The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy

Autophagy is an evolutionary conserved catabolic process involved in several physiological and pathological processes such as cancer and neurodegeneration. Autophagy initiation signaling requires both the ULK1 kinase and the BECLIN 1-VPS34 core complex to generate autophagosomes, double-membraned vesicles that transfer cellular contents to lysosomes. In this study, we show that the BECLIN 1-VPS34 complex is tethered to the cytoskeleton through an interaction between the BECLIN 1-interacting protein AMBRA1 and dynein light chains 1/2. When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from dynein. Its subsequent relocalization to the endoplasmic reticulum enables autophagosome nucleation. Therefore, AMBRA1 constitutes a direct regulatory link between ULK1 and BECLIN 1-VPS34, which is required for core complex positioning and activity within the cell. Moreover, our results demonstrate that in addition to a function for microtubules in mediating autophagosome transport, there is a strict and regulatory relationship between cytoskeleton dynamics and autophagosome formation.     

Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.     

Differential diagnosis between cutaneous lymphoma and pseudolymphoma

Differential diagnosis of cutaneous lymphoproliferative disorders represents one of the most vexing problems in dermatology and dermatopathology. For nearly a century the diagnosis has been based only upon clinicopathologic correlation. Immunohistochemical and molecular techniques developed during the last 3 decades added new criteria for the differentiation of these diseases. The purpose of this articles is to summarize the criteria for the differential diagnosis of benign vs. malignant lymphoid infiltrates of the skin. In this context, a proper classification of cutaneous lymphoproliferative disorders can be achieved only by a synthesis of clinical, histopathologic, immunophenotypic and molecular criteria, and in some cases only follow-up data allow a precise diagnosis to be made.     

The suitability of disintegrating force kinetics for studying the effect of manufacturing parameters on spironolactone tablet properties

The aim of this paper was to study the effect of the granulate properties and tablet compression force on disintegrating force behavior in order to investigate the capability of the disintegrating force to characterize tablets that have the same composition but were manufactured in different conditions. Several tablets containing spironolactone in the external or internal granulated mixture of calcium carbonate and maize starch differing in particle size distribution, were prepared at 3 compression levels. The force developed by tablets during water uptake and disintegration was measured and plotted versus time. The curves obtained were analyzed by the Weibull equation in order to calculate the parameters characterizing the tablet disintegration kinetics. The disintegrating force time parameter, the maximum force developed, and the area under the curve were determined. In general, the reduction of time parameter value and/or the increase in maximum force developed corresponded to an acceleration in tablet disintegration. In addition, the area under the force curve increased in stronger tablets, monitoring in a sensitive way the tablet structural changes introduced by compression force. The results showed that the disintegrating force measurement can detect small changes in the structure of the tablet that cannot be discriminated by pharmacopoeia tests. The effect of manufacturing, in particular compression force, on tablet properties was quantified by the parameters of disintegrating force kinetics.     

Theophylline-induced apoptosis is paralleled by protein kinase A-dependent tissue transglutaminase activation in cancer cells

beta(2)-Microglobulin (beta2M), the light chain of the type I major histocompatibility complex, is a major component of dialysis-related amyloid fibrils. beta2M in the native state has a typical immunoglobulin fold with a buried intrachain disulfide bond. The conformation and stability of recombinant beta2M in which the intrachain disulfide bond was reduced were studied by CD, tryptophan fluorescence, and one-dimensional NMR. The conformation of the reduced beta2M in the absence of denaturant at pH 8.5 was similar to that of the intact protein unless the thiol groups were modified. However, reduction of the disulfide bond decreased the stability as measured by denaturation in guanidine hydrochloride. Intact beta2M formed amyloid fibrils at pH 2.5 by extension reaction using sonicated amyloid fibrils as seeds. Under the same conditions, reduced beta2M did not form typical amyloid fibrils, although it inhibited fibril extension competitively, suggesting that the conformation defined by the disulfide bond is important for amyloid fibril formation of beta2M.     

Time trees and clock genes: a systematic review and comparative analysis of contemporary avian migration genetics

Timing is a crucial aspect for survival and reproduction in seasonal environments leading to carefully scheduled annual programs of migration in many species. But what are the exact mechanisms through which birds (class: Aves) can keep track of time, anticipate seasonal changes, and adapt their behaviour? One proposed mechanism regulating annual behaviour is the circadian clock, controlled by a highly conserved set of genes, collectively called ‘clock genes’ which are well established in controlling the daily rhythmicity of physiology and behaviour. Due to diverse migration patterns observed within and among species, in a seemingly endogenously programmed manner, the field of migration genetics has sought and tested several candidate genes within the clock circuitry that may underlie the observed differences in breeding and migration behaviour. Among others, length polymorphisms within genes such as Clock and Adcyap1 have been hypothesised to play a putative role, although association and fitness studies in various species have yielded mixed results. To contextualise the existing body of data, here we conducted a systematic review of all published studies relating polymorphisms in clock genes to seasonality in a phylogenetically and taxonomically informed manner. This was complemented by a standardised comparative re-analysis of candidate gene polymorphisms of 76 bird species, of which 58 are migrants and 18 are residents, along with population genetics analyses for 40 species with available allele data. We tested genetic diversity estimates, used Mantel tests for spatial genetic analyses, and evaluated relationships between candidate gene allele length and population averages for geographic range (breeding- and non-breeding latitude), migration distance, timing of migration, taxonomic relationships, and divergence times. Our combined analysis provided evidence (i) of a putative association between Clock gene variation and autumn migration as well as a putative association between Adcyap1 gene variation and spring migration in migratory species; (ii) that these candidate genes are not diagnostic markers to distinguish migratory from sedentary birds; and (iii) of correlated variability in both genes with divergence time, potentially reflecting ancestrally inherited genotypes rather than contemporary changes driven by selection. These findings highlight a tentative association between these candidate genes and migration attributes as well as genetic constraints on evolutionary adaptation.     

Profiling metabolites and lipoproteins in COMETA,an Italian cohort of COVID-19 patients

Metabolomics and lipidomics have been used in several studies to define the biochemical alterations induced by COVID-19 in comparison with healthy controls. Those studies highlighted the presence of a strong signature, attributable to both metabolites and lipoproteins/lipids. Here, ¹H NMR spectra were acquired on EDTA-plasma from three groups of subjects: i) hospitalized COVID-19 positive patients (≤21 days from the first positive nasopharyngeal swab); ii) hospitalized COVID-19 positive patients (>21 days from the first positive nasopharyngeal swab); iii) subjects after 2–6 months from SARS-CoV-2 eradication. A Random Forest model built using the EDTA-plasma spectra of COVID-19 patients ≤21 days and Post COVID-19 subjects, provided a high discrimination accuracy (93.6%), indicating both the presence of a strong fingerprint of the acute infection and the substantial metabolic healing of Post COVID-19 subjects. The differences originate from significant alterations in the concentrations of 16 metabolites and 74 lipoprotein components. The model was then used to predict the spectra of COVID-19>21 days subjects. In this group, the metabolite levels are closer to those of the Post COVID-19 subjects than to those of the COVID-19≤21 days; the opposite occurs for the lipoproteins. Within the acute phase patients, characteristic trends in metabolite levels are observed as a function of the disease severity. The metabolites found altered in COVID-19≤21 days patients with respect to Post COVID-19 individuals overlap with acute infection biomarkers identified previously in comparison with healthy subjects. Along the trajectory towards healing, the metabolome reverts back to the “healthy” state faster than the lipoproteome.