Results A stepwise data interpretation procedure that includes or excludes the types of amyloidosis group by group was established. The involvement of AFPs other than immunoglobulin was determined by P-score, as well as immunoglobulin light chain by variable of λ-κ, and immunoglobulin heavy chain by H-score. This achieved a total of 88% accuracy in 50 validation specimens. The AAPs showed significantly different expression levels between amyloidosis specimens and non-amyloid nephropathies. Each of the single AAP had a AUROC value more than 0.9 for diagnosis of amyloidosis from non-amyloid control, and the averaged level of the three AAPs showed the highest AUROC (0.966), which might be an alternative indicator for amyloidosis diagnosis. Conclusions The proteomic data interpretation procedure for LMD-MS based amyloidosis typing was established successfully that has a high practicability in clinical application.

The close interaction between the enteric nervous system, microbiome, and brain in vertebrates is an emerging topic of recent studies. Different species such as rat, mouse, and human are currently being used for this purpose, among others. The transferability of protocols for tissue isolation and sample collection is not always straightforward. Thus, the present work presents a new protocol for isolation and sample collection of rat myenteric plexus cells for in vivo as well as in vitro studies. With the methods and chemicals described in detail, a wide variety of investigations can be performed with regard to normal physiological as well as pathological processes in the postnatal developing enteric nervous system. The fast and efficient preparation of the intestine as the first step is particularly important. We have developed and described a LIENS chamber to obtain optimal tissue quality during intestinal freezing. Cryosections of the flat, snap-frozen intestine can then be prepared for histological examination of the various wall layers of the intestine, e.g. by immunohistochemistry. In addition, these cryosections are suitable for the preparation of defined regions, as shown here using the ganglia of the mesenteric plexus. This specific tissue was obtained by laser microdissection, making the presented methodology also suitable for subsequent analyses that require high quality (specificity) of the samples. Furthermore, we present here a fully modernized protocol for the cultivation of myenteric neurons from the rat intestine, which is suitable for a variety of in vitro studies. [ABSTRACT FROM AUTHOR]

White mold is caused by the fungal pathogen Sclerotinia sclerotiorum and leads to rapid and significant loss in plant yield. Among its many brassicaceous hosts, including Brassica napus (canola) and Arabidopsis, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll, and vascular leaf tissue layers of B. napus in response to S. sclerotiorum. High-throughput tissue-specific mRNA sequencing increased the total number of detected transcripts compared with whole-leaf assessments and provided novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. When subjected to pathogen infection, the epidermis, mesophyll, and vasculature activate both specific and shared gene sets. Putative defense genes identified through transcription factor network analysis were then screened for susceptibility against necrotrophic, hemi-biotrophic, and biotrophic pathogens. Arabidopsis deficient in PR5-like RECEPTOR KINASE (PR5K) mRNA levels were universally susceptible to all pathogens tested and were further characterized to identify putative interacting partners involved in the PR5K signaling pathway. Together, these data provide insight into the complexity of the plant defense response directly at the site of infection. [ABSTRACT FROM AUTHOR]

Background: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. Methods: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6–9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. Results: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. Conclusions: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS). [ABSTRACT FROM AUTHOR]

Since the discovery of striatal neurons expressing dopamine-synthesizing enzymes, researchers have attempted to identify their phenotype and functional significance. In this study, it was shown that in transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) gene promoter, (i) there are striatal neurons expressing only TH, only aromatic L-amino acid decarboxylase (AADC), or both enzymes of dopamine synthesis; (ii) striatal neurons expressing dopamine-synthesizing enzymes are not dopaminergic since they lack a dopamine transporter; (iii) monoenzymatic neurons expressing individual complementary dopamine-synthesizing enzymes produce this neurotransmitter in cooperation; (iv) striatal nerve fibers containing only TH, only AADC, or both enzymes project into the lateral ventricles, providing delivery pathways for L-3,4-dihydroxyphenylalanine and dopamine to the cerebrospinal fluid; and (v) striatal GFP neurons express receptor genes for various signaling molecules, i.e., classical neurotransmitters, neuropeptides, and steroids, indicating fine regulation of these neurons. Based on our data, it is assumed that the synthesis of dopamine by striatal neurons is a compensatory response to the death of nigral dopaminergic neurons in Parkinson's disease, which opens broad prospects for the development of a fundamentally novel antiparkinsonian therapy. [ABSTRACT FROM AUTHOR]

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals. [ABSTRACT FROM AUTHOR]

Background: Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-β (Aβ) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation.Methods: In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia.Results: First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain.Conclusions: Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aβ pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aβ-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aβ oligomer detection and in Aβ-plaque initiation, with increased contribution as the disease progresses. [ABSTRACT FROM AUTHOR]

SUMMARY: Piperine (1‐piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Piper nigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with a blend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzes the reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co‐localization of enzymes and products, but also potential transport of piperamides to the sink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS), provides experimental evidence that piperine and PS are co‐localized in specialized cells of the black pepper fruit perisperm. PS accumulates during early stages of fruit development and its level declines before the fruits are fully mature. The product piperine is co‐localized to PS and can be monitored at the cellular level by its strong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasing numbers of fluorescent cells within the perisperm. Signal intensities of individual laser‐dissected cells when monitored by LC‐ESI‐MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperine and additional piperamides were also detected in cells distributed in the cortex of black pepper roots. In summary, the data provide comprehensive experimental evidence of and insights into cell‐specific biosynthesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination of fluorescence microscopy and LC‐MS/MS analysis we localized the major piperidine alkaloids to specific cells of the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthases shows that enzymes are co‐localized with high concentrations of products in these idioblasts. [ABSTRACT FROM AUTHOR]

The assignment of specific patterns of gene expression to specific cells in a complex tissue facilitates the connection between genotype and phenotype. Single-cell sequencing of whole tissues produces single-cell transcript resolution but lacks the spatial information of the derivation of each cell, whereas techniques such as multiplex FISH localize transcripts to specific cells in a tissue but require a priori information of the target transcripts to examine. Laser dissection of tissues followed by transcriptome analysis is an efficient and cost-effective technique that provides both unbiased gene expression discovery together with spatial information. Here, we detail a laser dissection protocol for total RNA extraction from butterfly larval and pupal wing tissues, without the need of paraffin embedding or the use of a microtome, that could be useful to researchers interested in the transcriptome of specific areas of the wing during development. This protocol can bypass difficulties in extracting high quality RNA from thick fixed tissues for sequencing applications. [ABSTRACT FROM AUTHOR]

Background: Anti-phospholipase A2 receptor autoantibody (PLA2R Ab)-associated membranous nephropathy (MN) is the most common form of primary MN (pMN). On the other hand, bucillamine (BCL), an antirheumatic drug developed in Japan, was reported to cause a rare form of secondary MN (sMN). Between these MN forms, comparative proteomic analysis of glomerular proteins has not been performed. Methods: We used renal biopsy specimens from 6 patients with PLA2R Ab (+) pMN, 6 patients with PLA2R Ab (‒) pMN, 6 patients with BCL-induced sMN, and 5 control cases (time 0 transplant biopsies). Proteins were extracted from laser-microdissected glomeruli and analyzed using mass spectrometry. The quantification values of protein abundance in each MN group were compared with those in the control group. Results: More than 800 proteins with high confidence were identified. Principal component analysis revealed a different distribution between the pMN and sMN groups. For further analysis, 441 proteins matched with ≥ 3 peptides were selected. Among the pMN and sMN groups, we compared the profiles of several protein groups based on the structural and functional characteristics, such as immunoglobulins, complements, complement-regulating proteins, podocyte-associated proteins, glomerular basement membrane proteins, and several proteins that are known to be associated with kidney diseases, including MN. In all MN groups, increased levels of immunoglobulins (IgG, IgA, and IgM), complements (C3, C4, and C9), complement factor H-related protein 5, type XVIII collagen, calmodulin, polyubiquitin, and ubiquitin ligase were observed. For some proteins, such as type VII collagen and nestin, the fold-change values were significantly different between the pMN and sMN groups. Conclusions: Between the pMN and BCL-induced sMN groups, we observed common and different alterations in protein levels such as known disease-associated proteins and potential disease marker proteins. [ABSTRACT FROM AUTHOR]

