The DCR protein TTC3 affects differentiation and Golgi compactness in neurons through specific actin-regulating pathways.

In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation

The role of extracellular vesicles in the removal of aggregated TDP43 responsible for ALS/FTD diseases

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two related neurodegenerative diseases. ALS is caused by the death of both upper and lower motoneurons, while FTD is characterized predominantly by circumscribed atrophy of the frontal and temporal lobes. ALS and FTD overlap each other. This is demonstrated by the presence of cognitive and behavioral dysfunction in up to 50% of ALS patients and by the presence of frontotemporal atrophy in patients with ALS. Moreover, these diseases are both characterize by the presence of TAR DNA binding protein 43 (TDP43) inclusions in affected cells. These inclusions, observed in 97% of patients with ALS and 50% of patients with FTD, are composed by TDP43 and its C-terminal fragments of 35 kDa (TDP35) and 25 kDa (TDP25). These fragments are highly aggregation-prone and probably neurotoxic. Thus, their removal is protective for cells. The mechanism responsible for the clearance of aggregates and misfolded proteins is the intracellular protein quality control (PQC) system. It consists of molecular chaperones/co- chaperones and the degradative pathways. PQC controls the folding status of proteins and prevents the aggregation of misfolded proteins by refolding them or degrading. Recent data demonstrated that also extracellular secretory pathway, represented especially by exosomes (EXOs) and microvesicles (MVs), might be involved in the removal of misfolded proteins from affected cells. Thus, we evaluated the role of EXOs and MVs in the secretion of TDP43 and its C-terminal fragments, using neuronal cell models. We used ultracentrifugation, that allowed us to separate MVs from EXOs on the basis of their dimension. Then we analyzed them through i) Nanoparticle Tracking Analysis (NanoSight) to establish their number and sizes, and ii) western blot analysis, to characterize their protein content. Our preliminary results show that TDP43, TDP35 and TDP25 are all secreted, mainly by MVs. In particular, we found that MVs are enriched of insoluble forms of TDPs and also of superoxide dismutase 1 (SOD1), another ALS-related protein. Finally, both in EXOs and in MVs, we observed the presence of some important PQC-components, suggesting an interplay between the two pathways. GRANTS: Fondazione Cariplo, Italy (n. 2017_0747); Universit\ue0 degli Studi di Milano e piano di sviluppo UNIMI - linea B

Endoscopic-assisted transorbital surgery: Where do we stand on the scott’s parabola? personal considerations after a 10-year experience

Transorbital approaches are genuinely versatile surgical routes which show interesting potentials in skull base surgery. Given their "new" trajectory, they can be a very useful adjunct to traditional routes, even being a valid alternative to them in some cases, and add valuable opportunities in selected patients. Indications are constantly expanding, and currently include selected intraorbital, skull base and even intra-axial lesions, both benign and malignant. Given their relatively recent development and thus unfamiliarity among the skull base community, achieving adequate proficiency needs not only a personalized training and knowledge but also, above all, an adequate case volume and a dedicated setting. Current, but mostly future, applications should be selected by genetic, omics and biological features and applied in the context of a truly multidisciplinary environment

A t (9;11) translocation in childhood acute mixed leukemia

We present the case of a child with acute lymphoid leukemia (ALL) who was morphologically classified as FAB L1 (PAS and peroxidase were negative). Remission was achieved with an ALL-type protocol (GBTLI). Five months after the discontinuation of therapy, the patient presented mixed leukemia (CD10, CD19, CD13 and CD33 were positive) with t (9;11) (p21;q23) translocation. Unfortunately, as cytogenetic and immunophenotype studies were not performed at diagnosis, two possibilities could be considered for the relapse; secondary mixed leukemia with clonal chromosome changes, or mixed leukemia from the beginning.Apresentamos o caso de uma criança com leucemia aguda classificada morfologicamente como LLA (leucemia linfóide aguda) FAB L1 (PAS e peroxidase negativos) que alcançou remissão com protocolo para LLA (GBTLI). Após cinco meses fora de terapia desenvolveu uma leucemia mista (CD10, CD19, CD13 e CD33 positivos) com translocação t(9; 11)(p21;q23). Como, infelizmente, os estudos de citogenética e imunofenotipagem não foram realizados ao diagnóstico, pode-se considerar duas possibilidades para a recaída: uma leucemia mista secundária com alteração cromossômica clonal ou uma leucemia mista desde o início.Escola Paulista de Medicina Department of HematologyUniversidade de São Paulo Instituto da CriançaSt. Jude Children's Research Hospital Cytogenetic LaboratoryUNIFESP, EPM, Department of HematologySciEL

Upper and lower airway inflammation in severe asthmatics: a guide for a precision biologic treatment

Background and aims: Severe asthma may require the prescription of one of the biologic drugs currently available, using surrogate markers of airway inflammation (serum IgE levels and allergic sensitization for anti-IgE, or blood eosinophils for anti-IL5/IL5R). Our objective: to assess upper and lower airway inflammation in severe asthmatics divided according to the eligibility criteria for one of the target biologic treatments. Methods: We selected 91 severe asthmatics, uncontrolled despite high-dose ICS-LABA, and followed for >6 months with optimization of asthma treatment. Patients underwent clinical, functional and biological assessment, including induced sputum and nasal cytology. They were then clustered according to the eligibility criteria for omalizumab or mepolizumab/benralizumab. Results: Four clusters were selected: A (eligible for omalizumab, n = 23), AB (both omalizumab and mepolizumab, n = 26), B (mepolizumab, n = 22) and C (non-eligible for both omalizumab and mepolizumab, n = 20). There was no difference among clusters for asthma control (Asthma Control Test and Asthma Control Questionnaire 7), pre-bronchodilator forced expiratory volume in 1 s, serum IgE and fractional exhaled nitric oxide levels. Sputum eosinophils were numerically higher in clusters AB and B, in agreement with the higher levels of blood eosinophils. Allergic rhinitis was more frequent in clusters A and AB, while chronic rhinosinusitis with nasal polyps prevalence increased progressively from A to C. Eosinophils in nasal cytology were higher in clusters AB, B and C. Conclusion: Eosinophilic upper and lower airway inflammation is present in the large majority of severe asthmatics, independently from the prescription criteria for the currently available biologics, and might suggest the use of anti-IL5/IL5R or anti IL4/13 also in patients without blood eosinophilia. The reviews of this paper are available via the supplemental material section

Enhanced Clearance of Neurotoxic Misfolded Proteins by the Natural Compound Berberine and Its Derivatives

Background: Accumulation of misfolded proteins is a common hallmark of several neurodegenerative disorders (NDs) which results from a failure or an impairment of the proteinquality control (PQC) system. The PQC system is composed by chaperones and the degradative systems (proteasome and autophagy). Mutant proteins that misfold are potentially neurotoxic, thus strategies aimed at preventing their aggregation or at enhancing their clearance are emerging as interesting therapeutic targets for NDs. Methods: We tested the natural alkaloid berberine (BBR) and some derivatives for their capability to enhance misfolded protein clearance in cell models of NDs, evaluating which degradative pathway mediates their action. Results: We found that both BBR and its semisynthetic derivatives promote degradation of mutant androgen receptor (ARpolyQ) causative of spinal and bulbar muscular atrophy, acting mainly via proteasome and preventing ARpolyQ aggregation. Overlapping effects were observed on other misfolded proteins causative of amyotrophic lateral sclerosis, frontotemporal-lobar degeneration or Huntington disease, but with selective and specific action against each different mutant protein. Conclusions: BBR and its analogues induce the clearance of misfolded proteins responsible for NDs, representing potential therapeutic tools to counteract these fatal disorders

Ret/PTC activation does not influence clinical and pathological features of adult papillary thyroid carcinomas

OBJECTIVE: RET proto-oncogene rearrangements (ret/PTCs) represent the most common genetic alterations found in papillary thyroid carcinomas (PTCs). Correlation of ret/PTC expression with clinical outcome is controversial. The aim of the present study was to analyze the frequency of RET rearrangements in adult PTCs, and to investigate if ret/PTCs influence biological behavior and clinical features of the cancers. DESIGN: Ret/PTC rearrangements were looked for in tIssue samples of 48 PTCs collected at our institution. Data about clinical and pathological features of the tumors were also reviewed. Three separate association analyses were carried out on the cohort evaluating the effects of, respectively, ret/PTC positivity, preferential RET tyrosine kinase domain (RET-TK) expression, and ret/PTC plus RET-TK positivity, on age, sex, tumor size, staging, number of neoplastic foci, and histological subtype. METHODS: The genetic study was conducted with the RT-PCR-Southern blot technique. Standard Student's t-test and Fisher exact test were applied for the association analyses. RESULTS: The molecular genetic study demonstrated the positivity of ret/PTC1 and ret/PTC3 in 13 of 48 tumors (27.1%), and an exclusive or preferential RET-TK expression in 17 cases (35.4%). None of the three genetico-clinical analyses showed any significant association between ret/PTC expression and the clinical and pathological features of the cancers. CONCLUSIONS: These data indicate that RET rearrangements may not play any distinctive role in driving histotype development and cancer progression in these neoplasms. Moreover, they weaken the possibility of using ret/PTC as a prognostic marker for papillary thyroid carcinomas

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells

Aim: Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. Methods: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. Results: We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. Conclusion: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars