Immunocytochemical Analysis Research Articles

Abstract TP307: oxLDL Preconditioning Intensifies Ischemic-Like Injury Mediated Alterations in Human Male Brain Endothelial Cell Tight Junction Protein Levels and Proinflammatory Mediators

Introduction: Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular function and integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular dysfunction and injury is elevated levels of oxidized low density lipoprotein (oxLDL). We hypothesized that oxLDL would exacerbate acute ischemic injury mediated alteration of endothelial proinflammatory mediator and integral barrier protein levels. Methods: Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with human oxLDL (50 or 100ug/dL; 6, 12, 24, or 36h) or vehicle, using a serum dose reported in AIS patients. HBMECs were then exposed to normoxia (21% O 2 ) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O 2 ), for 3 or 6h. HBMEC levels or expression of barrier markers, adhesion molecules, and inflammatory mediators were examined using qRT-PCR, in cell western, and FIJI mediated immunocytochemical analysis. Secondary analysis was performed on RNA sequencing of adult male aortic endothelial cells (HAoECs) exposed to oxLDL (50ug/mL; 3, 6, 12, 24h) obtained from GEO dataset GSE13139. Results: HGD injury concomitantly decreased HBMEC claudin-5, occludin, and ZO-1 as well as increased IL-1b, ICAM-1, VCAM-1, iNOS, and TNF-a. OxLDL exacerbated an HGD mediated decrease in HBMEC barrier levels and increase in proinflammatory levels in a dose and time dependent manner. HAoEC barrier, adhesion, and proinflammatory levels were not altered by oxLDL. Conclusion: OxLDL may preferentially alter brain endothelial barrier and proinflammatory levels, as opposed to aortic endothelial cells in dose and time dependent manner. Mitigating the effect of oxLDL on brain endothelial permeability and inflammation, possibly via receptor inhibition may be a novel, viable therapeutic target in the treatment of AIS.

Immunocytochemical Analysis of Crocin against Oxidative Stress in Trigeminal Sensory Neurons Innervating the Cornea.

Corneal diseases are a major cause of vision loss, often associated with aging, trauma and disease. Damage to corneal sensory innervation leads to discomfort and pain. Environmental stressors, such as short-wavelength light, can induce oxidative stress that alters mitochondrial function and affects cell and tissue homeostasis, including corneal innervation. Cellular antioxidant mechanisms may attenuate oxidative stress. This study investigates crocin, a derivative of saffron, as a potential antioxidant therapy. In vitro rat trigeminal sensory ganglion neurons were exposed to both sodium azide and blue light overexposure as a model of oxidative damage. Crocin was used as a neuroprotective agent. Mitochondrial and cytoskeletal markers were studied by immunofluorescence analysis to determine oxidative damage and neuroprotection. In vivo corneal innervation degeneration was evaluated in cornea whole mount preparations using Sholl analyses. Blue light exposure induces oxidative stress that affects trigeminal neuron mitochondria and alters sensory axon dynamics in vitro, and it also affects corneal sensory innervation in an in vivo model. Our results show that crocin was effective in preserving mitochondrial function and protecting corneal sensory neurons from oxidative stress. Crocin appears to be a promising candidate for the neuroprotection of corneal innervation.

Role of cellular interaction and cell phenotype in azacytidine to doxorubicin sensitivity in breast cancer cell-derived clones

BackgroundBreast malignancies are the most diverse tumors, having the highest level of cellular heterogeneity at various stages of development. This heterogeneous nature of cells is even observed in a controlled growth environment. Cellular phenotype, expression of cell surface markers, epithelial to mesenchymal transition, and cellular niche play a pivotal role in the heterogeneity of breast cancer cells. Heterogeneity in breast cancer cells is one of the leading causes of drug resistance where only a portion of cells respond to the therapeutic drugs while the other cells escape from therapies. Furthermore, cellular heterogeneity is one of the major barriers to designing therapeutic strategies. Therefore, it is important to design multidimensional strategies that consider various aspects, including cell type, cellular microenvironment, and the type of anticancer drugs to treat breast cancer. AimThis study aimed to investigate the responses of cells from various single-cell-derived breast cancer clones to 5-azacytidine (AzaC) and doxorubicin (Dox). Furthermore, the role of AzaC in the sensitization of breast cancer cells to Dox was studied in breast cancer clones. Materials and methodsSingle cell-derived clones were established from four distinct breast cancer cell lines including MCF7, MDA-MB-231, MCF7-GAPDH-RFP and MDA-GAPDH-RFP. Following one week of clonal development, the clones were subjected to separate or combined treatments of 5 μM AzaC and 500 nM Dox for 48 h. In the sequential group (AzaC/Dox), clones were exposed to 5 μM AzaC for one week, followed by 500 nM Dox for 48 h. Morphological and immunocytochemical analyses were performed by using crystal violet staining and immunolabeling, respectively. Furthermore, the expression levels of specific protein markers, including alpha smooth muscle actin (α-SMA), vimentin (VIM), cytokeratin-8 (CK-8), cytokeratin-19 (CK-19) and platelet endothelial cell adhesion molecule-1 (PECAM-1), were evaluated in response to the drugs. In addition, the expressed target protein markers were quantified in five independent sets of clones from each treatment group (n = 5). ResultsAfter 48 h of treatment, the sizes of the colonies in all treatment groups were markedly decreased as compared to the control group. In the AzaC group, colonies exhibited irregular shapes and scattered cell patterns. In Dox- and AzaC+Dox-treated clones, the cell number in each colony evidently decreased, while in AzaC/Dox-treated clones, a fraction of clonal cells showed a similar phenotype to mesenchymal-like cells. Immunocytochemistry showed a significant decrease in the number of α-SMA- and VIM-expressing cells in the drug-treated groups. Notably, in MCF7- GAPDH-RFP and MDA-GAPDH-RFP clones, VIM expression remained high despite the low cell number. Overall, CK-8 was down-regulated while CK-19 was strongly expressed in AzaC-treated MDA-MB-231 clones. Despite the small cell number, both markers exhibited up-regulation in the drug-treated MCF7- and MDA-GAPDH-overexpressed clones. Likewise, there was a notable increase in the expression of PECAM-1 in drug-treated MCF7- and MDA-RFP clones. ConclusionThis study demonstrates that AzaC-sensitizes breast cancer cells to Dox in single cell-derived clones. Furthermore, the high expression pattern of the target protein markers in drug-treated clones reflects their role in cell survival and drug resistance. Further studies are needed to uncover the detailed molecular mechanism involved in cell survival and drug resistance against AzaC and Dox in breast cancer cell-derived clones.

In vitro effects of heparin-binding epidermal growth factor on adhesion stage of implantation.

A member of the epidermal growth factor (EGF) family, the heparin-binding EGF (HB-EGF) is expressed in the uteri of both humans and mice during the implantation process. To study the effects of HB-EGF on adhesion stage, we developed an in vitro implantation model employing Ishikawa cell line and JAR cell line, which may attach to Ishikawa cells. For 1, 6, 12, and 24 hours, co-cultures of JAR spheroids grown on Ishikawa monolayers were treated with 1, 10, and 100 ng∕mL doses of HB-EGF. Using immunocytochemistry and Western blot analysis, the effects of HB-EGF on the protein expressions of E-cadherin, Erb-B2 receptor tyrosine kinase 4 (ErbB4), and integrin ανβ3 in Ishikawa and JAR cells were examined semi-quantitatively and quantitatively. Ultrastructural changes of in vitro implantation model were investigated by transmission electron microscopy. We revealed that HB-EGF influenced trophoblast cell adhesion to endometrial cells by upregulating the expression of the proteins ErbB4 and trophoblastic integrin ανβ3. Decrease in trophoblastic E-cadherin expression and increase in endometrial E-cadherin expression were demonstrated accompanying morphological variations in cells required for the invasion. We discovered ultrastructurally that Ishikawa cells acquired uterodome-like appearance, including the organelles, when 10 and 100 ng∕mL dosages of HB-EGF were administered for 12 and 24 hours. However, following additional hours of adhesion and invasion, their intercellular spaces enlarged. The trafficking of vesicular transport was enhanced by JAR spheroids. We therefore discovered that in this implantation paradigm, HB-EGF may enhance the receptivity of Ishikawa cells and the adherence of JAR cells.

Cell wall epitope distribution in the functional compartments of galls induced by Palaeomystella oligophaga (Lepidoptera) in Macairea radula (Melastomataceae)

Context The parasitic interaction between the galling insect Palaeomystella oligophaga (Lepidoptera) and the host plant tissues of Macairea radula (Melastomataceae) leads to the formation of globoid galls. These galls have storage and typical nutritive tissues (outer and inner compartments, respectively), whose functions may be related to differential cell wall component distributions and to the stage of development of the gall inducer. Aims We evaluated whether the cell wall composition of the gall compartments (storage and nutritive tissues) changes according to the developmental stage of the gall inducer P. oligophaga. Methods The galls occupied by the gall inducer in different stages of development (i.e. larval and pupal stages, as well as empty galls) were collected and submitted to histological examination, immunocytochemical analyses using monoclonal antibodies for pectins, hemicelluloses, and glycoproteins, and histochemical analyses using phloroglucinol for lignins. Key results The histological results showed that nutritive tissues have smaller cells compared with storage ones when the galling insect is in the larval stage. Immunocytochemical analyses also showed the occurrence of both methyl-esterified and unesterified pectins, as well hemicelluloses in the nutritive tissue during the larval stage. The dynamic of some cell wall components changes when the gall inducer is in the pupal stage, or the galls are empty. Conclusions The changes in the composition of the cell walls, especially regarding homogalacturonans and xyloglucans, support the high metabolism and resource availability in cell walls of nutritive tissue for the gall inducer. Implications The cell wall composition may indicate important metabolic steps during gall formation and galling insect diet.

SUPPRESSION OF PROLIFERATION AND INCREASED OF PRO-APOPTOTIC PROTEINS EXPRESSION IN HUMAN BREAST CANCER CELLS AFTER THEIR CO-CULTIVATION WITH BIFIDOBACTERIUM ANIMALIS IN VITRO

Summary. Aim: to determine the viability, proliferative activity, and reactive oxygen species (ROS) production in human breast cancer (BC) cells after their co-cultivation with a human microbiota member Bifidobacterium animalis. Object and methods: the study was conducted in vitro on human BC cells of MCF7, T47D, and MDA-MB-231 lines. Cells were co-cultured with live B. animalis bacteria in direct or metabolite-mediated (contactless co-cultivation in the Insert system) interaction between eukaryotic and bacterial cells. ROS production was assessed by flow cytometry. The expression of proteins associated with the regulation of proliferation and apoptosis in BC cells was evaluated by immunocytochemical analysis. Results: The most pronounced changes in the growth characteristics of human BC cells in vitro are caused by the direct interaction of bacterial and malignant cells, compared to the bacterial metabolite-mediated action. MCF-7 cells of the luminal subtype are most sensitive to the effects of B. animalis. Co-cultivation of BC cells with B. animalis leads to a decrease of Ki-67 expression in malignant cells, an increase in ROS production, and the expression of pro-apoptotic proteins Bax and p21WAF1. Translocalization of p21WAF1 expression from the cytoplasm to the cell nucleus was detected in T47D cells after exposure to B. animalis. Conclusions: the interaction of human BC cells with B. animalis and their secreted metabolites led to inhibition of the malignant cells proliferation, induction of oxidative stress, and apoptosis program activation in BC cells.

Deletion of the Sodium Glucose Cotransporter 1 (Sglt-1) impairs mouse sperm movement.

The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.

Investigating comparative polymerase chain reaction for antigen receptor rearrangement analysis in different types of feline lymphoma samples.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

The CB2-PKC pathway is involved in esketamine-induced anti-inflammation in BV-2 microglial cells exposed to lipopolysaccharides.

Esketamine (ESK), an intravenous anesthetic, exerts antidepressant effects; however, the antidepression mechanism is not clear. The aim of this study was to explore whether microglial cannabinoid type 2 (CB2) receptor and protein kinase C (PKC) are involved in the antidepressant effects of ESK. In this investigation, lipopolysaccharide (LPS) was used to stimulate BV-2 microglia to mimic neuroinflammation. An enzyme-linked immunosorbent assay (ELISA) and Griess reagent kits were used to determine cytokine and nitrite concentrations in the medium. CB2, inducible nitric oxide synthase (iNOS) and nuclear factor (NF)-κB (p65) protein expression were evaluated by immunocytochemistry and western blot analysis. Compared with the control, LPS enhanced proinflammatory factor and nitrite concentration in the medium, upregulated iNOS and NF-κB (p65) expressions, and coadministration of ESK decreased proinflammatory cytokine and nitrite levels, and downregulated iNOS and NF-κB (p65) expression. Moreover, ESK exposure enhanced CB2 receptor expression; coadministration of the CB2 receptor antagonist AM630 or the PKC inhibitor chelerythrine (Che), however, markedly blocked the anti-inflammatory effect of ESK in reducing cytokine and nitrite concentration, and downregulating iNOS and NF-κB (p65) expression. These observations demonstrated that the microglial CB2-PKC pathway mediates ESK-induced anti-inflammation in LPS-stimulated microglial cells.

A novel effect of plasma rich in growth factors in the small leucine‐rich proteoglycans regulation in trabecular meshwork

Aims/Purpose: To analyse the effect of plasma rich in growth factors (PRGF) on the activity of small leucine‐rich proteoglycans (SRLPs) involved in extracellular matrix regulation in a trabecular meshwork fibrosis in vitro model.Methods: Trabecular meshwork cells were obtained from cadaveric donors in collaboration with the Blood and Tissue Centre of the Principality of Asturias (CEImPA2022.359). The cells were exposed to TGFβ2, which induces the expression of fibrosis markers. After 24 h in the presence of TGFβ2, the cells were treated with PRGF or human serum (control group) for 7 days. After the treatment period, samples were processed for immunocytochemistry or gene expression analysis by qPCR. Immunocytochemical techniques were used to examine the markers α‐sma, collagen I and collagen IV markers. The gene expression analysis included the genes lumican (LUM), decorin (DCN), biglycan (BGN) and fibromodulin (FMOD).Results: The induction of fibrosis in trabecular meshwork cells, characterized by an increase in extracellular matrix components such as α‐sma, collagen I and collagen IV, led to an increase in the expression of several SRLPs analysed, such as lumican, decorin, biglycan and fibromodulin. Treatment with PRGF for 7 days significantly reduced the expression of lumican, decorin and biglycan. In contrast, fibromodulin expression levels were slightly increased in the PRGF‐treated group.Conclusions: SRLPs play an important role in the regulation of the extracellular matrix in several ocular tissues. In glaucoma, altered expression of SRLPs such as lumican, decorin and biglycan leads to a glaucomatous phenotype in animal models. Changes in the expression of SRLPs have also been reported in the aqueous humour of glaucoma patients. PRGF treatment regulates the expression of SRLPs, as well as the expression and deposition of other fibrotic markers, and may promote aqueous humour flow and intraocular pressure control.

"MeiQuant": An Integrated Tool for Analyzing Meiotic Prophase I Spread Images.

Immunocytochemical analysis of meiotic proteins on mouse chromosome spreads is one method of choice to study prophase I chromosome organization and homologous recombination. In recent decades, the development of microscopic approaches led to the production of a large number of images that monitor fluorescent proteins, defined as fluorescent objects, and a major challenge facing the community is the deep analysis of these fluorescent objects (measurement of object length, intensity, distance between objects, as well as foci identification, counting, and colocalization). We propose a set of tools designed from the macro language of the widely used image analysis software ImageJ (Schindelin et al., Nat Methods 9: 676-682, 2012), embedded in the "MeiQuant" macro, which are specifically designed for analyzing objects in the field of meiosis. Our aim is to propose a unified evolutive common tool for image analysis, with a specific focus on mouse prophase I meiotic events.

Identification of intracellular activation mechanism of rhamnogalacturonan-I type polysaccharide purified from Panax ginseng leaves in macrophages and roles of component sugar chains on activity.

This study aimed to investigate the mechanisms underlying intracellular signaling pathways in macrophages in relation to the structural features of rhamnogalacturonan (RG) I-type polysaccharide (PGEP-I) purified from Panax ginseng leaves. For this investigation, we used several specific inhibitors and antibodies against mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and pattern recognition receptors (PRRs). Furthermore, we investigated the roles of component sugar chains on immunostimulating activity through a sequential enzymatic and chemical degradation steps. We found that PGEP-I effectively induced the phosphorylation of several MAPK- and NF-κB-related proteins, such as p38, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p65. Particularly, immunocytochemistry analysis confirmed the PGEP-I-induced translocation of p65 into the nucleus. Furthermore, the breakdown of PGEP-I side chains and main chain during sequential enzymatic and chemical degradation reduced the PGEP-I-induced macrophage cytokine secretion activity. IL-6, TNF-α, and NO secreted by macrophages are associated with several signaling pathway proteins such as ERK, JNK, and NF-κB and several PRRs such as dectin-1, CD11b, CD14, TLR2, TLR4, and SR. Thus, these findings suggest that PGEP-I exerts potent macrophage-activating effects, which can be attributed to its typical RG-I structure comprising arabinan, type II arabinogalactan, and rhamnose-galacturonic acid repeating units in the main chain.

