Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2.

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαß+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors' laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

Response to Treatment with an Anti-Interleukin-6 Receptor Antibody (Tocilizumab) in a Patient with Hemophagocytic Syndrome Secondary to Immune Checkpoint Inhibitors.

Background. Immunotherapy represents one of the fundamental treatments in the management of some types of cancer, especially malignant melanoma. Toxicity derived from increased immune system activity can manifest in multiple organs and systems. We present a case of hematological toxicity, manifested as hemophagocytic syndrome (HPS), which was successfully treated with an anti-interleukin-6 antibody (tocilizumab). Case Report. This case presents a 75-year-old woman diagnosed with metastatic choroidal melanoma, refractory to several lines of treatment. After the failure of the previous lines, ipilimumab was started. After the third dose, she developed grade 2 thrombocytopenia and anemia accompanied by elevated levels of ferritin, triglycerides, and decreased fibrinogen. Hemophagocytosis was observed in the bone marrow biopsy, and a PET-CT showed splenomegaly with increased metabolism. Treatment was based on high doses of corticosteroids and tocilizumab. Four days after the start of treatment, progressive clinical and analytical improvement was observed, achieving total remission of the condition. Discussion. HPS induced by immunotherapy is due to an immunorelated cytokine storm syndrome (CSS). The administration of the anti-interleukin-6 receptor antibody drug acted on this cytokine cascade, leading to stabilization and subsequent remission. For this reason, the use of tocilizumab should be part of the immunotherapy-induced HPS treatment algorithm.

Bioelectrical impedance vector values in a Spanish healthy newborn population for nutritional assessment.

INTRODUCTION: Vector bioimpedance analysis (BIVA) can be very useful for the evaluation of body composition, hydration, and nutritional status in infants and newborns. The objective of this study was to determine the impedance vector distribution for a group of healthy newborn Spanish children. METHODS: This was a cross-sectional, descriptive study conducted with 154 healthy, Spanish newborns (gestational age: 37-41 weeks) aged 24 to 72 hours (79 males, 75 females). Weight, height, and cephalic-circumference were determined. Resistance and reactance were measured with a single-frequency impedance analyzer at 50 kHz (tetrapolar analysis). The newborns' specific 95% confidence intervals of the mean vectors and the 95%, 75%, and 50% tolerance intervals for the individual vector measurements were plotted using R and Xc components standardized by the subjects' lengths. The mean impedance vectors were compared with Hotelling's-T2 test for vector analysis (significance level: P < .05). RESULTS: The newborns exhibited gender-related differences in the mean impedance vector (mean [SD] R/H: 833.6 [97.5] Ohm/m in males vs 918.2 [107.7] Ohm/m in females; mean [SD] Xc/H: 91.3 [34.7] Ohm/m in males vs 95.6 [23.2] Ohm/m in females). No statistically significant differences in the mean impedance vectors were observed according to days of life. Lower values of resistance and slightly higher reactance values were observed in the healthy Spanish newborns compared to Italian newborns. CONCLUSIONS: New tolerance ellipses were constructed for healthy Spanish newborns. These data allow detecting alterations in the hydration status and cell mass in term newborns in the first 3 days of life.

Differential NtcA Responsiveness to 2-Oxoglutarate Underlies the Diversity of C/N Balance Regulation in Prochlorococcus.

Previous studies showed differences in the regulatory response to C/N balance in Prochlorococcus with respect to other cyanobacteria, but no information was available about its causes, or the ecological advantages conferred to thrive in oligotrophic environments. We addressed the changes in key enzymes (glutamine synthetase, isocitrate dehydrogenase) and the ntcA gene (the global nitrogen regulator) involved in C/N metabolism and its regulation, in three model Prochlorococcus strains: MED4, SS120, and MIT9313. We observed a remarkable level of diversity in their response to azaserine, a glutamate synthase inhibitor which increases the concentration of the key metabolite 2-oxoglutarate, used to sense the C/N balance by cyanobacteria. Besides, we studied the binding between the global nitrogen regulator (NtcA) and the promoter of the glnA gene in the same Prochlorococcus strains, and its dependence on the 2-oxoglutarate concentration, by using isothermal titration calorimetry, surface plasmon resonance, and electrophoretic mobility shift. Our results show a reduction in the responsiveness of NtcA to 2-oxoglutarate in Prochlorococcus, especially in the MED4 and SS120 strains. This suggests a trend to streamline the regulation of C/N metabolism in late-branching Prochlorococcus strains (MED4 and SS120), in adaptation to the rather stable conditions found in the oligotrophic ocean gyres where this microorganism is most abundant.

Psychopathic traits mediate the effects of neighbourhood risk on juvenile antisocial behaviour.

BACKGROUND: Research of the indirect effects of neighbourhood risk through psychopathic personality traits (i.e., impulsiveness, narcissism and callous-unemotional traits) on juvenile antisocial behaviour has become relevant. METHODS: In the current study, mediation effects were assessed using a sample composed of 406 young people between 14 and 21 years old, 82.7% males, from juvenile centres in Galicia and Andalucía (Spain). RESULTS: The results showed significant mediation effects of neighbourhood risk on antisocial behaviour through impulsiveness and callous-unemotional traits.  Conclusions: The implications of the mediation effects for prevention, risk assessment, and risk management in juvenile offenders are discussed.

MODE OF ATTACHMENT AND PATHOLOGY CAUSED BY PARORCHITES ZEDERI IN THREE SPECIES OF PENGUINS: PYGOSCELIS PAPUA, PYGOSCELIS ADELIAE, AND PYGOSCELIS ANTARCTICA IN ANTARCTICA.

We identified and compared gross and microscopic lesions associated with the cestode, Parorchites zederi, in the digestive tracts of three species of penguins (Spheniscidae): the Chinstrap ( Pygoscelis antarctica ), Gentoo ( Pygoscelis papua ), and Adélie penguins ( Pygoscelis adeliae ). The gastrointestinal tracts of 79 recently dead individuals (71 chicks and eight adults) were collected in locations throughout the Antarctic Peninsula during summer field trips in 2006-09. Parorchites zederi was found in the small intestine of 37 animals (47%), and 23 (62%) of these had parasite-associated lesions. The cestodes were either free in the intestinal lumen, clustered within mucosal ulcers, or deeply embedded in the intestinal wall. Histopathologic changes were most severe in adult Gentoo Penguins and included transmural fibrogranulomatous enteritis, hemorrhage, and edema. This report of pathology associated with P. zederi in the digestive tracts of penguins can serve as reference to monitor health in Antarctic birds associated with environmental changes.

Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum.

BACKGROUND: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg(2+) and Mn(2+). RESULTS: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. CONCLUSIONS: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Mitral valve repair in a patient with mitral regurgitation and osteogenesis imperfecta tarda.

We present the case of a 62-year-old female with a diagnosis of osteogenesis imperfecta and mitral valve regurgitation. The patient underwent a mitral valve repair without complications. We describe the case and our surgical technique.

Spontaneous closure of pseudoaneurysm after transapical aortic valve implantation.

Transcatheter aortic valve implantation by an apical approach has been developed as an alternative to conventional aortic valve replacement. Complications with these relatively new procedures are being reported. We report a case of transapical transcatheter aortic valve implantation, in which a pseudoaneurysm at the apex of the left ventricle as a complication of the procedure developed in the patient and was treated without surgery. The defect spontaneously closed.

Mitroaortic valve replacement after aortic transapical approach failure in a patient with essential thrombocytosis.

There is a lack of published information about intraoperative and postoperative course of cardiac surgery in patients with essential thrombocytosis using cardiopulmonary bypass. Both risks of intraoperative thrombosis of extracorporeal conduits or uncontrolled postoperative bleeding are present, but its incidence and treatment are not well known. Here, we present a rare case of a patient with essential thrombocytosis, moderate mitral regurgitation and severe aortic stenosis who had a transapical aortic valve implantation with short-term severe periprosthetic regurgitation, who needed a mitroaortic replacement on cardiopulmonary bypass with no complications.

Video-assisted cardioscopy for removal of primary left ventricular fibroma.

