Major Cell Research Articles

Regulation of the mouse ventral tegmental area by melanin-concentrating hormone.

Melanin-concentrating hormone (MCH) acts via its sole receptor MCHR1 in rodents and is an important regulator of homeostatic behaviors like feeding, sleep, and mood to impact overall energy balance. The loss of MCH signaling by MCH or MCHR1 deletion produces hyperactive mice with increased energy expenditure, and these effects are consistently associated with a hyperdopaminergic state. We recently showed that MCH suppresses dopamine release in the nucleus accumbens, which principally receives dopaminergic projections from the ventral tegmental area (VTA), but the mechanisms underlying MCH-regulated dopamine release are not clearly defined. MCHR1 expression is widespread and includes dopaminergic VTA cells. However, as the VTA is a neurochemically diverse structure, we assessed Mchr1 gene expression at glutamatergic, GABAergic, and dopaminergic VTA cells and determined if MCH inhibited the activity of VTA cells and/or their local microcircuit. Mchr1 expression was robust in major VTA cell types, including most dopaminergic (78%) or glutamatergic cells (52%) and some GABAergic cells (38%). Interestingly, MCH directly inhibited dopaminergic and GABAergic cells but did not regulate the activity of glutamatergic cells. Rather, MCH produced a delayed increase in excitatory input to dopamine cells and a corresponding decrease in GABAergic input to glutamatergic VTA cells. Our findings suggested that MCH may acutely suppress dopamine release while disinhibiting local glutamatergic signaling to restore dopamine levels. This indicated that the VTA is a target of MCH action, which may provide bidirectional regulation of energy balance.Significance Statement Role of melanin-concentrating hormone (MCH) on energy balance may converge on the dopamine system via the mesolimbic pathway, as loss of MCH or MCH receptor (MCHR1) signaling increases hyperactivity and energy expenditure associated with a hyperdopaminergic state. MCH can suppress dopamine release within the mesolimbic pathway, but its underlying mechanism is not known. We thus determined if MCH could inhibit dopamine release through direct actions within the ventral tegmental area (VTA). We found that MCH directly inhibited dopaminergic VTA cells, but MCH also disinhibited excitatory input to dopamine cells. Therefore, we showed that the VTA is a putative target site supporting dopamine-dependent actions of MCH.

Brain cell-type shifts in Alzheimer's disease, autism, and schizophrenia interrogated using methylomics and genetics.

Few neuropsychiatric disorders have replicable biomarkers, prompting high-resolution and large-scale molecular studies. However, we still lack consensus on a more foundational question: whether quantitative shifts in cell types-the functional unit of life-contribute to neuropsychiatric disorders. Leveraging advances in human brain single-cell methylomics, we deconvolve seven major cell types using bulk DNA methylation profiling across 1270 postmortem brains, including from individuals diagnosed with Alzheimer's disease, schizophrenia, and autism. We observe and replicate cell-type compositional shifts for Alzheimer's disease (endothelial cell loss), autism (increased microglia), and schizophrenia (decreased oligodendrocytes), and find age- and sex-related changes. Multiple layers of evidence indicate that endothelial cell loss contributes to Alzheimer's disease, with comparable effect size to APOE genotype among older people. Genome-wide association identified five genetic loci related to cell-type composition, involving plausible genes for the neurovascular unit (P2RX5 and TRPV3) and excitatory neurons (DPY30 and MEMO1). These results implicate specific cell-type shifts in the pathophysiology of neuropsychiatric disorders.

#2069 Elucidating the function of novel Arg1+/Clec4d+ scar-associated monocyte-derived macrophage population in driving fibrosis in kidney disease models

