Beta-adrenergic stimulation promotes an endoplasmic reticulum stress-dependent inflammatory program in salivary gland epithelial cells.

The effect of beta-adrenergic stimulation on human labial minor salivary gland epithelial cells (LMSGEC) on IL-6 production, and its dependency to endoplasmic reticulum (ER) stress were investigated. Primary LMSGEC from Sjögren's syndrome (SS) patients and controls in culture were stimulated with epinephrine and IL-6 expression was evaluated by qPCR and ELISA. The expression of ß-ARs in cultured LMSGEC was tested by qPCR, while adrenoceptors and cAMP levels were examined in LMSGs by immunofluorescence. ER evaluation was performed by Transmission electron microscopy (TEM) and ER stress by Western blot. Adrenergic induced IL-6 production by cultured LMSGEC was evaluated after alleviation of the ER stress by applying Tauroursodeoxycholic acid (TUDCA) and silencing of PKR-like ER kinase (PERK) and activating transcription factor 4 (ATF4) RNAs. Expression of IL-6 by LMSGEC was upregulated after ß-adrenergic stimulation, while the silencing of adrenoreceptors downregulated IL-6. The amelioration of ER stress, as well as the silencing of PERK/ATF4, prevented epinephrine-induced upregulation of IL-6. Adrenergic stimulation led to higher and sustained IL-6 levels secreted by LMSGEC of SS patients compared to controls. Adrenergic signaling was endogenously enhanced in LMSGEC of SS patients (expression of ß-ARs in situ, intracellular cAMP in cultured LMSGEC). In parallel, SS-LMSGEC expressed dilated ER (TEM) and higher levels of GRP78/BiP. PERK/ATF4 pathway of the ER stress emerged a considerable mediator of adrenergic stimulation for IL-6 production by the LMSGEC. An enhanced endogenous adrenergic activation and a stressed ER observed in SS-LMSGEC may contribute to a sustained IL-6 production by these cells after adrenergic stimulation.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory system can progress to a multisystemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named senescence-associated secretory phenotype (SASP). We investigated whether coronavirus disease 2019 (COVID-19) is associated with cellular senescence and SASP. METHODS: Autopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID-19 controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients. RESULTS: SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed angiotensin-converting enzyme 2 and exhibited increased senescence (p16INK4A and SenTraGor positivity) and interleukin (IL)-1ß and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGor), DNA damage (γ-H2AX) and increased cytokine (IL-1ß, IL-6, CXCL8) and apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extrapulmonary sites (kidney and liver) of a COVID-19 patient. CONCLUSIONS: We demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.

Key biological mechanisms involved in high-LET radiation therapies with a focus on DNA damage and repair.

DNA damage and repair studies are at the core of the radiation biology field and represent also the fundamental principles informing radiation therapy (RT). DNA damage levels are a function of radiation dose, whereas the type of damage and biological effects such as DNA damage complexity, depend on radiation quality that is linear energy transfer (LET). Both levels and types of DNA damage determine cell fate, which can include necrosis, apoptosis, senescence or autophagy. Herein, we present an overview of current RT modalities in the light of DNA damage and repair with emphasis on medium to high-LET radiation. Proton radiation is discussed along with its new adaptation of FLASH RT. RT based on α-particles includes brachytherapy and nuclear-RT, that is proton-boron capture therapy (PBCT) and boron-neutron capture therapy (BNCT). We also discuss carbon ion therapy along with combinatorial immune-based therapies and high-LET RT. For each RT modality, we summarise relevant DNA damage studies. Finally, we provide an update of the role of DNA repair in high-LET RT and we explore the biological responses triggered by differential LET and dose.

The O-Linked N-Acetylglucosamine Containing Epitope H (O-GlcNAcH) is Upregulated in the Trophoblastic and Downregulated in the Fibroblastic Cells in Missed Miscarriage Human Chorionic Villi With Simple Hydropic Degeneration.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.

Silymarin Enriched Extract (Silybum marianum) Additive Effect on Doxorubicin-Mediated Cytotoxicity in PC-3 Prostate Cancer Cells.

