Nucleophagy delays aging and preserves germline immortality.

Marked alterations in nuclear ultrastructure are a universal hallmark of aging, progeroid syndromes and other age-related pathologies. Here we show that autophagy of nuclear proteins is an important determinant of fertility and aging. Impairment of nucleophagy diminishes stress resistance, germline immortality and longevity. We found that the nematode Caenorhabditis elegans nuclear envelope anchor protein, nuclear anchorage protein 1 (ANC-1) and its mammalian ortholog nesprin-2 are cleared out by autophagy and restrict nucleolar size, a biomarker of aging. We further uncovered a germline immortality assurance mechanism, which involves nucleolar degradation at the most proximal oocyte by ANC-1 and key autophagic components. Perturbation of this clearance pathway causes tumor-like structures in C. elegans, and genetic ablation of nesprin-2 causes ovarian carcinomas in mice. Thus, autophagic recycling of nuclear components is a conserved soma longevity and germline immortality mechanism that promotes youthfulness and delays aging under conditions of stress.

The Use of Dietary Supplements and Their Association with COVID-19-Related Anxiety among Non-Institutionalized Elderly in Northern Greece.

The elderly constitute a vulnerable group for increased anxiety and poor diet during the COVID-19 pandemic. There is limited research on the levels of COVID-19-related anxiety and dietary habits including dietary supplementation practices among the elderly and very elderly in Greece. A cross-sectional survey was conducted in 364 non-institutionalized elderly (65-74 y) and very elderly (≥75 y) living in northern Greece, with the aim to investigate the use of dietary supplements and their association with other factors, particularly the COVID-19-related anxiety. Levels of anxiety were assessed with the use of the Coronavirus Anxiety Scale (CAS-5). The percentage of dietary supplement users was 62.6%. The most popular dietary supplements used were vitamin D followed by vitamin C and multivitamin and mineral supplements. Multivariate analysis showed that the very elderly and overweight individuals were less likely to consume vitamin D supplements. Approximately a third of the participants (33.8%) exhibited signs of COVID-19-related anxiety but only 8% showed dysfunctional levels of anxiety. Regression analysis indicated that women, former smokers, and people exhibiting any sign of COVID-19 anxiety were approximately two times more likely to consume dietary supplements of any kind (Gender: OR 2.34, 95% CI 1.30-4.19; Smoking: OR 2.15, 95% CI: 1.08-4.26; COVID-19 anxiety: OR 2.16, 95 % CI: 1.20-3.91). Our results provide useful insights into the current practices of dietary supplement use in this population group and could be used by dietetic and medical associations as well as public authorities in the formulation of targeted, safe, and effective interventions for the protection of public health.

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

Aging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

Molecular Basis of Neuronal Autophagy in Ageing: Insights from Caenorhabditis elegans.

Autophagy is an evolutionarily conserved degradation process maintaining cell homeostasis. Induction of autophagy is triggered as a response to a broad range of cellular stress conditions, such as nutrient deprivation, protein aggregation, organelle damage and pathogen invasion. Macroautophagy involves the sequestration of cytoplasmic contents in a double-membrane organelle referred to as the autophagosome with subsequent degradation of its contents upon delivery to lysosomes. Autophagy plays critical roles in development, maintenance and survival of distinct cell populations including neurons. Consequently, age-dependent decline in autophagy predisposes animals for age-related diseases including neurodegeneration and compromises healthspan and longevity. In this review, we summarize recent advances in our understanding of the role of neuronal autophagy in ageing, focusing on studies in the nematode Caenorhabditis elegans.

Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin.

Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery of small molecules targeting these receptors may yield insights into their biology. However, due to their intrinsic properties, membrane protein targets often cannot be identified by means of established approaches, in particular affinity-based proteomics, calling for the exploration of new methods. Here, we report the identification of indophagolin as representative member of an indoline-based class of autophagy inhibitors through a target-agnostic phenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified the purinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolin targets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable the de novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.

RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein.

Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation.Abbreviations: ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle's balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5'-[ß,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5'-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type.

Autophagy of the Nucleus in Health and Disease.