The endolymphatic sac is a small sac-shaped organ at the end of the membranous labyrinth of the inner ear. The endolymphatic sac absorbs the endolymph, in which the ion balance is crucial for inner ear homeostasis. Of the three sections of the endolymphatic sac, the intermediate portion is the center of endolymph absorption, particularly sodium transport, and is thought to be regulated by aldosterone. Disorders of the endolymphatic sac may cause an excess of endolymph (endolymphatic hydrops), a histological observation in Meniere's disease. A low-salt diet is an effective treatment for Meniere's disease, and is based on the assumption that the absorption of endolymph in the endolymphatic sac abates endolymphatic hydrops through a physiological increase in aldosterone level. However, the molecular basis of endolymph absorption in each portion of the endolymphatic sac is largely unknown because of difficulties in gene expression analysis, resulting from its small size and intricate structure. The present study combined reverse transcription-quantitative polymerase chain reaction and laser capture microdissection techniques to analyze the difference of gene expression of the aldosterone-controlled epithelial Na+ channel, thiazide-sensitive Na+-Cl− cotransporter, and Na+, K+-ATPase genes in the three individual portions of the endolymphatic sac in a rat model. A low-salt diet increased the expression of aldosterone-controlled ion transporters, particularly in the intermediate portion of the endolymphatic sac. Our findings will contribute to the understanding of the physiological function of the endolymphatic sac and the pathophysiology of Meniere's disease. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proteomes of acetabular glands in Trichobilharzia szidati cercariae greatly differ. • Postacetabular glands (PAg) possess 40 unique proteins and are rich in hydrolases. • Circumacetabular glands (CAg) have six unique proteins and are rich in VAL proteins. • Peptidases make up 8% of PAg and 6% of CAg proteins, elastase is unique to CAg. • Metallopeptidases of M8 and M16 families are the most abundant gland endopeptidases. Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum , the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati , employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 106 μm3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins. [ABSTRACT FROM AUTHOR]

Main conclusion: Glandular trichomes of Artemisia argyi H. Lév. & Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév. & Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography–mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4–6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi. [ABSTRACT FROM AUTHOR]