Ablation of DGKα facilitates α-smooth muscle actin expression via the Smad and PKCδ signaling pathways during the acute phase of CCl4 -induced hepatic injury.

Expression of α-smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ-induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second-messenger DG, thereby controlling components of DG-mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα-knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively.

OsSHMT4 Is Required for Synthesis of Rice Storage Protein and Storage Organelle Formation in Endosperm Cells.

Storage proteins are essential for seed germination and seedling growth, as they provide an indispensable nitrogen source and energy. Our previous report highlighted the defective endosperm development in the serine hydroxymethyltransferase 4 (OsSHMT4) gene mutant, floury endosperm20-1 (flo20-1). However, the alterations in storage protein content and distribution within the flo20-1 endosperm remained unclear. Here, the immunocytochemistry analyses revealed a deficiency in storage protein accumulation in flo20-1. Electron microscopic observation uncovered abnormal morphological structures in protein bodies (PBI and PBII) in flo20-1. Immunofluorescence labeling demonstrated that aberrant prolamin composition could lead to the subsequent formation and deposition of atypical structures in protein body I (PBI), and decreased levels of glutelins and globulin resulted in protein body II (PBII) malformation. Further RNA-seq data combined with qRT-PCR results indicated that altered transcription levels of storage protein structural genes were responsible for the abnormal synthesis and accumulation of storage protein, which further led to non-concentric ring structural PBIs and amorphous PBIIs. Collectively, our findings further underscored that OsSHMT4 is required for the synthesis and accumulation of storage proteins and storage organelle formation in endosperm cells.

SARS-CoV-2 impact on ACE2 expression in NSCLC: mRNA and protein insights COVID-19 associated (ACE2) expression in non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) is a pervasive and challenging global health concern. This research delves into the intricate relationship between NSCLC and ACE2 expression, exploring the potential impact of COVID-19 history on this interaction. Tissue samples were meticulously gathered from a cohort of 32 NSCLC patients, 18 of whom had a documented history of COVID-19 infection. The methodology included extensive investigations, such as cell dissociation, histopathological analysis, immunohistochemistry, cell culture, adhesion assays, immunocytochemistry, RNA isolation, and RT-PCR analysis. The results of this comprehensive study unearthed intriguing findings regarding ACE2 expression patterns within NSCLC tissues. Notably, variations were observed in ACE2 profiles between individuals with and without a prior record of COVID-19 infection, hinting at a dynamic interplay. These discoveries carry profound implications for both the understanding of NSCLC progression and the response to COVID-19 in patients with pre-existing NSCLC. The interrelationship between ACE2 expression, NSCLC, and COVID-19, as revealed in this study, may significantly influence patient outcomes and, potentially, therapeutic strategies. In summary, this research serves as an essential contribution to the growing body of knowledge on NSCLC, offering unique insights into the intricate connections between ACE2, COVID-19, and NSCLC. This information may open new avenues for tailored treatment approaches and clinical management strategies, ultimately benefiting patients grappling with NSCLC in the background of the current COVID-19 pandemic.

Nutritive tissue rich in reserves in the cell wall and protoplast: the case of Manihot esculenta (Euphorbiaceae) galls induced by Iatrophobia brasiliensis (Diptera, Cecidomyiidae).

The galls can offer shelter, protection, and an adequate diet for the gall-inducing organisms. Herein, we evaluated the structure of Manihot esculenta leaves and galls induced by Iatrophobia brasiliensis in order to identify metabolic and cell wall composition changes. We expected to find a complex gall with high primary metabolism in a typical nutritive tissue. Non-galled leaves and galls were subjected to anatomical, histochemical, and immunocytochemical analyses to evaluate the structural features, primary and secondary metabolites, and glycoproteins, pectins, and hemicelluloses in the cell wall. The gall is cylindric, with a uniseriate epidermis, a larval chamber, and a parenchymatic cortex divided into outer and inner compartments. The outer compartment has large cells with intercellular spaces and stocks starch and is designated as storage tissue. Reducing sugars, proteins, phenolic compounds, and alkaloids were detected in the protoplast of inner tissue cells of galls, named nutritive tissue, which presents five layers of compact small cells. Cell walls with esterified homogalacturonans (HGs) occurred in some cells of the galls indicating the continuous biosynthesis of HGs. For both non-galled leaves and galls, galactans and xyloglucans were broadly labeled on the cell walls, indicating a cell growth capacity and cell wall stiffness, respectively. The cell wall of the nutritive tissue had wide labeling for glycoproteins, HGs, heteroxylans, and xyloglucans, which can be used as source for the diet of the galling insect. Manihot esculenta galls have compartments specialized in the protection and feeding of the galling insect, structured by nutritive tissue rich in resource compounds, in the cell walls and protoplast.

Single-Cell Transcriptomics and In Vitro Lineage Tracing Reveals Differential Susceptibility of Human iPSC-Derived Midbrain Dopaminergic Neurons in a Cellular Model of Parkinson's Disease.

Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson's disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson's disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP+), we showed a preferential reduction of BFP+ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP+ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling.

Prostate-specific antigen: An unfamiliar protein in the human salivary glands.

The presence of prostate-specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin-biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive-like secretory pathways.

Inhibitor of Wnt receptor 1 suppresses the effects of Wnt1, Wnt3a and β‑catenin on the proliferation and migration of C6 GSCs induced by low‑dose radiation.

The radioresistance of glioma is an important cause of treatment failure and tumor aggressiveness. In the present study, under performed with linear accelerator, the effects of 0.3 and 3.0Gy low‑dose radiation (LDR) on the proliferation and migration of C6 glioma stem cells invitro were examined by flow cytometric analysis, immunocytochemistry and western blot analysis. It was found that low‑dose ionizing radiation (0.3Gy) stimulated the proliferation and migration of these cells, while 3.0Gy ionizing radiation inhibited the proliferation of C6 glioma stem cells, which was mediated through enhanced Wnt/β‑catenin signaling, which is associated with glioma tumor aggressiveness. LDR treatment increased the expression of the DNA damage marker γ‑H2AX but promoted cell survival with a significant reduction in apoptotic and necrotic cells. When LDR cells were also treated with an inhibitor of Wnt receptor 1 (IWR1), cell proliferation and migration were significantly reduced. IWR1 treatment significantly inhibited Wnt1, Wnt3a and β‑catenin protein expression. Collectively, the current results demonstrated that IWR1 treatment effectively radio‑sensitizes glioma stem cells and helps to overcome the survival advantages promoted by LDR, which has significant implications for targeted treatment in radioresistant gliomas.

Inhibition of BMP signaling delays tauopathy and cognitive deficits in P301S mouse model

AbstractBackgroundAging is the highest risk factor for sporadic Alzheimer’s disease (AD). Expression of bone morphogenic protein 4 (BMP4) increases 10‐fold with age, while expression of the BMP antagonist, noggin, decreases. We have observed that activating BMP signaling in induced human neurons increased levels of phosphorylated tau. The increase of p‐tau has been correlated with the activated non‐canonical BMP pathway leading to p38 activity. We hypothesized that modulating BMP signaling in an AD mouse model of tau would rescue disease phenotype.MethodP301S (PS19) mice were injected with lentivirus overexpressing noggin (LV‐noggin) or lentivirus mcherry (LV‐mcherry) control in the dentate gyrus. Cognitive behavioral testing was run at 7 months of age and brain tissue was collected for protein and immunofluorescent imaging.ResultWe observe that levels of BMP4, as well as BMP signaling, are increased in the brains of PS19 mice compared to littermate wildtype mice. Analysis of noggin injected PS19 brains show downregulation of BMP signaling in the DG shown by decreased pSMAD1/5/8, as well as non‐canonical signaling through p38 kinase activity. Inhibition of BMP signaling through overexpression of noggin improves cognitive ability shown through Barnes maze testing. Downregulation of BMP signaling in P301S mice led to a decrease in tau phosphorylation seen both in immunoblotting, as well as immunocytochemical analysis in the DG.ConclusionPS19 model of tauopathy show increased levels of BMP signaling. We have shown that increased levels of BMP signaling lead to increased tau phosphorylation and reduced degradation of p‐tau. We show that by inhibiting BMP signaling in vivo we can delay cognitive behavioral deficits seen in these mice by decreasing tau phosphorylation. This suggests that the age‐related increase in BMP signaling may participate in the initiation and progression of AD pathology and help to explain why age is the largest risk factor for AD.