Nucleophagy is an organelle-selective subtype of autophagy that targets nuclear material for degradation. The macroautophagic delivery of micronuclei to the vacuole, together with the nucleus-vacuole junction-dependent microautophagic degradation of nuclear material, were first observed in yeast. Nuclear pore complexes and ribosomal DNA are typically excluded during conventional macronucleophagy and micronucleophagy, indicating that degradation of nuclear cargo is tightly regulated. In mammals, similarly to other autophagy subtypes, nucleophagy is crucial for cellular differentiation and development, in addition to enabling cells to respond to various nuclear insults and cell cycle perturbations. A common denominator of all nucleophagic processes characterized in diverse organisms is the dependence on the core autophagic machinery. Here, we survey recent studies investigating the autophagic processing of nuclear components. We discuss nucleophagic events in the context of pathology, such as neurodegeneration, cancer, DNA damage, and ageing.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Tuberpulchrosporum sp. nov., a black truffle of the Aestivum clade (Tuberaceae, Pezizales) from the Balkan peninsula.

Knowledge on the diversity of hypogeous sequestrate ascomycetes is still limited in the Balkan Peninsula. A new species of truffle, Tuberpulchrosporum, is described from Greece and Bulgaria. Specimens were collected from habitats dominated by various oak species (i.e. Quercusilex, Q.coccifera, Q.robur) and other angiosperms. They are morphologically characterised by subglobose, ovoid to irregularly lobed, yellowish-brown to dark brown ascomata, usually with a shallow basal cavity and surface with fissures and small, dense, almost flat, trihedral to polyhedral warts. Ascospores are ellipsoid to subfusiform, uniquely ornamented, crested to incompletely reticulate and are produced in (1-)2-8-spored asci. Hair-like, hyaline to light yellow hyphae protrude from the peridium surface. According to the outcome of ITS rDNA sequence analysis, this species forms a distinct well-supported group in the Aestivum clade, with T.panniferum being the closest phylogenetic taxon.

Identification of Novel Autophagy Inhibitors via Cell-Based High-Content Screening.

Autophagy is a fundamental cellular catabolic pathway mediating the recycling of cellular components. Autophagy has been implicated in pathogenesis of diverse diseases such as neurodegeneration and cancer. Due to the therapeutic potential, the autophagy-modulating agents have profoundly enriched the spectrum of tools used to investigate autophagy. However, many of these compounds have additional off-target effects that may confound elucidation of autophagy in certain contexts. There remains high demand for highly specific and novel chemotypes that can be used to study the regulation mechanism of autophagy and contribute novel pharmacophores for therapeutic purposes. Here, we describe a cell-based quantitative high-content screening (HCS) for autophagy inhibitors using a human breast adenocarcinoma MCF7 cell line stably expressing EGFP-LC3, a bona fide marker of autophagy.

Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I.

Macroautophagy is a conserved eukaryotic process for degradation of cellular components in response to lack of nutrients. It is involved in the development of diseases, notably cancer and neurological disorders including Parkinson's disease. Small molecule autophagy modulators have proven to be valuable tools to dissect and interrogate this crucial metabolic pathway and are in high demand. Phenotypic screening for autophagy inhibitors led to the discovery of the novel autophagy inhibitor aumitin. Target identification and confirmation revealed that aumitin inhibits mitochondrial respiration by targeting complex I. We show that inhibition of autophagy by impairment of mitochondrial respiration is general for several mitochondrial inhibitors that target different mitochondrial complexes. Our findings highlight the importance of mitochondrial respiration for autophagy regulation.

Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34.

Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

Discovery of Novel Cinchona-Alkaloid-Inspired Oxazatwistane Autophagy Inhibitors.

The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely-related oxazatricyclo[4.4.0.0]decane ("oxazatwistane") scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state-of-the-art C-H functionalization, and metal-catalyzed cross-coupling reactions as key late diversity-generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.

Incidence, location and classification of glenoid labrum meniscoid folds.

