Perinuclear Area Research Articles

Expression of LOXL3, NES, and SNAI1 in Melanoma Genesis and Progression.

Melanoma is the most severe type of skin cancer and among the most malignant neoplasms in humans. With the growing incidence of melanoma, increased numbers of therapeutic options, and the potential to target specific proteins, understanding the basic mechanisms underlying the disease's progression and resistance to treatment has never been more important. LOXL3, SNAI1, and NES are key factors in melanoma genesis, regulating tumor growth, metastasis, and cellular differentiation. In our study, we explored the potential role of LOXL3, SNAI1, and NES in melanoma progression and metastasis among patients with dysplastic nevi, melanoma in situ, and BRAF+ and BRAF- metastatic melanoma, using immunofluorescence and qPCR analysis. Our results reveal a significant increase in LOXL3 expression and the highest NES expression in BRAF+ melanoma compared to BRAF-, dysplastic nevi, and melanoma in situ. As for SNAI1, the highest expression was observed in the metastatic melanoma group, without significant differences among groups. We found co-expression of LOXL3 and SNAI1 in the perinuclear area of all investigated subgroups and NES and SNAI1 co-expression in melanoma cells. These findings suggest a codependence or collaboration between these markers in melanoma EMT, suggesting new potential therapeutic interventions to block the EMT cascade that could significantly affect survival in many melanoma patients.

Pituitary Crooke cell neuroendocrine tumor of adrenocorticotropic hormone differentiation-specific transcription factor lineage: a clinicopathological analysis of six cases

Objective: To investigate the clinicopathological features of Crooke cell tumor of adrenocorticotropic hormone differentiation specific transcription factor (TPIT, also known as transcription factor 19, TBX19) lineage neuroendocrine tumors. Methods: Six cases of Crooke cell tumor diagnosed at the First Affiliated Hospital of University of Science and Technology of China, Hefei, China from October 2019 to October 2023 were collected. The clinical and pathological features of these cases were analyzed. Results: Among the six cases, one was male and five were female, with ages ranging from 26 to 75 years, and an average age of 44 years. All tumors occurred within the sella turcica. Clinical presentations included visual impairment in two cases, menstrual disorders in one case, Cushing's syndrome in one case, headache in one case, and one asymptomatic case discovered during a physical examination. Preoperative serum analyses revealed elevated levels of cortisol and adrenocorticotropic hormones in two cases, elevated cortisol in two cases, elevated adrenocorticotropic hormone in one case, and one case with a mild increase in prolactin due to the pituitary stalk effect. Magnetic resonance imaging revealed uneven enhancement of masses with maximum diameters ranging from 1.7 to 3.2 cm, all identified as macroadenomas. Microscopically, tumor cells exhibited irregular polygonal shapes, solid sheets, or pseudo-papillary arrangements around blood vessels. The cell nuclei were eccentric or centrally located, varying in size, with abundant cytoplasm. Some tumor cells showed perinuclear halo. Immunohistochemistry demonstrated diffuse strong positivity for TPIT in five cases, focal weak positivity for TPIT in one case, diffuse strong positivity for adrenocorticotropic hormone in all cases, and faint staining around the nuclei in a few cells. CK8/18 showed a strong positive ring pattern in more than 50% of tumor cells, focal weak positive expression of p53, and the Ki-67 positive index ranged 1%-5%. Periodic acid-Schiff staining revealed positive cytoplasm and negative perinuclear areas. Conclusions: Crooke cell tumor is a rare type of pituitary neuroendocrine tumors. Its pathological characteristics include a distinctive perinuclear clear zone and immunohistochemical markers, such as CK8/18 exhibiting a ring or halo pattern. This entity represents a high-risk subtype among pituitary neuroendocrine tumors, displaying a high risk of invasion and a propensity for recurrence. Accurate diagnosis is crucial for the postoperative follow-up and multimodal treatment planning.

Mechanisms of mitotic inhibition in human aorta endothelial cells: Molecular and morphological in vitro spectroscopic studies

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments.Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug’s action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells.The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells.

