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Photoactivatable green fluorescent protein (paGFP) exhibits peculiar photo-physical properties making it an invaluable tool for protein/cell tracking in living cells/organisms. paGFP is normally excited in the violet range (405 nm), with an emission peak centred at 520 nm. Absorption cross-section at 488 nm is low in the not-activated form. However, when irradiated with high-energy fluxes at 405 nm, the protein shows a dramatic change in its absorption spectra becoming efficiently excitable at 488 nm. Confocal microscopes allow to control activation in the focal plane. Unfortunately, irradiation extends to the entire illumination volume, making impracticable to limit the process in the 3D (three-dimensional) space. In order to confine the process, we used two advanced intrinsically 3D confined optical methods, namely: total internal reflection fluorescence (TIRF) and two-photon excitation fluorescence (2PE) microscopy. TIRF allows for spatially selected excitation of fluorescent molecules within a thin region at interfaces, i.e. cellular membranes. Optimization of the TIRF optical set-up allowed us to demonstrate photoactivation of paGFP fused to different membrane localizing proteins. Exploitation of the penetration depth showed that activation is efficiently 3D confined even if limited at the interface. 2PE microscopy overcomes both the extended excitation volume of the confocal case and the TIRF constraint of operating at interfaces, providing optical confinement at any focal plane in the specimen within subfemtoliter volumes. The presented results emphasize how photoactivation by non-linear excitation can provide a tool to increase contrast in widefield and confocal cellular imaging.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs’ biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

THC2, an autosomal-dominant thrombocytopenia described so far in only two families, has been ascribed to mutations in MASTL or ACBD5. Here, we show that ANKRD26, another gene within the THC2 locus, and neither MASTL nor ACBD5, is mutated in eight unrelated families. ANKRD26 was also found to be mutated in the family previously reported to have an ACBD5 mutation. We identified six different ANKRD26 mutations, which were clustered in a highly conserved 19 bp sequence located in the 5' untranslated region. Mutations were not detected in 500 controls and are absent from the 1000 Genomes database. Available data from an animal model and Dr. Watson's genome give evidence against haploinsufficiency as the pathogenetic mechanism for ANKRD26-mediated thrombocytopenia. The luciferase reporter assay suggests that these 5' UTR mutations might enhance ANKRD26 expression. ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, which have been recently identified as a family of proapoptotic proteins. Dysregulation of apoptosis might therefore be the pathogenetic mechanism, as demonstrated for another thrombocytopenia, THC4. Further investigation is needed to provide evidence supporting this hypothesis.

The SRC gene was the first proto-oncogene to be discovered. Its product SRC is a nonreceptor protein tyrosine kinase that is the prototype, and a ubiquitously expressed member, of the SRC family kinases. SRC has been investigated for decades in mouse and in vitro models: these studies have indicated that SRC signalling has a central role in many cellular functions and in oncogenesis. 1 In platelets, SRC mediates signal activation pathways downstream different integrins and G protein-coupled receptors.2 However, much remains to be understood about SRC functions in human megakaryocytes and platelets. Recently, the first germline mutation in SRC causing human disease was reported in two families. Here we report the investigation of a new unrelated individual carrying the p.E527K variant that provides additional information on the clinical and pathogenetic features of the disorder and the role of SRC in human megakaryocytes.

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response., The nucleus is a mechanically stiff organelle of the cell and the DNA damage response protein ATR can localize to the nuclear envelope upon mechanical stress. Here, the authors show that ATR may contribute to the integrity of the nuclear envelope and may play a role in cell migration.

Essentials Thrombocytopenia 2 (THC2) is an inherited thrombocytopenia (IT) with dysmegakaryopoiesis. Physicians often do not suspect the genetic origin of thrombocytopenia in patients with THC2. We report two THC2 patients misdiagnosed with myelodysplasia and treated with chemotherapy. IT should be always considered in patients with isolated thrombocytopenia and dysmegakaryopoiesis. Summary Thrombocytopenia 2 (THC2) is an autosomal-dominant disorder caused by point substitutions in the 5′UTR of the ANKRD26 gene. Patients have congenital thrombocytopenia, normal platelet morphology and function, and dysmegakaryopoiesis. Thrombocytopenia is frequently discovered only in adulthood and physicians often do not suspect its genetic origin. We describe two unrelated patients referred to two different institutions for investigation of thrombocytopenia. Based on the finding of dysmegakaryopoiesis at bone marrow examination, patients were diagnosed with myelodysplastic syndrome (MDS) (refractory thrombocytopenia) and treated with several courses of 5-azacytidine. Subsequently, demonstration of thrombocytopenia in their relatives eventually led to molecular diagnosis of THC2 in both families. These cases highlight that patients with THC2 are at risk of being misdiagnosed with MDS and receiving undue myelosuppressive treatments. Because dysmegakaryopoiesis is a feature also of other forms of inherited thrombocytopenia, a genetic disorder must always be considered when a patient presents with isolated thrombocytopenia and dysmegakaryopoiesis.

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

Summary Objectives The aim of this study was to determine the prevalence and clinical characteristics of voice disorders in a large group of patients seeking voice therapy. Study design This is a prospective prevalence study. Methods A total of 821 patients were enrolled. Each patient was evaluated following a multidimensional protocol including videolaryngostroboscopy, perception, acoustics, aerodynamics, and self-rating by the patient. Data regarding age, gender, tobacco use, gastroesophageal reflux disease (GERD), and professional voice use were collected and analyzed. Results Based on videolaryngoscopic findings, the sample group was divided into patients with functional dysphonia (n = 155), patients with organic dysphonia (n = 359), and patients with dysphonia due to movement disorders (n = 307). The most frequently detected pathologies were vocal fold paralysis, muscle tension dysphonia, and vocal fold edema. Children (n = 41) and adolescents (n = 43) represented a minority of the sample group. Dysphonia was significantly more common in women. Organic dysphonia was more common in children and adolescents. GERD was suspected in 382 patients and confirmed in 83 of them; 164 patients were smokers. Professional voice users composed the large majority of the working population and were more frequently affected by organic dysphonia. Patients with dysphonia due to movement disorders presented a worse voice quality and voice-related quality of life. Conclusions In patients seeking voice therapy, there are more females than males, children and adolescents represent a minority of the sample, professional voice users more commonly present organic dysphonia, and patients with dysphonia due to movement disorders show significantly worse voice quality.