Studies on the assembly of rotavirus viroplasmas

The processes that regulate Rotavirus replication are not fully understood and the lack of a reverse genetic approach represent an obstacle for the investigations in Rotavirus biology. Viroplasms are cytoplasmic structures that form soon after infection, and constitute the site of virus replication. Structural proteins like the viral RNA-dependent RNA -polymerase VP1, the capping enzyme VP3, the scaffolding protein VP2,and the middle layer VP6 localize in viroplasms; in addition, also the non-structural proteins NSP5 and NSP2 have been demonstrated to be essential components for viroplasm formation. Following the characterization of the interaction between NSP5 and VP1, we characterized the relationships between NSP5 and the structural protein VP2. In this work, interaction of NSP5 with VP2 was investigated by coexpression of the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation of NSP5 and in the formation of viroplasm like structures (VLS). The behaviour of NSP5 in the presence of VP2 is very similar to that induced by NSP2 and already described (1), (60). Therefore, a comparison between the phosphorylation degree of NSP5 and VLS formation induced either by VP2 or by NSP2 was conducted. In both cases VLS formation was shown to assemble independently of the phosphorylation degree of NSP5, and to recruit the viroplasm-resident proteins VP1. However, VP6 (the protein forming the middle layer of the virion) was shown to be recruited only into VLS induced by VP2 (VLS(VP2i)), while it remains organized in tubular structures when VLSinduced by NSP2 (VLS(NSP2i)) were formed. Attempts to coimmunoprecipitate NSP5 and VP2 failed both from infected and co-transfected cells. However, promising preliminary results were obtained with a recently isolated monoclonal Ab specific for NSP5. Altogether, these data showed that two different viral proteins induced the same kind of modifications in NSP5, suggesting that these modifications may have a fundamental role for virus replication. Moreover, these data suggest that NSP5 plays a key role in architectural assembly of viroplasms and in recruitment of the other viroplasmic proteins

An assay system to evaluate riboflavin/UV-A corneal phototherapy efficacy in a porcine corneal organ culture model

The purpose of this study is to investigate the response of a porcine corneal organ cultures to the riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model has been established. Corneal alterations in the stroma were valuated setting an assay system, based on an automated image analysis method able to quantify the damaged (brightness values), within of the 24 regions of interest (ROIs) in which the corneal section has been divided and integrating the data analysis with a multi-aspect approach. Three group of corneas have been analyzed: (healthy, injured and injured-and-treated group). Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p< 0.00) and healthy (p<0.001) corneas whereas the treated and healthy corneas showed not significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in the restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic method such as the OCT imaging for in vivo damaged ocular structures investigations

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations

AMPLICON-BASED NGS: AN EFFECTIVE APPROACH FOR THE MOLECULAR DIAGNOSIS OF EPIDERMOLYSIS BULLOSA

Background: Epidermolysis Bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS), which are labour intensive and expensive clinical pathways. Objectives: According to the recently published recommendations for the EB diagnosis and treatment, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. Next-Generation Sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. Methods: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on the Ion Torrent PGM platform. The panel was performed on ten patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. Results: The panel was successful in finding the causative mutations in all ten of the patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity, and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. Conclusions: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a costand time-effective 72-hour procedure

Amoebic splenic abscess: a case report

Objective: Report a clinical case of amoebic splenic abscess and discuss its clinical characteristics, its difficult diagnosis, its clinical and surgical management. Case presentation: A 39-year-old male patient was admitted complaining of abdominal pain in the left flank region, nausea and lack of appetite, denying fever, vomiting and changes in bowel habits. Physical abdominal examination showed a tense, reactive abdomen, with pain on palpation of the left flank, with a sign of positive sudden decompression and a positive Giordano on the left. In admission, a computed tomography scan of the abdomen was performed, which showed hypodense collections in the spleen and the presence of free fluid in the abdominal cavity, with a hypothesis of splenic abscess. Splenectomy and caudal pancreatectomy were performed, and ceftriaxone and metronidazole were started. After the surgery, a secretion culture with a positive result was requested on July 05th, 2019 for direct research of Entamoeba histolytica, defining the diagnosis of splenic abscess by amoeba. The patient was discharged on July 07th, 2019 with a prescription for metronidazole to end up treatment. Conclusion: The amoebic splenic abscess is a rare pathology with few data in the literature, with difficulty and delay in the diagnosis and treatment of this pathology, which directly affects the patient’s prognosis. Therefore, it is necessary that more cases on the pathology be reported for a better understanding of the diagnosis and management of amebic splenic abscess.Objetivo: Relatar um caso clínico de abscesso esplênico amebiano e discutir suas características clínicas, seu difícil diagnóstico, seu manejo clínico e cirúrgico. Apresentação do caso: Paciente masculino 39 anos foi admitido com queixa de dor abdominal em região de flanco esquerdo, náuseas e inapetência, negando febre, vômitos e alterações do hábito intestinal. Ao exame físico abdominal evidenciou um abdome tenso, reativo, com dor à palpação de flanco esquerdo, com sinal de descompressão brusca positiva e Giordano positivo à esquerda. Na admissão foi realizada tomografia computadorizada de abdome que evidenciou coleções hipodensas no baço e presença de líquido livre na cavidade abdominal, com hipótese de abscesso esplênico. Foram realizadas esplenectomia e pancreatectomia caudal e iniciado ceftriaxona e metronidazol. Após a cirurgia foi solicitada cultura da secreção com resultado positivo no dia 05/07/2019 para pesquisa direta de Entamoeba histolytica, definindo o diagnóstico de abscesso esplênico por ameba. Paciente recebeu alta no dia 06/07/2019 com receita de metronidazol para término do tratamento. Considerações finais: O abscesso esplênico amebiano é uma patologia raríssima com poucos dados na literatura, havendo uma dificuldade e demora no diagnóstico e no tratamento dessa patologia o que implica diretamente no prognostico do paciente. Portanto, é necessário que mais casos sobre a patologia sejam relatados para melhor entendimento sobre o diagnóstico e manejo do abscesso esplênico amebiano

Gamma-Ray Burst observations by the high-energy charged particle detector on board the CSES-01 satellite between 2019 and 2021

In this paper we report the detection of five strong Gamma-Ray Bursts (GRBs) by the High-Energy Particle Detector (HEPD-01) mounted on board the China Seismo-Electromagnetic Satellite (CSES-01), operational since 2018 on a Sun-synchronous polar orbit at a $\sim$ 507 km altitude and 97$^\circ$ inclination. HEPD-01 was designed to detect high-energy electrons in the energy range 3 - 100 MeV, protons in the range 30 - 300 MeV, and light nuclei in the range 30 - 300 MeV/n. Nonetheless, Monte Carlo simulations have shown HEPD-01 is sensitive to gamma-ray photons in the energy range 300 keV - 50 MeV, even if with a moderate effective area above $\sim$ 5 MeV. A dedicated time correlation analysis between GRBs reported in literature and signals from a set of HEPD-01 trigger configuration masks has confirmed the anticipated detector sensitivity to high-energy photons. A comparison between the simultaneous time profiles of HEPD-01 electron fluxes and photons from GRB190114C, GRB190305A, GRB190928A, GRB200826B and GRB211211A has shown a remarkable similarity, in spite of the different energy ranges. The high-energy response, with peak sensitivity at about 2 MeV, and moderate effective area of the detector in the actual flight configuration explain why these five GRBs, characterised by a fluence above $\sim$ 3 $\times$ 10$^{-5}$ erg cm$^{-2}$ in the energy interval 300 keV - 50 MeV, have been detected.Comment: Accepted for publication in The Astrophysical Journal (ApJ