Silymarin-enriched extract (SEE) is obtained from Silybum marianum (Asteraceae). Doxorubicin (DXR) is a widely used chemotherapeutical yet with severe side effects. The goal of the present study was to assess the pharmacologic effect of SEE and its bioactive components silibinin and silychristine when administrated alone or in combination with DXR in the human prostate cancer cells (PC-3). PC-3 cells were treated with SEE, silibinin (silybins A and B), silychristine, alone, and in combination with DXR, and cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis, and autophagy rate were assessed by flow cytometry. Expression levels of autophagy-related genes were quantified by qRT-PCR, ELISA and western blot while transmission electron microscopy was performed to reveal autophagic structures. Finally, NMR spectrometry was used to identify specific metabolites related to autophagy. SEE inhibited PC-3 cell proliferation in a dose-dependent manner while the co-treatment (DXR-SEE) revealed an additive cytotoxic effect. Cell cycle, apoptosis, and autophagy variations were observed in addition to altered expression levels of autophagy related genes (LC3, p62, NBR1, Beclin1, ULK1, AMBRA1), while several modifications in autophagic structures were identified after DXR-SEE co-treatment. Furthermore, treated cells showed a different metabolic profile, with significant alterations in autophagy-related metabolites such as branched-chain amino acids. In conclusion, the DXR-SEE co-treatment provokes perturbations in the autophagic mechanism of prostate cancer cells (PC-3) compared to DXR treatment alone, causing an excessive cell death. These findings propose the putative use of SEE as an adjuvant cytotoxic agent.

Monitoring Autophagy Immunohistochemically and Ultrastructurally during Human Head and Neck Carcinogenesis. Relationship with the DNA Damage Response Pathway.

Autophagy is a catabolic process that preserves cellular homeostasis. Its exact role during carcinogenesis is not completely defined. Specifically in head and neck cancer, such information from clinical settings that comprise the whole spectrum of human carcinogenesis is very limited. Towards this direction, we examined the in situ status of the autophagy-related factors, Beclin-1, microtubule-associated protein 1 light chain 3, member B (LC3B) and sequestosome 1/p62 (p62) in clinical material covering all histopathological stages of human head and neck carcinogenesis. This material is unique as each panel of lesions is derived from the same patient and moreover we have previously assessed it for the DNA damage response (DDR) activation status. Since Beclin-1, LC3B and p62 reflect the nucleation, elongation and degradation stages of autophagy, respectively, their combined immunohistochemical (IHC) expression profiles could grossly mirror the autophagic flux. This experimental approach was further corroborated by ultrastructural analysis, applying transmission electron microscopy (TEM). The observed Beclin-1/LC3B/p62 IHC patterns, obtained from serial sections analysis, along with TEM findings are suggestive of a declined authophagic activity in preneoplastic lesions that was restored in full blown cancers. Correlating these findings with DDR status in the same pathological stages are indicative of: (i) an antitumor function of autophagy in support to that of DDR, possibly through energy deprivation in preneoplastic stages, thus preventing incipient cancer cells from evolving; and (ii) a tumor-supporting role in the cancerous stage.

Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa.

BACKGROUND/AIM: Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress. MATERIALS AND METHODS: In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps. RESULTS: In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH. CONCLUSION: O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.

Chromogranin A and vesicular monoamine transporter 2 immunolocalization in protein bodies of human locus coeruleus neurons.

Our previous histochemical and ultrastructural studies have identified, in human catecholamine neurons, abundant spherical acidophilic protein bodies (pb), which originate from regular mitochondria, retaining their double membrane. In locus coeruleus (LC) neurons, pb have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline, as demonstrated by immunolocalization of Dopamine-ß-Hydroxylase. In the present study, in order to reinforce the identity of pb as monoamine storage sites in human LC, and to assess their potential of somatodendritic release, we studied the subcellular immunolocalization of chromogranin A (CgA) and vesicular monoamine transporter 2 (VMAT2), given the fact that their localization defines the vesicles capacity of filling with monoamine and hence exocytotic release. The data provided in the present study, demonstrate the novel ultrastructural immunolocalization of both CgA and VMAT2 in protein bodies, supporting their involvement in somatodendritic storage and release of noradrenaline in human LC. Since the molecular mechanism of LC somatodendritic exocytosis remains largely elusive, the present study may shed light to a better understanding of this mechanism.

A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer.

BACKGROUND: Prostate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed. METHODS: Androgen-dependent (LNCaP) and -independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy. RESULTS: We demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression. CONCLUSIONS: We identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.

Nanoparticle-Mediated Radiotherapy: Unraveling Dose Enhancement and Apoptotic Responses in Cancer and Normal Cell Lines.

Cervical cancer remains a pressing global health concern, necessitating advanced therapeutic strategies. Radiotherapy, a fundamental treatment modality, has faced challenges such as targeted dose deposition and radiation exposure to healthy tissues, limiting optimal outcomes. To address these hurdles, nanomaterials, specifically gold nanoparticles (AuNPs), have emerged as a promising avenue. This study delves into the realm of cervical cancer radiotherapy through the meticulous exploration of AuNPs' impact. Utilizing ex vivo experiments involving cell lines, this research dissected intricate radiobiological interactions. Detailed scrutiny of cell survival curves, dose enhancement factors (DEFs), and apoptosis in both cancer and normal cervical cells revealed profound insights. The outcomes showcased the substantial enhancement of radiation responses in cancer cells following AuNP treatment, resulting in heightened cell death and apoptotic levels. Significantly, the most pronounced effects were observed 24 h post-irradiation, emphasizing the pivotal role of timing in AuNPs' efficacy. Importantly, AuNPs exhibited targeted precision, selectively impacting cancer cells while preserving normal cells. This study illuminates the potential of AuNPs as potent radiosensitizers in cervical cancer therapy, offering a tailored and efficient approach. Through meticulous ex vivo experimentation, this research expands our comprehension of the complex dynamics between AuNPs and cells, laying the foundation for their optimized clinical utilization.

RGD binding to integrin alphavbeta3 affects cell motility and adhesion in primary human breast cancer cultures.

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.

Biological Response of Human Cancer Cells to Ionizing Radiation in Combination with Gold Nanoparticles.

In the context of improving radiation therapy, high-atomic number (Z) metallic nanoparticles and, more importantly, gold-based nanostructures are developed as radiation enhancers/radiosensitizers. Due to the diversity of cell lines, nanoparticles, as well as radiation types or doses, the resulting biological effects may differ and remain obscure. In this multiparameter study, we aim to shed light on these effects and investigate them further by employing X-irradiation and three human cancer cell lines (PC3, A549, and U2OS cells) treated by multiple techniques. TEM experiments on PC3 cells showed that citrate-capped AuNPs were found to be located mostly in membranous structures/vesicles or autophagosomes, but also, in the case of PEG-capped AuNPs, inside the nucleus as well. The colony-forming capability of cancer cells radiosensitized by AuNPs decreased significantly and the DNA damage detected by cytogenetics, γH2AX immunostaining, and by single (γH2AX) or double (γH2AX and OGG1) immunolocalization via transmission electron microscopy (TEM) was in many cases higher and/or persistent after combination with AuNPs than upon individual exposure to ionizing radiation (IR). Moreover, different cell cycle distribution was evident in PC3 but not A549 cells after treatment with AuNPs and/or irradiation. Finally, cellular senescence was investigated by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13) which showed that based on the AuNPs' concentration, an increased number of senescent cells might be observed after exposure to IR. Even though different cell lines or different types and concentrations of AuNPs may alter the levels of radiosensitization, our results imply that the complexity of damage might also be an important factor of AuNP-induced radiosensitization.

MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

The bright and dark side of skin senescence. Could skin rejuvenation anti-senescence interventions become a "bright" new strategy for the prevention of age-related skin pathologies?

The number of senescent cells in the skin is increasing with age. Numerous studies have attempted to elucidate the role of these cells in normal aging of the skin as well as in age-related skin conditions. In recent years, attempts have also been made to find treatments that aim either to cleanse the skin tissues of senescent cells or to neutralize their effects (referred to as senolytics and senomorphics respectively) and thus prevent the consequences, particularly on the skin's appearance in advanced age. Through this review, we have tried to gather data on the role of senescent cells in the skin, in treatments aimed at removing them, and we are asking a reasonable question as to whether anti-senescence treatments may contribute to the protection against age-related skin pathologies, including skin cancer, such as non-melanoma skin cancer, in addition to their involvement in skin rejuvenation.

A Guide for Using Transmission Electron Microscopy for Studying the Radiosensitizing Effects of Gold Nanoparticles In Vitro.

The combined effects of ionizing radiation (IR) with high-z metallic nanoparticles (NPs) such as gold has developed a growing interest over the recent years. It is currently accepted that radiosensitization is not only attributed to physical effects but also to underlying chemical and biological mechanisms' contributions. Low- and high-linear energy transfer (LET) IRs produce DNA damage of different structural types. The combination of IR with gold nanoparticles may increase the clustering of energy deposition events in the vicinity of the NPs due to the production mainly of photoelectrons and Auger electrons. Biological lesions of such origin for example on DNA are more difficult to be repaired compared to isolated lesions and can augment IR's detrimental effects as shown by numerous studies. Transmission electron microscopy (TEM) offers a unique opportunity to study the complexity of these effects on a very detailed cellular level, in terms of structure, including nanoparticle uptake and damage. Cellular uptake and nanoparticle distribution inside the cell are crucial in order to contribute to an optimal dose enhancement effect. TEM is mostly used to observe the cellular localization of nanoparticles. However, it can also provide valuable insights on the NPs' radiosensitization pathways, by studying the biochemical mechanisms through immunogold-labelling of antigenic sites at ultrastructural level under high resolution and magnification. Here, our goal is to describe the possibilities, methodologies and proper use of TEM in the interest of studying NPs-based radiosensitization mechanisms.

Chromatin alterations in leukocytes of first-episode schizophrenic patients.

Studies of peripheral blood leukocytes of schizophrenic patients have shown in electron microscopy (EM) that decondensation of the chromatin constitutes a biological marker indicating increased genomic expression. Since this increase depends on chromatin relaxation by dissociation of lysine-rich histone H1 from nucleosomes, with exposure of arginine residues of core histones, the ratio of arginine to lysine residues in each nucleus represents a reliable measure of activation. Lysine- and arginine-rich proteins are demonstrable in light microscopy (LM), differentially, as yellow and black, respectively, with the ammoniacal silver reaction (ASR). Application of ASR on leukocyte pellets before they are dehydrated and embedded in epoxy resins gives reliable results in semithin sections. In thin sections the ASR method localizes only the amino acid arginine by forming deposits of electron-opaque particles, visualized in the EM. Leukocytes of 12 first-episode schizophrenic patients and 5 controls were used. Light micrographs of the semithin sections were inserted in a personal computer. The percentage of lysine and arginine was measured in 300 nuclei per subject. Morphometry showed that lymphocytes of schizophrenic patients have increased ratios of arginine to lysine, compared to controls, indicating activation; neutrophils of the patients have even a higher ratio, indicating an abnormal condition of the genome. Chromatin conformational changes are also evident by phosphotungstic acid hematoxylin (PTAH) block staining, which reveals condensed chromatin as an electron-lucent area in the nuclei, and decondensed chromatin as an electron-dense area. Because decondensed chromatin is a biological marker of schizophrenia, the efficacy of these methods to demonstrate this particular state offers a tool for early diagnosis, since first-episode schizophrenic patients have a better prognosis when treatment is started promptly, at the beginning of the disease.

mTORC1-dependent protein synthesis and autophagy uncouple in the regulation of Apolipoprotein A-I expression.

BACKGROUND: Apolipoprotein A-I (ApoA-I) is involved in reverse cholesterol transport as a major component of HDL, but also conveys anti-thrombotic, anti-oxidative, anti-inflammatory and immune-regulatory properties that are pertinent to its protective roles in cardiovascular, inflammatory and malignant pathologies. Despite the pleiotropy in ApoA-I functions, the regulation of intracellular ApoA-I levels remains poorly explored. METHODS: HepG2 hepatoma cells and primary mouse hepatocytes were used as in vitro models to study the impact of genetic and chemical inhibitors of autophagy and the proteasome on ApoA-I by immunoblot, immunofluorescence and electron microscopy. Different growth conditions were implemented in conjunction with mTORC inhibitors to model the influence of nutrient scarcity versus sufficiency on ApoA-I regulation. Hepatic ApoA-I expression was also evaluated in high fat diet-fed mice displaying blockade in autophagy. RESULTS: Under nutrient-rich conditions, basal ApoA-I levels in liver cells are sustained by the balancing act of autophagy and of mTORC1-dependent de novo protein synthesis. ApoA-I proteolysis occurs through a canonical autophagic pathway involving Beclin1 and ULK1 and the receptor protein p62/SQSTM1 that targets ApoA-I to autophagosomes. However, upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis. CONCLUSION: These data underscore the major contribution of post-transcriptional mechanisms to ApoA-I levels which differentially involve mTORC1-dependent signaling to protein synthesis and autophagy, depending on nutrient availability. Given the established role of ApoA-I in HDL-mediated reverse cholesterol transport, this mode of ApoA-I regulation may reflect a hepatocellular response to the organismal requirement for maintenance of cholesterol and lipid reserves under conditions of nutrient scarcity.

Unique Spatial Immune Profiling in Pancreatic Ductal Adenocarcinoma with Enrichment of Exhausted and Senescent T Cells and Diffused CD47-SIRPα Expression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is resistant to single-agent immunotherapies. To understand the mechanisms leading to the poor response to this treatment, a better understanding of the PDAC immune landscape is required. The present work aims to study the immune profile in PDAC in relationship to spatial heterogeneity of the tissue microenvironment (TME) in intact tissues. METHODS: Serial section and multiplex in situ analysis were performed in 42 PDAC samples to assess gene and protein expression at single-cell resolution in the: (a) tumor center (TC), (b) invasive front (IF), (c) normal parenchyma adjacent to the tumor, and (d) tumor positive and negative draining lymph nodes (LNs). RESULTS: We observed: (a) enrichment of T cell subpopulations with exhausted and senescent phenotype in the TC, IF and tumor positive LNs; (b) a dominant type 2 immune response in the TME, which is more pronounced in the TC; (c) an emerging role of CD47-SIRPα axis; and (d) a similar immune cell topography independently of the neoadjuvant chemotherapy. CONCLUSION: This study reveals the existence of dysfunctional T lymphocytes with specific spatial distribution, thus opening a new dimension both conceptually and mechanistically in tumor-stroma interaction in PDAC with potential impact on the efficacy of immune-regulatory therapeutic modalities.