Backgrounds: Systemic amyloidosis is classified according to the deposited amyloid fibril protein (AFP), which determines its best therapeutic scheme. The most common type of AFP found are immunoglobulin light chains. The laser microdissection combined with mass spectrometry (LMD-MS) technique is a promising approach for precise typing of amyloidosis, however, the major difficulty in interpreting the MS data is how to accurately identify the precipitated AFP from background.Objectives: The objective of the present study is to establish a complete data interpretation procedure for LMD-MS based amyloidosis typing.Methods: Formalin-fixed paraffin-embedded specimens from patients with renal amyloidosis and non-amyloid nephropathies (including diabetic nephropathy, fibrillary glomerulonephritis, IgA nephropathy, lupus nephritis, membranous nephropathy, and normal tissue adjacent to tumors) were analyzed by LMD-MS. Forty-two specimens were used to train the data interpretation procedure, which was validated by another 50 validation specimens. Area under receiver operating curve (AUROC) analysis of amyloid accompanying proteins (AAPs, including apolipoprotein A-IV, apolipoprotein E and serum amyloid P-component) for discriminating amyloidosis from non-amyloid nephropathies was performed.Results: A stepwise data interpretation procedure that includes or excludes the types of amyloidosis group by group was established. The involvement of AFPs other than immunoglobulin was determined by P-score, as well as immunoglobulin light chain by variable of λ-κ, and immunoglobulin heavy chain by H-score. This achieved a total of 88% accuracy in 50 validation specimens. The AAPs showed significantly different expression levels between amyloidosis specimens and non-amyloid nephropathies. Each of the single AAP had a AUROC value more than 0.9 for diagnosis of amyloidosis from non-amyloid control, and the averaged level of the three AAPs showed the highest AUROC (0.966), which might be an alternative indicator for amyloidosis diagnosis.Conclusions: The proteomic data interpretation procedure for LMD-MS based amyloidosis typing was established successfully that has a high practicability in clinical application. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LUAD) shows heterogeneous morphological features and the stepwise progression from adenocarcinoma in situ to minimally invasive adenocarcinoma to invasive LUAD. Although multiple genetic alterations have been linked to the progression, the differences between the gene expression profiles of non-invasive lesions (non-ILs) and adjacent histologically normal lung (aNL) tissues within invasive LUAD have not been investigated. Herein, we analyzed differentially expressed genes (DEGs) specific to early-stage carcinogenesis in LUAD. Invasive LUAD tissue samples containing both non-ILs and aNL tissues were obtained from seven patients with pathological stage I LUAD, and each component was subjected to microdissection. Gene expression profiles of each component were determined using targeted RNA-sequencing. In total, 2536 DEGs, including 863 upregulated and 1673 downregulated genes, were identified in non-ILs. In non-ILs, the expression of SLC44A5, a choline transporter-like protein-coding gene, was significantly upregulated, and that of TMEM100, a gene encoding a transmembrane protein, was significantly downregulated. Reportedly, SLC44A5 plays an important role in the development and progression of hepatocellular carcinoma, whereas TMEM100 functions as a tumor suppressor in non-small cell lung cancer. Gene set enrichment analysis showed that DEGs in non-ILs were negatively enriched in cell death and immune response. Immunohistochemical analysis revealed that increased SLC44A5 expression and decreased TMEM100 expression were maintained in ILs. A protein–protein interaction (PPI) network analysis identified several upregulated and downregulated hub genes with high degrees in non-ILs. In conclusion, several new DEGs and key PPI network hub genes were identified in non-ILs, contributing to understanding of early-stage carcinogenesis in LUAD. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicine is made from the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae), known as Cangzhu. In this study, gas chromatography‐mass spectrometry was used to identify and quantify the volatile oils of different organs of A. lancea from four regions of China: Jiangsu, Anhui, Henan, and Hubei provinces. The volatile oils of A. lancea were qualitatively and quantitatively characterized using gas chromatography‐mass spectrometry combined with laser microdissection. The results identified 21 components in A. lancea, the majority of the components were found in the rhizomes, followed by the fibrous roots, flowers, leaves, and stems. According to the contents of volatile oils in A. lancea, it was divided into Dabieshan (mainly includes hinesol and β‐eudesmol) and Maoshan types (mainly includes atractylon and atractylodin), and the ratios of hinesol:β‐eudesmol:atractylon:atractylodin were 17.06:4.55:0:1, 12.66:11.71:0.99:1, 7.43:6.23:0:1, and 0.13:0.16:1.52:1 in A. lancea from AH, HN, HB, and JS, respectively. Tissue‐specific study indicated that Dabieshan type mainly includes elemol, hinesol, and β‐eudesmol in the periderm and secretory cavities of A. lancea, whereas Maoshan type mainly includes atractylon, atractylodin, little hinesol, and β‐eudesmol in the secretory cavities. Conversely, no volatile oils were detected in the cortex, phloem, xylem, vascular ray, or pith. This study provides a foundation for further evaluation and utilization of A. lancea. [ABSTRACT FROM AUTHOR]