Asprosin as a potential new paracrine regulator in the heart

Abstract Funding Acknowledgements None. Background Asprosin was recently described as a novel hormone that is released by white adipose tissue upon fasting. However, a functional role of asprosin in human cardiovascular tissues has not been yet investigated at the tissue level. Asprosin is the C-terminal propeptide of the extracellular matrix (ECM) glycoprotein fibrillin-1 and is cleaved off from the pro-fibrillin-1 precursor by the endopeptidase furin. Fibrillin-1 is expressed in the heart and aorta, and FBN1 mutations are causative for aortic aneurysm formation and cardiomyopathy. Whether asprosin is also present in cardiac tissues and utilized by cardiac resident cells remains unknown. Purpose In the present study, we investigated the localization and distribution of asprosin protein in human atrial and ventricular heart tissue. Methods Human heart tissue was obtained from patients undergoing aortic valve replacement. In addition, we used ventricular tissue from mice. Cryosections from human (n=8) and mouse (n=8) heart samples were analysed by immunohistochemistry. The anti-asprosin antibodies were raised against human or mouse asprosin respectively and applied in combination with anti-CD31/PECAM, an endothelial cell marker, or anti-sarcomeric alpha-actinin, a Z-line marker. The immunofluorescence of incubated specimens was examined by confocal microscopy. Results Our data suggest that asprosin is localized in myocardium in different cell types: cardiomyocytes (Figure 1 a-d), arterial smooth muscle cells (Figure 1 a), micro- and vascular endothelial cells (Figure 1 d), and with the lowest intensity, in periarterial located epicardial adipocytes (Figure 1 e). In cardiomyocytes, incubation with anti-asprosin resulted in a high signal intensity. Asprosin immunoreactivity was observed: in intercalary discs (Figure 1 b-d), as striated pattern in the sarcoplasm, and as weak signal in the perinuclear area (Figure 1 b, c). In human tissue, the anti-asprosin signal was about four times higher in intercalated discs than in the sarcoplasm. The distribution pattern in ventricular cardiomyocytes of mice were similar. Conclusion Our results show that in human myocardium, asprosin localises mainly intracellular. Asprosin accumulations were found in intercalary discs. In short, asprosin was found at locations that are known to be crucial for regulation of cardiac function. Intracellular presence of asprosin may indicate cellular utilization by uptake from extracellular asprosin pools. Whether these pools are replenished exclusively by resident cells or by other tissues such as WAT is currently unknown. However, further research is needed to prove this hypothesis. Figure. 1. Confocal microscopy of asprosin localization in human myocardium. Cryosections incubated with anti-asprosin (red) or combined with anti-alpha actinin (sarcomeric) or anti-CD31/PECAM (green) antibodies and DAPI (blue, nuclei). SM: vascular smooth muscle cells, BV: blood vessels, Arrowheads: Intercalated disks.

Esophageal squamous cell carcinoma with pagetoid spread: a clinicopathologic study.

Pagetoid spread in esophageal squamous epithelium associated with underlying esophageal adenocarcinoma (EAC) has been well studied. Case reports describing pagetoid spread of esophageal squamous cell carcinomas (ESCC) also exist in the literature. The latter, however, has not been systematically studied. In this study, we report seven cases of pagetoid spread associated with ESCC. The clinical, morphologic, and immunophenotypic profiles of pagetoid spread in the context of ESCC and EAC are compared. Cases of pagetoid spread of ESCC were identified through computerized search of pathology archives at five institutions. Additional cases were identified through manual review of surgical resection cases of treatment naive ESCC in Mass General Brigham (MGB) pathology archive. Clinical history was collected via chart review. Immunohistochemistry for CK7, CK20, CDX2, p53, p63, and p40 was performed on selected cases. A computerized search of pathology archives of five institutions revealed only two cases. A manual review of 76 resected untreated ESCC revealed five additional cases with unequivocal pagetoid spread of ESCC, indicating the condition was not uncommon but rarely reported. Patient age ranged from 54 to 78years (median, 65). There were six women and one man. One case had in situ disease, five had pT1 (1 pT1a and 4 pT1b), and one had pT3 disease. One of the patients with pT1 tumor had a positive lymph node, while the remaining six patients were all N0. Four tumors were in the proximal to mid esophagus, and three in the distal esophagus. Patient survival ranged from 25months to more than 288months. The pagetoid tumor cells demonstrated enlarged, hyperchromatic nuclei with variable amounts of eosinophilic cytoplasm. The cytoplasm was often condensed to the perinuclear area, creating peripheral clearing. By immunohistochemistry, the pagetoid cells were positive for p40 (6/6) and p63 (7/7) and negative for CDX2 (7/7). The tumor cells showed mutant-type staining for p53 in five of seven cases. One of the patients had pagetoid tumor cells at the resection margin and subsequently had recurrent disease 2years later. All other patients had negative resection margins and did not have local recurrence. Four cases of pagetoid spread in the context of EAC were used as a comparison group. Previously published studies were also analyzed. These tumors were all located in the distal esophagus or gastroesophageal junction. All cases were associated with underlying invasive EAC. Pagetoid spread associated with EAC often had cytoplasmic vacuoles or mucin. They were more frequently positive for CK7 than pagetoid ESCC (p = 0.01). Both ESCC and EAC may give rise to pagetoid spread of tumor cells within surface squamous epithelium. Pagetoid spread from ESCC and EAC have overlapping morphologic features. P40 and p63 immunostains can facilitate the distinction between ESCC and EAC. P53 immunostain can aid in confirmation of malignancy. Understanding their overlapping pathologic features will help pathologists avoid pitfalls and diagnose these lesions correctly on biopsy specimens.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Opposing actions of JIP4 and RILPL1 provide antagonistic motor force to dynamically regulate membrane reformation during lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Stromal Cell-Derived Factor 1α Inhibits Chondrocyte Apoptosis and Promotes Autophagy Through the Akt Signaling Pathway

To investigate the effects of stromal cell-derived factor 1α (SDF-1α) on the apoptosis and autophagy of chondrocytes and the underlying mechanisms. Chondrocytes were isolated from the knee joints of neonatal mice. The chondrocytes were then stimulated with 0 (the control group), 50, 100, and 200 ng/mL of SDF-1α. CCK-8 assay was performed to determine the effects of SDF-1α stimulation for 24 h, 48 h, and 72 h on the viability of the chondrocytes. Wound healing assay was conducted to determine the effects of SDF-1α stimulation for 12 h and 24 h on chondrocyte migration. The changes in the expression of Akt signaling pathway proteins in chondrocytes were determined by Western blot assay. Chondrocytes were stimulated with 0 (the control group) and 200 ng/mL of SDF-1α. Flow cytometry was performed to determine the effect of SDF-1α on the apoptosis of chondrocytes. Transmission electron microscope was used to examine the effect of SDF-1α on chondrocyte autophagy. Immunofluorescence staining assays were performed to visualize the differences in p-Akt expression and distribution in chondrocytes treated with SDF-1α. Compared with the control group, findings for the experimental groups showed that SDF-1α at the concentrations of 50, 100, and 200 ng/mL did not decrease chondrocyte activity at any time point (P<0.01) and it consistently promoted chondrocyte migration at 24 h (P<0.05). Western blot results revealed that, in comparison to the control group, SDF-1α at concentrations of 50, 100, and 200 ng/mL significantly up-regulated the protein expression of p-Akt in chondrocytes, while no significant difference in Akt expression was observed. Flow cytometry demonstrated that SDF-1α could inhibit chondrocyte apoptosis (P<0.05) and transmission electron microscopic observation showed that SDF-1α promoted chondrocyte autophagy (P<0.05). Immunofluorescence staining showed that the expression of p-Akt in chondrocytes was concentrated in the perinuclear area of the cells and this expression was further enhanced in the perinuclear area of the chondrocytes after treatment with SDF-1α. SDF-1α inhibits chondrocyte apoptosis and promotes chondrocyte migration and autophagy through activating the Akt signaling pathway.

Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.

Addition of granulocyte macrophage colony stimulating factor (GM-CSF) during in vitro oocyte maturation improves embryo development in a mouse model of advanced maternal age.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12-14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62; P < 0.01) and total cell number (42.98 vs. 48.78; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

Evaluation of the Moonlighting Histone H3 Specific Protease (H3ase) Activity and the Dehydrogenase Activity of Glutamate Dehydrogenase (GDH).

The N-terminus of Histone H3 is proteolytically processed in aged chicken liver. A histone H3 N-terminus specific endopeptidase (named H3ase) has been purified from the nuclear extract of aged chicken liver. By sequencing and a series of biochemical methods including the demonstration of H3ase activity in bacterially expressed GDH, it was established that the H3ase activity was a moonlighting protease activity of glutamate dehydrogenase (GDH). However, the active site for the H3ase in the GDH remains elusive. Here, using cross-linking studies of the homogenously purified H3ase, we show that the GDH and the H3ase remain in the same native state. Further, the H3ase and GDH activities could be uncoupled by partial denaturation of GDH, suggesting strong evidence for the involvement of different active sites for GDH and H3ase activities. Through densitometry of the H3ase clipped H3 products, the H3ase activity was quantified and it was compared with the GDH activity of the chicken liver nuclear GDH. Furthermore, the H3ase mostly remained distributed in the perinuclear area as demonstrated by MNase digestion and immuno-localization of H3ase in chicken liver nuclei, as well as cultured mouse hepatocyte cells, suggesting that H3ase demonstrated regulated access to the chromatin. The present study thus broadly compares the H3ase and GDH activities of the chicken liver GDH.

Sars-CoV2 ORF7a Protein Stimulates Synthesis and Secretion of Endothelial Von Willebrand Factor

Introduction: Patients with SARS-CoV-2 show significantly elevated plasma levels of von Willebrand factor (VWF) and increased thrombosis. However, the molecular mechanism underlying increased plasma levels of VWF in COVID-19 patients is not fully understood. Methods. Drosophila genetic screen, recombinant expression, endothelial culture, confocal microscopy, flow cytometry, and a microfluidic assay were employed for the study. Results. Drosophila genetic screen for SARS-CoV-2 proteins allowed us to identify Orf7a to be the key factor that induces Hml (drosophila homolog of human VWF)-dependent hypercoagulability. Recombinant Orf7a tagged with mCherry (mCh) and FLAG was expressed transiently and stably in a murine endothelial cell line SVEC4-10 and a HEK293 cell line. The ORF7a protein is localized predominantly to the Golgi and trans-Golgi network (TGN) in HEK293 cells ( Fig. 1A) but shows its expression in perinuclear area and cell surface in SVEC4-10 cells ( Fig. 1B). Significantly higher numbers of VWF-strings were formed in the ORF7a transfected SVEC4-10 cell line under static state than in the non-transfected control cells. These cell surface or extracellular VWF strings were readily removed by a human recombinant ADAMTS13 ( Fig. 1C &amp; 1D). Flow cytometry analysis revealed that significant higher levels of intracellular VWF signal in the cells following transient expression of Orf7a than in those non-expressed controls ( Fig. 2A &amp; 2B). Microfluidic shear-based assay and confocal imaging analysis further demonstrated that dramatically increased rate and surface coverage of fluorescent labeled murine platelets over a histone-stimulated SVEC surface expressing Orf7 comparing with over the same cells without expression of Orf7, following perfusion of Adamts13 -/- murine whole blood (anticoagulated with PPACK) under arterial shear ( Fig. 2C &amp; 2D). Using proteomics, we identified conserved host proteins ARF4, GBF1 and ERGIC-53 (a COPII pathway component) as direct interacting factors for Orf7a and demonstrated that these proteins have conserved roles in VWF secretion. Conclusions:Our results demonstrate that ORF7a may upregulate VWF synthesis and secretion in endothelial cells, which enhances thrombus formation. Recombinant ADAMTS13 may rapidly remove endothelial surface ultra large VWF strings and exhibit therapeutic potential for COVID-19 associated thrombosis.

Intracellular remodeling associated with endoplasmic reticulum stress modifies biomechanical compliance of bladder cells

Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades.AnQ6eoaFZ-G6Rs7Kzp4dpwVideo

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Role of osteopontin in oral epithelial dysplasia, oral submucous fibrosis and oral squamous cell carcinoma.

Inflammatory cells and cytokines in the chronically injured mucosa promote fibrosis in the oral submucous fibrosis (OSF) fibrotic milieu. Osteopontin (OPN) is a wound-healing mediator that upregulates the inflammatory response and is involved in the malignancy and fibrosis of multiple organ systems. We investigated the expression of OPN in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinomas (OSCCs) to determine its role in the malignant transformation and fibrosis of oral tissues. The expression of OPN in OPMDs and OSCCs was compared and correlated, and the role of OPN as a fibrotic mediator in OSF was explained. A total of 30 cases of normal mucosa and OPMDs (mild dysplasia, severe dysplasia, OSF and OSCCs) were studied by purposive sampling. In these groups, OPN immunoreactivity was examined and correlated with clinical findings. In mild dysplasia, OPN expression was restricted to the basal cell layer with moderate staining intensity. In severe dysplasia, it was extremely intense and extended throughout the epithelium. In the OSF, OPN expression was moderate in the perinuclear areas of the basal cell layer. The expression of OPN was very strong in OSCC. A flow diagram explaining the profibrotic role of OPN in OSF has been provided. A positive role of OPN in both pathogenesis and malignant transformation of OPMDs and OSCC has been demonstrated.

The Poly-Arginine Peptide R18D Interferes with the Internalisation of α-Synuclein Pre-Formed Fibrils in STC-1 Enteroendocrine Cells.

In Parkinson's disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 μM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology.

Characters of KRT80 and its roles in neoplasms diseases.

KRT80 is a human epithelial intermediate filament type II gene; its expression product is a component of intracellular intermediate filaments (IFs) and is involved in the assembly of the cytoskeleton. There is evidence that IFs form a dense network mainly in the perinuclear area, but they can also reach the cortex. They are essential for mechanical cushioning of cells, organelle positioning, cell apoptosis, migration, adhesion, and interactions with other cytoskeletal components. Humans possess 54 functional keratin genes, and KRT80 is one of the more unique genes. It is widely expressed in almost all epithelial cells, although it is structurally more similar to type II hair keratins than to type II epithelial keratins. In this review, we summarize the basic facts about the keratin family and KRT80, the essential role of KRT80 in neoplasms, and its potential as a therapeutic target. We hope that this review will inspire researchers to at least partially focus on this area. In many neoplastic diseases, the high expression status of KRT80 and its role in regulating the biological functions of cancer cells have been well established. KRT80 can effectively enhance the proliferation, invasiveness and migration of cancer cells. However, the effects of KRT80 on prognosis and clinically relevant indices in patients with various cancers have not been extensively studied, and even opposite conclusions have been reached in different studies of the same cancer. Based on this, we should add more clinically relevant studies to clarify the prospect of clinical application of KRT80. Many researchers have made great progress in studying the mechanism of action of KRT80. However, their studies should be extended to more cancers to find common regulators and signaling pathways of KRT80 in different cancers. KRT80 may have far-reaching effects on the human body, and this marker may play a crucial role in the function of cancer cells and the prognosis of cancer patients, so it has a promising future in the field of neoplasms. In neoplastic diseases, KRT80 is overexpressed in many cancers and plays an essential role in promoting proliferation, migration, invasiveness and poor prognosis. The mechanisms of KRT80 functions in cancer have been partially elucidated, suggesting that KRT80 is a potentially useful cancer therapeutic target. However, more systematic, in-depth and comprehensive studies are still needed in this field.

Cellular Localization of Selected Porphyrins and Their Effect on the In Vitro Motility of Human Colon Tumors and Normal Cells.

Standard therapies for colorectal cancer cannot eliminate or sufficiently reduce the metastasis process. Photodynamic therapy (PDT) may be an alternative to minimizing this problem. Here, we examined the cellular localization of selected porphyrins and determined whether free-base and manganese (III) metallated porphyrins may limit colon cancer cells' (HT29) or normal colon epithelial cells' (CCD 841 CoTr) motility in vitro. White light irradiation was used to initiate the photodynamic effect. Porphyrin uptake by the cells was determined by porphyrin fluorescence measurements through the use of confocal microscopy. Free-base porphyrin was found in cells, where it initially localized at the edge of the cytoplasm and later in the perinuclear area. The concentrations of porphyrins had no effect on cancer cell migration but had a significant effect on normal cell motility. Due to the low concentrations of porphyrins used, no changes in F-actin filaments of the cellular cytoskeleton were detected. Signal transmission via connexons between neighbouring cells was limited to a maximum of 40 µm for HT29 and 30 µm for CCD 841 CoTr cells. The tested porphyrins differed in their activity against the tumor and normal cells' migration capacity. Depending on the porphyrin used and the type of cells, their migration changed in relation to the control sample. The use of white light may change the activity of the porphyrins relative to the migratory capacity of the cells. The aim of the present study was to analyse the intracellular localization of tested porphyrins and their influence on the mobility of cells after irradiation with harmless white light.