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.

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.

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.

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.

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.

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.

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.

Regulation of myosin light chain function by BMP signaling controls actin cytoskeleton remodeling.

BACKGROUND/AIMS: Actin cytoskeleton dynamics support and coordinate signaling events that control cell proliferation, differentiation and migration. Growth factors provide essential signals that act on multi-protein complexes that regulate actin assembly with myosin. We previously analyzed the action of the transforming growth factor ß (TGF-ß) and now extend our studies to the bone morphogenetic protein (BMP) 7, an important regulator of stem cell function and bone differentiation. METHODS: Using a well-established cell model of actin dynamics, Swiss3T3 fibroblasts, we applied cell biological and biochemical approaches to monitor the pathway that links the BMP-7 receptors to the acto-myosin complex. RESULTS: We demonstrate that BMP-7 induces actin and focal adhesion remodeling in starved fibroblasts as potently as TGF-ß. BMP-7 mediates changes of actin dynamics via the kinase ROCK1 and induces rapid activation of RhoA and RhoB with concomitant inactivation of Cdc42. These molecular events correlate well with induction of phosphorylation on Ser19 of the myosin light chain, but not with LIMK1 kinase activation. Depletion of endogenous myosin light chain inhibits actin remodeling induced by BMP-7. This novel pathway regulates fibroblast migration without affecting cell proliferation. CONCLUSION: We establish a BMP-Rho-ROCK1 pathway, which targets myosin light chain to control actin remodeling in fibroblasts.

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.

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.

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.

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.

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.

Regional differences in maturation of germ cells of cryptorchid testes: role of environment.

OBJECTIVE: To investigate differences in maturation of germ cells in cryptorchid testes in three different regions. PATIENTS AND METHODS: A total of 103 consecutive patients were operated for unilateral undescended testis in Vojvodina, from March 2006 until September 2007, and had a testicular biopsy performed. Germ cells were counted, and the presence of Ad spermatogonia was noted. Biopsies were compared to biopsies of similar patients from two different regions: Philadelphia, USA (130), and Liestal, Switzerland (55 patients). RESULTS: In Vojvodina, 84.5% of patients had Sertoli cells only, or some spermatogonia, but no Ad spermatogonia, and 15.5% had Ad spermatogonia. In Philadelphia, 59.3% of patients had poor testicular histology, and 40.7% had Ad spermatogonia. In Liestal, 61.8% of patients had no, or some, spermatogonia, but no Ad spermatogonia, and 38.2% had Ad spermatogonia. There was a difference (p = 0.000025) between the patients with normal testicular histology from Philadelphia and those from Vojvodina, as well as between the patients from Vojvodina and Liestal (p = 0.0027). CONCLUSION: The reduction in the number of germ cells in patients with cryptorchidism from Vojvodina is more pronounced than patients from either Switzerland or USA. This is a unique observation, since such a study has not been published yet.

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.

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.

Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU145 prostate cancer cells.

BACKGROUND: Recently we have reported membrane androgen receptors-induced apoptotic regression of prostate cancer cells regulated by Rho/ROCK/actin signaling. In the present study we explored the specificity of these receptors and we analyzed downstream effectors controlling survival and apoptosis in hormone refractory DU145-prostate cancer cells stimulated with membrane androgen receptor-selective agonists. RESULTS: Using membrane impermeable conjugates of serum albumin covalently linked to testosterone, we show here down-regulation of the activity of pro-survival gene products, namely PI-3K/Akt and NF-kappaB, in DU145 cells. Testosterone-albumin conjugates further induced FasL expression. A FasL blocking peptide abrogated membrane androgen receptors-dependent apoptosis. In addition, testosterone-albumin conjugates increased caspase-3 and Bad protein activity. The actin cytoskeleton drug cytochalasin B and the ROCK inhibitor Y-27632 inhibited FasL induction and caspase-3 activation, indicating that the newly identified Rho/Rock/actin signaling may regulate the downstream pro-apoptotic effectors in DU145 cells. Finally, other steroids or steroid-albumin conjugates did not interfere with these receptors indicating testosterone specificity. CONCLUSION: Collectively, our results provide novel mechanistic insights pointing to specific pro-apoptotic molecules controlling membrane androgen receptors-induced apoptotic regression of prostate cancer cells and corroborate previously published observations on the potential use of membrane androgen receptor-agonists as novel anti-tumor agents in prostate cancer.