Abstract Background and Aims Fibrosis is the final common pathway in all progressive kidney disease. Macrophages are a major myeloid cell component of the renal mononuclear phagocyte system, with roles in defence against infection, renal injury, and repair. Using single-cell RNA sequencing, we identified a novel myeloid cell subset exclusively present in acute injury of the unilateral ureteric obstruction (UUO) model of kidney fibrosis. This population transcriptomically aligns to monocytes but is enriched for both Arginase-1 (Arg1) and the C-type lectin 4d (Clec4d) and a large number of pro-inflammatory and pro-fibrotic genes. We hypothesise that this novel Arg1+/Clec4d+ population contributes to fibrosis deposition in progressive kidney disease. Methods Monocyte-macrophage populations were characterised in different pre-clinical models; UUO, unilateral ischaemic reperfusion injury (uIRI) and subtotal nephrectomy with flow cytometry. Arg1+/Clec4d+ cells were identified by RT-qPCR, flow cytometry, and immunofluorescence. The origin and consequence of Arg1 deletion in monocytes on renal fibrosis was determined by utilising Ccr2-ERT2-TdTomato and Ccr2-ERT2-TdTomato; Arg1 fl/fl mice, respectively. Results The presence of the Arg1+/Clec4d+ cells was validated in the UUO, uIRI and subtotal nephrectomy models of kidney injury. Analysis of intra-renal inflammation revealed CD45+CD11b+Arg1+ cells persisted and increased in number across 7 days. Post-injury, Arg1+ cells were confirmed to be Clec4dhi. To confirm that Arg1+/Clec4d+ cells were derived from monocytes (Ccr2+), we administered tamoxifen to Ccr2CreERT2-TdTomato mice under a single or multiple-dose regimen following UUO surgery. At day 7 post-injury, ∼50% and ∼90% of the Arg1+ cells were TdTomato+ following a single or multiple doses of tamoxifen, respectively, suggesting that these cells are primarily monocyte derived. Moreover, the Arg1+/Clec4d+ cells were found to localise to areas of scarring (Fig. 1). To investigate the therapeutic potential of targeting Clec4d+ expressed on the Arg1+/Clec4d+ macrophages, mice underwent UUO surgery and given multiple doses of a Clec4d-neutralising antibody. A decrease in Arg1 and fibrosis-related gene expression was evident by day 7 post-injury. To understand the role of increased Arg1 expression in the cells we generated Ccr2-ERT2-TdTomato; Arg1 fl/fl mice. These mice had as expected a reduced Arg1 expression in the Ccr2+ population and metabolic profiling by SCENITH of the tissue monocytes revealed that the loss of Arg1 in the cells reduced their glucose dependence. The loss of Arg1 in these cells reduced Havcr1 gene expression in the kidney. Conclusion As fibrosis is a pathological process that can affect any organ, the work included here, in addition to the proposed future studies, has the potential to develop novel therapeutics that limit fibrosis and enhance repair to halt the progression of disease in CKD.

#161 Single-Nucleus RNA-Seq of Human Kidney Biopsy Tissue Reveals Microstructure-Specific Similarities and Differences in Various Glomerulonephritis

Abstract Background and Aims Transcriptomic alterations in kidney diseases may lead to the discovery of novel biomarkers. Single-nucleus RNA-seq (SnSeq) is a valuable method for profiling the transcriptome of kidney-residing cells. Additional SnRNA-seq data is necessary, including cases of human biopsy-confirmed glomerulonephritis. Method We collected snap-frozen kidney biopsy tissues from 6 cases of IgA nephropathy (IgAN), 6 cases of minimal change disease, 6 cases of PLA2R-Ab positive membranous nephropathy, 3 cases of diabetic kidney disease, and 7 nephrectomy control cases. SnSeq was performed on the kidney tissues, and kidney cells were identified in the dataset through UMAP clustering. The proportions of each cell subcluster were compared between the study groups. Differentially expressed genes (DEGs) were identified. K-means clustering and Weighted Gene Co-expression Network Analysis (WGCNA) were performed. Results We successfully collected transcriptomic profiles of 55 615 cells from IgAN, 53 370 from minimal change disease, 53 495 from membranous nephropathy, 34 343 from diabetic kidney disease, and 66 161 from control cases. The SnSeq data successfully identified major kidney cell clusters, including a considerable number of glomerular cells such as podocytes, mesangial cells, endothelial cells, and others. DEGs were largely identified in podocytes and proximal tubule cells, as well as in other glomerular cells. K-means clustering identified diverse similarities and differences between the study groups for each cell type. We identified diverse cell type-specific but disease-common DEGs, particularly in the downregulated genes, suggesting that the studied diseases share similar destruction in kidney microstructure. In comparison, cell type-common but disease-specific DEGs were also identified, considering a common pathophysiologic mechanism may occur in total kidney cortex. Diseases that can feature nephrotic syndrome showed high expression of AVP receptors, suggesting the association with edema, or highly expressed renin-angiotensin-aldosterone pathway was noted in IgAN or diabetic kidney disease, which was not evident in minimal change disease or membranous nephropathy patients. The studied disease shared certain types of inflammatory pathways (e.g. interferon-alpha or TGF-beta) but some pathways were differently enriched according to the disease types. WGCNA analysis revealed gene modules that were highly correlated with age, proteinuria, and estimated GFR, regardless of the diagnosis subtype. Conclusion The current SnSeq data is, to date, the largest one, including biopsy-confirmed human kidney glomerulonephritis cases. The results identified disease-specific changes in specific cell types. Additionally, common transcriptomic alterations occurring following changes in estimated GFR, age, and proteinuria were investigated. The data may be a valuable resource for future research on glomerulonephritis biomarker development.

Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Cabergoline Targets Multiple Pathways to Inhibit PRL Secretion and Increases Stromal Fibrosis.

Unravel potential mechanism(s) of the on- and off-target actions of dopamine agonist therapy in both human prolactinoma tumor and neighboring stromal and immune cells. Five surgically resected prolactinomas from 3 cabergoline (CBG)-treated and 2 treatment naive patients were analyzed by single cell RNA sequencing (scRNA-seq) to compare the cellular composition and transcriptional landscape. Six major cell populations that included tumor (88.2%), immune (5.6%), stromal (4.9%), progenitor cells (0.6%), proliferating cells (0.4%), and erythrocytes (0.2%) were observed. Tumor cells from CBG-treated patients expressed lower levels of genes that regulated hormone secretion, such as SCG2, VGF, TIMP1, NNAT, and CALD1, consistent with the inhibitory effects of CBG on hormone processing and secretion. Interestingly, we also observed an increased number of CD8+ T cells in the CBG-treated tissues. These cytotoxic CD8+ T cells expressed killing granule components, such as perforin and the granzymes GZMB, GNLY and KLRD1 as well as the inflammatory cytokine CCL5. Immune cell activation of these CD8+ T cells was further analyzed in a compartment-specific manner, and increased CD25 (IL2R) expression was noted in the CD8+ T cells from CBG-treated samples. Additionally, and confirming prior reports, we noted a higher stromal cell population in CBG-treated samples. Our scRNAseq studies revealed key differences in the transcriptomic features of CBG-treated and untreated PRLomas in both tumor and microenvironment cellular constituents, and for the first time describe previously unknown activation of CD8+ T cells following CBG-treatment which may play a role in the tumoricidal actions of CBG.

CD44 and its implication in neoplastic diseases.

CD44, a nonkinase single span transmembrane glycoprotein, is a major cell surface receptor for many other extracellular matrix components as well as classic markers of cancer stem cells and immune cells. Through alternative splicing of CD44 gene, CD44 is divided into two isoforms, the standard isoform of CD44 (CD44s) and the variant isoform of CD44 (CD44v). Different isoforms of CD44 participate in regulating various signaling pathways, modulating cancer proliferation, invasion, metastasis, and drug resistance, with its aberrant expression and dysregulation contributing to tumor initiation and progression. However, CD44s and CD44v play overlapping or contradictory roles in tumor initiation and progression, which is not fully understood. Herein, we discuss the present understanding of the functional and structural roles of CD44 in the pathogenic mechanism of multiple cancers. The regulation functions of CD44 in cancers-associated signaling pathways is summarized. Moreover, we provide an overview of the anticancer therapeutic strategies that targeting CD44 and preclinical and clinical trials evaluating the pharmacokinetics, efficacy, and drug-related toxicity about CD44-targeted therapies. This review provides up-to-date information about the roles of CD44 in neoplastic diseases, which may open new perspectives in the field of cancer treatment through targeting CD44.

Integrated analysis of single-cell and bulk RNA sequencing data reveals a myeloid cell-related regulon predicting neoadjuvant immunotherapy response across cancers

BackgroundImmunotherapy has brought about a paradigm shift in the treatment of cancer. However, the majority of patients exhibit resistance or become refractory to immunotherapy, and the underlying mechanisms remain to be explored.MethodsSing-cell RNA sequencing (scRNA‑seq) datasets derived from 1 pretreatment and 1 posttreatment achieving pathological complete response (pCR) patient with lung adenocarcinoma (LUAD) who received neoadjuvant immunotherapy were collected, and pySCENIC was used to find the gene regulatory network (GRN) between cell types and immune checkpoint inhibitor (ICI) response. A regulon predicting ICI response was identified and validated using large‑scale pan-cancer data, including a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, The Cancer Genome Atlas (TCGA) pan-cancer cohort, and 5 ICI transcriptomic cohorts. Symphony reference mapping was performed to construct the myeloid cell map.ResultsThirteen major cluster cell types were identified by comparing pretreatment and posttreatment patients, and the fraction of myeloid cells was higher in the posttreatment group (19.0% vs. 11.8%). A PPARG regulon (containing 23 target genes) was associated with ICI response, and its function was validated by a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, TCGA pan-cancer cohort, and 5 ICI transcriptomic cohorts. Additionally, a myeloid cell map was developed, and cluster I, II, and III myeloid cells with high expression of PPARG were identified. Moreover, we constructed a website called PPARG (https://pparg.online/PPARG/ or http://43.134.20.130:3838/PPARG/), which provides a powerful discovery tool and resource value for researchers.ConclusionsThe PPARG regulon is a predictor of ICI response. The myeloid cell map enables the identification of PPARG subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

Keratin 19 (Krt19) is a novel marker gene for epicardial cells

Epicardial cells regulate heart growth by secreting numerous growth factors and undergoing lineage specification into other cardiac lineages. However, the lack of specific marker genes for epicardial cells has hindered the understanding of this cell type in heart development. Through the analysis of a cardiac single cell mRNA sequencing dataset, we identified a novel epicardial gene named Keratin 19 (Krt19). Further analysis of the expression patterns of Krt19 and Wt1, a well-known epicardial gene, revealed their preferences in major cardiac cell types. Using lineage-tracing analysis, we analyzed Krt19-CreER labeled cells at multiple time windows and found that it labels epicardial cells at both embryonic and neonatal stages. Furthermore, we studied the function of epicardial cells using a diphtheria toxin A chain (DTA)-based cell ablation system. We discovered that Krt19-CreER labeled cells are essential for fetal heart development. Finally, we investigated the function of Krt19-CreER and Wt1-CreER labeled cells in neonatal mouse development. We observed that the Krt19-CreER; Rosa-DTA mice displayed a smaller size after tamoxifen treatment, suggesting the potential importance of Krt19-CreER labeled cells in neonatal mouse development. Additionally, we found that Wt1-CreER; Rosa-DTA mice died at early stages, likely due to defects in the kidney and spleen. In summary, we have identified Krt19 as a new epicardial cell marker gene and further explored the function of epicardial cells using the Krt19-CreER and Wt1-CreER-mediated DTA ablation system.

A Prospective Study Investigating Immune Checkpoint Molecule and CD39 Expression on Peripheral Blood Cells for the Prognostication of COVID-19 Severity and Mortality

In patients with COVID-19, broad panels of immune checkpoint molecules (ICPMs) and the purinergic signaling have not been studied in parallel. We aimed to perform in-depth immunophenotyping of major cell subsets present in human peripheral blood of COVID-19 patients and controls using PD1, TIM3, LAG3, TIGIT, and CD200R, as well as CD39, as markers for the purinergic signaling pathway. We studied 76 COVID-19 patients and 12 healthy controls using peripheral blood mononuclear cells on flow cytometry. Univariable and multivariable statistics were performed. All ICPMs studied were significantly overexpressed on different cell subsets of COVID-19 patients when compared with healthy controls. Elevated lactate dehydrogenase; C-reactive protein; age; and high expression of CD45+, CD39+CD45+, TIM3+CD39+CD4+CD45+, and TIM3+CD39+CD8+CD3+CD4+ cells were significantly associated with severe COVID-19. On multivariable analysis, however, only high expression of CD39+CD45+ (OR 51.4, 95% CI 1.5 to 1763) and TIM3+CD39+CD4+CD3+CD45+ (OR 22.6, 95% CI 1.8 to 277) cells was an independent predictor for severe COVID-19. In conclusion, numerous ICPMs are overexpressed in COVID-19 patients when compared with healthy controls, suggesting a pathophysiological role of these molecules in SARS-CoV-2 infection. However, only TIM3 in co-expression with CD39 remained as a significant independent prognostic ICPM on multivariable analysis. The flow cytometric evaluation of TIM3+CD39+CD4+CD3+CD45+, as well as CD39+CD45+, is a powerful tool for the prognostication of COVID-19 patients on hospital admission.

O15 Transcriptomic profiling reveals regulators of dermal fibroblast state switching

Abstract Introduction and aims Fibroblasts, the major cell population in connective tissue, are the guardians of the extracellular matrix homeostasis. In adult tissue they exist in a quiescent state that can persist for years, but upon tissue injury, they become rapidly activated and re-enter the cell cycle, followed by deposition and reorganization of the dermal matrix. The fundamental mechanism by which fibroblasts can maintain quiescence and be primed for prompt activation is currently unknown. Deregulations of this process in the skin and other organs is associated with several pathologies including fibrosis or chronic wounds. Notably, rapid fibroblast state switching is also observed during skin development, where a tightly controlled change from a highly proliferative state to a quiescent state promotes extracellular matrix deposition and maturation of the dermal architecture. Understanding the shared mechanisms and differences between development and wound healing will help in the development of tissue regenerative therapies. Methods Using dermal development as a model for fibroblast state switching, we integrated several single-cell RNA-Seq and assay for transposase-accessible chromatin-Seq datasets of whole skin during embryonic and postnatal growth. Our novel integrated multiomics single-cell dataset can recapitulate skin developmental changes observed in vivo and reveal the major regulatory networks. Results We identified the activating protein (AP)-1 member cJun as the key regulator of fibroblast state switching in skin development and wound repair. Mechanistically, in quiescent adult fibroblasts cJun is highly transcribed, but the protein is constantly ubiquitinated and degraded, priming fibroblasts for rapid activation when needed. Upon injury cJun ubiquitination is reduced, allowing an accumulation of cJun leading to a change in AP-1 dimer composition at the chromatin level, inhibiting fibroblast quiescence and promoting proliferation and activation. Conclusions In normal wounds cJun accumulation is inhibited during the advanced (remodelling) wounding phase; however, chronic wounds and fibrotic disease conditions show a continuous and pathological cJun stabilization, which can be targeted through pharmacological intervention.

The capsular polysaccharide obstructs wall teichoic acid functions in Staphylococcus aureus.

The cell envelope of Staphylococcus aureus contains two major secondary cell wall glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Both the CP and the WTA are attached to the cell wall and play distinct roles in S. aureus colonization, pathogenesis, and bacterial evasion of host immune defenses. We aimed to investigate whether CP interferes with WTA-mediated properties. Strains with natural heterogeneous expression of CP, strains with homogeneous high CP expression and CP-deficient strains were compared to WTA deficient controls regarding WTA dependent phage binding, cell adhesion, IgG deposition, and virulence in vivo. WTA-mediated phage adsorption, specific antibody deposition and cell adhesion were negatively correlated with CP expression. WTA, but not CP, enhanced the bacterial burden in a mouse abscess model, while CP overexpression resulted in intermediate virulence in vivo. CP protects the bacteria from WTA-dependent opsonization and phage binding. This protection comes at the cost of diminished adhesion to host cells. The highly complex regulation and mostly heterogeneous expression of CP has probably evolved to ensure the survival and optimal physiological adaptation of the bacterial population as a whole.

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Deciphering the cross-talk between miRNA and tight junctions in hepatocellular carcinoma

The most common type of liver cancer is hepatocellular carcinoma (HCC) causes a lower survival rate even after systemic treatment. Previous studies have shown evidence that various molecular and epigenetic mechanisms are involved in the transition of HCC from normal liver cells. Epigenetics plays an important role in maintaining genomic stability in normal liver cells. Apart from the mutation of genes, epigenetic factors are involved in HCC progression. miRNA tends to be a major epigenetic factor involved in regulating major cell cycle pathways. miRNA regulates the HCC progression by inhibiting the major apoptotic pathways and favors angiogenesis and tumor microenvironment. Apart from regulating major pathways, miRNA appears to regulate tight junction integrity. Tight junction proteins appear to be strong barrier proteins involved in cell adhesion and integrity. Disturbance in cell adhesion and integrity leads to the major dysregulation of cell cycle resulting in cancer progression. Moreover, dysregulation of tight junction integrity was observed in the pathogenesis of HCC. The regulation of tight junction proteins via miRNA were studied in various diseases. Here, we discussed the regulation of tight junction proteins via miRNA in HCC progression. This review may focus on the significance of miRNA in cellular function and its role in regulating tight junction proteins that impact in HCC progression which opens a new approach to develop a new diagnostic marker for an early detection of HCC and novel therapeutic approach against HCC.

Zinc about it - zinc and calf immunity.

Micronutrients, such as vitamins and trace minerals, are critical for supporting growth, performance, health and maintaining redox balance. Zinc (Zn), an essential micronutrient, aids the functioning of innate and adaptive immune cells. This scoping review aims to assemble and evaluate the evidence available for the role of Zn within calf immunity. Relevant literature was identified within Web of Science, PubMed, and CABI using search terms specific to the major innate and adaptive immune cell populations. There was no evidence that Zn supplementation altered neutrophil, natural killer cell, or T-cell functions. However, there was limited evidence to support Zn supplementation with reduced monocyte numbers, but there was no evidence to associate the monocytopenia with improvements in monocyte function. There is moderate evidence to suggest that Zn supplementation was beneficial for maintaining epithelial barriers of integumental and mucosal surfaces. The evidence supports supplementation above the current industry recommendations for improving immunoglobulin (Ig) production, with the strongest results being observed for IgG and IgM. Moreover, Zn supplementation was associated with reduced proinflammatory cytokine production, which may reduce inflammation-associated hypophagia and warrants further investigation. Furthermore, Zn reduced the duration of clinical signs in animals facing respiratory disease and diarrhea. However, consensus is needed about the optimal dose, route, and Zn formulation most appropriate for supporting immunity. In conclusion, while the literature supports that Zn could enhance calf immunity, there is insufficient evidence to adequately determine the extent to which Zn impacts innate immune cell and T-cell functions. Determination of the immune cell functions susceptible to modification by Zn supplementation is an important knowledge gap for enhancing the understanding of Zn and calf immunity.

Integrating spatial and single-cell transcriptomics reveals tumor heterogeneity and intercellular networks in colorectal cancer

Single cell RNA sequencing (scRNA-seq), a powerful tool for studying the tumor microenvironment (TME), does not preserve/provide spatial information on tissue morphology and cellular interactions. To understand the crosstalk between diverse cellular components in proximity in the TME, we performed scRNA-seq coupled with spatial transcriptomic (ST) assay to profile 41,700 cells from three colorectal cancer (CRC) tumor-normal-blood pairs. Standalone scRNA-seq analyses revealed eight major cell populations, including B cells, T cells, Monocytes, NK cells, Epithelial cells, Fibroblasts, Mast cells, Endothelial cells. After the identification of malignant cells from epithelial cells, we observed seven subtypes of malignant cells that reflect heterogeneous status in tumor, including tumor_CAV1, tumor_ATF3_JUN | FOS, tumor_ZEB2, tumor_VIM, tumor_WSB1, tumor_LXN, and tumor_PGM1. By transferring the cellular annotations obtained by scRNA-seq to ST spots, we annotated four regions in a cryosection from CRC patients, including tumor, stroma, immune infiltration, and colon epithelium regions. Furthermore, we observed intensive intercellular interactions between stroma and tumor regions which were extremely proximal in the cryosection. In particular, one pair of ligands and receptors (C5AR1 and RPS19) was inferred to play key roles in the crosstalk of stroma and tumor regions. For the tumor region, a typical feature of TMSB4X-high expression was identified, which could be a potential marker of CRC. The stroma region was found to be characterized by VIM-high expression, suggesting it fostered a stromal niche in the TME. Collectively, single cell and spatial analysis in our study reveal the tumor heterogeneity and molecular interactions in CRC TME, which provides insights into the mechanisms underlying CRC progression and may contribute to the development of anticancer therapies targeting on non-tumor components, such as the extracellular matrix (ECM) in CRC. The typical genes we identified may facilitate to new molecular subtypes of CRC.

Validation of an Organ Mapping Antibody Panel for Cyclical Immunofluorescence Microscopy on Normal Human Kidneys.

The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for 2-D and 3-D Cyclical Immunofluorescence (CyCIF) to provide a more detailed method to evaluate of tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills, opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types and how these are altered in disease.

Brain organoids engineered to give rise to glia and neural networks after 90 days in culture exhibit human-specific proteoforms.

Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However, it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein, we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation, and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months in vitro. Under these optimized conditions, unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male-female siblings are similar in growth rate, size, and total protein content, and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal, microglial, and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples, providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus, our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain, and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g., isoforms, splice variants) as well as modulate glial and neuronal processes in an in situ-like environment.

Lipidomics of Microplasma-Irradiated Cells at Optimized Discharge Conditions for the Absorption of High-Molecule Drug

Microplasma irradiation is a promising technique for the transdermal delivery of high-molecular-weight drugs. In this technique, microplasma components interact with the skin surface or cell membranes, allowing the drugs to penetrate. For efficient and safe drug delivery, it is crucial to understand these interactions. To this end, this study investigated the effects of microplasma irradiation on cellular lipids, particularly those associated with cell membranes. Rat intestinal epithelial cells were treated with microplasma irradiation at two different voltages (4.0 kV or 4.5 kV). An untargeted lipidomic was conducted using liquid chromatography–mass spectrometry (LC/MS) technique. The results revealed that microplasma irradiation at 4.0 kV induces a significant increase in cell membrane lipids within 10 min post-irradiation. All major cell membrane lipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin, exhibited increases of over 25% within this short timeframe. Notably, this effect is transient, as lipid levels return to their baseline after 12 h. Furthermore, no significant differences in live and apoptotic cell percentages were observed between the control and 12 h post-irradiated cells. In contrast, irradiation at 4.5 kV did not elicit significant changes in cell membrane lipids, correlating with the absence of drug absorption under this condition. Hence, our study unveiled a correlation between the rapid increase in cell membrane lipids and enhanced drug absorption in microplasma-irradiated cells. This lipid augmentation potentially enhances membrane fluidity and permeability, thus facilitating drug absorption. Beyond elucidating the mechanisms and safety of microplasma-based drug delivery, our research provides valuable insights for advancing various microplasma-based biomedical technologies.

Dissecting microenvironment in cystadenomas and hepatic cysts based on single nucleus RNA-sequencing data

Hepatic cystadenoma is a rare disease, accounting for about 5% of all cystic lesions, with a high tendency of malignant transformation. The preoperative diagnosis of cystadenoma is difficult, and some cystadenomas are easily misdiagnosed as hepatic cysts at first. Hepatic cyst is a relatively common liver disease, most of which are benign, but large hepatic cysts can lead to pressure on the bile duct, resulting in abnormal liver function. To better understand the difference between the microenvironment of cystadenomas and hepatic cysts, we performed single-nuclei RNA-sequencing on cystadenoma and hepatic cysts samples. In addition, we performed spatial transcriptome sequencing of hepatic cysts. Based on nucleus RNA-sequencing data, a total of seven major cell types were identified. Here we described the tumor microenvironment of cystadenomas and hepatic cysts, particularly the transcriptome signatures and regulators of immune cells and stromal cells. By inferring copy number variation, it was found that the malignant degree of hepatic stellate cells in cystadenoma was higher. Pseudotime trajectory analysis demonstrated dynamic transformation of hepatocytes in hepatic cysts and cystadenomas. Cystadenomas had higher immune infiltration than hepatic cysts, and T cells had a more complex regulatory mechanism in cystadenomas than hepatic cysts. Immunohistochemistry confirms a cystadenoma-specific T-cell immunoregulatory mechanism. These results provided a single-cell atlas of cystadenomas and hepatic cyst, revealed a more complex microenvironment in cystadenomas than in hepatic cysts, and provided new perspective for the molecular mechanisms of cystadenomas and hepatic cyst.