We present a case of a cardiac fibroma affecting the base of the anterior papillary muscle resected under cardiopulmonary bypass with cardioscopy and video-assisted thoracic surgery (VATS) instruments through the mitral valve. The surgical approach and instrumentation of previous case reports are reviewed.

Aortic valve endocarditis by Capnocytophaga haemolytica.

We report a case of a 51-year-old patient with suspected prosthetic valve endocarditis. Capnocytophaga haemolytica was isolated in blood cultures and the repeated echocardiograms showed growth of vegetations and disruption of the proximal prosthetic valve suture line with progressive aortic regurgitation. The patient underwent a root debridement and replacement with a stentless bioprosthesis.

[Cutaneous myiasis over tumor-lesions: presentation of three cases]. / Miasis cutáneas sobre lesiones tumorales: presentación de tres casos.

We report three cases of myiasis in patients with tumor-lesions. The first patient, a 54-year-old male, presented with laryngeal carcinoma with extensive local involvement, forming a large tumorous mass on the front of the neck that had been irradiated, where several active larvae were seen. The other two patients, females aged 101 and 87 years, respectively, presented with skin tumors on the scalp and face, and several active larvae could be seen in these tumors. After microbiological examination of the larvae removed, the species in the first case was identified as Chrysomya, while the species in the other two cases was Sarcophaga. Several risk factors for developing myiasis have been described, such as advanced age, poor hygiene, diabetes mellitus, venous insufficiency, etc. These manifestations are self-limited, but in most cases treatment is necessary. This consists of cleaning and mechanical removal of visible larvae, with or without occlusion methods.

Miasis cutáneas sobre lesiones tumorales: presentación de tres casos / Cutaneous myiasis over tumor-lesions: presentation of three cases

Comunicamos 3 casos de miasis en pacientes con lesiones tumorales. El primer paciente, de 54 años, presentaba un carcinoma laríngeo con gran afectación local que formaba una gran masa tumoral en la cara anterior del cuello, previamente irradiada, donde se visualizaron varias larvas móviles. Las 2 pacientes restantes, de 101 y 87 años, respectivamente, presentaban tumores cutáneos en cuero cabelludo y cara, y en ellos se objetivaron numerosas larvas móviles. El examen microbiológico de las larvas extraídas identificó en el primer caso la especie Chrysomyia y en los otros dos la especie Sarcophaga. Se han descrito una serie de factores de riesgo de adquirir una miasis, como edad avanzada, higiene deficiente, diabetes mellitus, insuficiencia venosa, etc. Estos cuadros son autolimitados, pero en la mayoría de los casos es necesario realizar tratamiento, que consiste en la limpieza y eliminación mecánica de las larvas visibles, asociado o no a métodos de oclusión

We report three cases of myiasis in patients with tumor-lesions. The first patient, a 54-year-old male, presented with laryngeal carcinoma with extensive local involvement, forming a large tumorous mass on the front of the neck that had been irradiated, where several active larvae were seen. The other two patients, females aged 101 and 87 years, respectively, presented with skin tumors on the scalp and face, and several active larvae could be seen in these tumors. After microbiological examination of the larvae removed, the species in the first case was identified as Chrysomyia, while the species in the other two cases was Sarcophaga. Several risk factors for developing myiasis have been described, such as advanced age, poor hygiene, diabetes mellitus, venous insufficiency, etc. These manifestations are self-limited, but in most cases treatment is necessary. This consists of cleaning and mechanical removal of visible larvae, with or without occlusion methods

Cutis laxa adquirida localizada asociada a traquioniquia / Localized acquired cutis laxa associated with trachyonychia

La cutis laxa adquirida es una rara enfermedad de causa desconocida que afecta al metabolismo de la elastina. Clínicamente se caracteriza por piel redundante e hiperelasticidad, y el estudio histológico muestra reducción o ausencia de fibras elásticas en la dermis. Presentamos un caso de cutis laxa adquirida localizada asociado con traquioniquia

Acquired cutis laxa is a rare disease of unknown cause, which affects elastin metabolism. Clinically, it is characterized by redundant skin and hyperelasticity, while the histological study shows a reduction in or absence of elastic fibers in the dermis. We present a case of localized acquired cutis laxa associated with trachyonychia