INTRODUCTION: The purpose of the present study was to arthroscopically investigate the incidence and location of labral meniscoid folds of the shoulder joint, as well as to classify them into types and detect any possible correlation with gender, side and age of patients. MATERIALS AND METHODS: The shoulder joint of 59 patients who underwent arthroscopic surgery for different reasons was examined for meniscoid folds. We classified all meniscoid folds into slim or thick and large or small. The location and area of labral folds were assessed according to o'clock position and were defined by the center of the fold. RESULTS: The incidence of labral meniscoid folds in shoulder joint was 62.7 %. Meniscoid folds were more frequently found at 2-o'clock position in right shoulders and at 10-o'clock position in left shoulders. Most of them were located in anterior and superior rim of labrum. Statistically significant difference (p = 0.018) was only detected between location of meniscoid folds and gender. In male patients meniscoid folds were mostly located in a more anterior position than women, whose meniscoid folds were found more superiorly. Older patients presented a higher rate of meniscoid folds. CONCLUSION: Meniscoid folds are quite common in shoulder joint. Labral meniscoid folds are located more often at an anterosuperior position of shoulder joints and their incidence tends to be higher in older patients, while in male ones they are located in a more anterior position in comparison to female patients where they are located more superiorly.

A bioorthogonal small-molecule-switch system for controlling protein function in live cells.

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP' (SLF') linked to trimethoprim (TMP). The SLF' moiety binds to the F36V mutant of FK506-binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF'-TMP-induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 µM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

Comparative validation of the WOMAC osteoarthritis and Lequesne algofunctional indices in Greek patients with hip or knee osteoarthritis.

PURPOSE: To comparatively evaluate the reliability and validity of the Western Ontario and McMaster (WOMAC) and the Lequesne algofunctional indices in Greek patients with hip or knee osteoarthritis (OA). METHODS: The Greek versions of WOMAC LK 3.1 and Lequesne indices were administered to 97 outpatients with OA. Internal consistency reliability was assessed by Cronbach's alpha and item-scale correlations. Test-retest reliability was examined with intraclass correlations. Patients were also asked to complete the Short Form 36 (SF-36) and a Visual Analog Scale capturing strength of pain, in order to assess construct validity. Additional demographic and clinical data were also recorded to evaluate further associations. RESULTS: Cronbach's alpha values of the WOMAC ranged between 0.92 and 0.98 for hip and 0.89-0.97 for knee OA. The respective values for Lequesne were 0.63-0.74 and 0.74-0.80. Item-scale correlations confirmed the superiority of WOMAC with respect to internal consistency reliability. Intraclass correlations were 0.79-0.97 and 0.57-0.98 for hip and 0.86-0.97 and 0.82-0.97 for knee OA, for WOMAC and Lequesne, respectively. The two indices showed high correlations with comparable subscales of SF-36 and the Visual Analog Scale. Significant relationships were identified for age, body mass index, duration of disease, duration of stiffness and radiographic classification. CONCLUSIONS: Our findings, in samples of knee and hip OA patients, indicate that the WOMAC index demonstrates better internal consistency reliability than the Lequesne counterpart, as well as equivalent test-retest reliability and construct validity.

Pancreas divisum: analysis and therapeutic alternatives with a case report.

INTRODUCTION: Pancreas divisum is a relatively common pancreatic duct anatomic variant, firstly described in the 17th century. CASE REPORT: We reported a 2-year-old child admitted to the Pediatric Clinic with breathing difficulties and abdominal pains. Examination and X-ray image, showed a vast right hydrothorax containing rusty coloured solution with a high degree of amylase. Ultrasound and computed tomography examination revealed pancreatic polycyclic pseudocysts; following magnetic resonance cholangiopancreatography (MRCP), the diagnosis of pancreas divisum was confirmed. The general condition of the patient worsened, requiring an urgent operation. External drainage of the perforated pancreatic pseudocyst was performed. Following external fistula maturation, a change from external to internal drainage was performed using Roux-en-Y fistulojejunostomy. A 3-year postoperative period was uneventful. CONCLUSION: Pancreas divisum cases are unique requiring clinical experience, rational approach, and complex multimodal management. MRCP is a valuable diagnostic method. Amongst therapeutic options, outer and internal drainage can be seen as reliable methods. Further investigations are absolutely required to determine practical and appropriate conclusions.

Apoptotic circulating tumor cells in early and metastatic breast cancer patients.

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P = 0.0001; M30-antibody: 32.1% vs. 76.63%; P = 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics.