Kaposi sarcoma-associated herpesvirus complement control protein (KCP) and glycoprotein K8.1 are not required for viral infection in vitro or in vivo.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE: Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection.

Patient derived tumoroids of high grade neuroendocrine neoplasms for more personalized therapies.

There are no therapeutic predictive biomarkers or representative preclinical models for high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN), a highly aggressive, fatal, and heterogeneous malignancy. We established patient-derived (PD) tumoroids from biobanked tissue samples of advanced high-grade GEP-NEN patients and applied this model for targeted rapid ex vivo pharmacotyping, next-generation sequencing, and perturbational profiling. We used tissue-matched PD tumoroids to profile individual patients, compared ex vivo drug response to patients' clinical response to chemotherapy, and investigated treatment-induced adaptive stress responses.PD tumoroids recapitulated biological key features of high-grade GEP-NEN and mimicked clinical response to cisplatin and temozolomide ex vivo. When we investigated treatment-induced adaptive stress responses in PD tumoroids in silico, we discovered and functionally validated Lysine demethylase 5 A and interferon-beta, which act synergistically in combination with cisplatin. Since ex vivo drug response in PD tumoroids matched clinical patient responses to standard-of-care chemotherapeutics for GEP-NEN, our rapid and functional precision oncology approach could expand personalized therapeutic options for patients with advanced high-grade GEP-NEN.

Dengue and Zika Virus Capsid Proteins Contain a Common PEX19-Binding Motif.

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.

Pulmonary Administration: Strengthening the Value of Therapeutic Proximity.

In recent years inhaled systems have shown momentum as patient-personalized therapies emerge. A significant improvement in terms of therapeutic efficacy and/or reduction adverse systemic effects is anticipated from their use owing these systems regional accumulation. Nevertheless, whatever safety and efficacy evidence required for inhaled formulations regulatory approval, it still poses an additional hurdle to gaining market access. In contrast with the formal intravenous medicines approval, the narrower adoption of pulmonary administration might rely on discrepancies in pre-clinical and clinical data provided by the marketing authorization holder to the regulatory authorities. Evidences of a diverse and inconsistent regulatory framework led to concerns over toxicity issues and respiratory safety. However, an overall trend to support general concepts of good practices exists. Current regulatory guidelines that supports PK/PD (pharmacokinetics/pharmacodynamic) assessment seeks attention threatening those inhaled formulations set to be approved in the coming years. A more complex scenario arises from the attempt of implementing nanomedicines for pulmonary administration. Cutting-edge image techniques could play a key role in supporting diverse stages of clinical development facilitating this pharmaceutics take off and speed to patients. The ongoing challenge in adapting conventional regulatory frameworks has proven to be tremendously difficult in an environment where market entry relies on multiple collections of evidence. This paper intention is to remind us that an acceptable pre-clinical toxicological program could emerge from, but not only, an accurate and robust data imaging collection. It is our conviction that if implemented, inhaled nanomedicines might have impact in multiple severe conditions, such as lung cancer, by fulfilling the opportunity for developing tailored treatments while solving dose-related toxicity issues; the most limiting threat in conventional lung cancer clinical management.

The interplay between Mn and Fe in Deinococcus radiodurans triggers cellular protection during paraquat-induced oxidative stress.

The bacterium Deinococcus radiodurans is highly resistant to several stress conditions, such as radiation. According to several reports, manganese plays a crucial role in stress protection, and a high Mn/Fe ratio is essential in this process. However, mobilization of manganese and iron, and the role of DNA-binding-proteins-under-starved-conditions during oxidative-stress remained open questions. We used synchrotron-based X-ray fluorescence imaging at nano-resolution to follow element-relocalization upon stress, and its dependency on the presence of Dps proteins, using dps knockout mutants. We show that manganese, calcium, and phosphorus are mobilized from rich-element regions that resemble electron-dense granules towards the cytosol and the cellular membrane, in a Dps-dependent way. Moreover, iron delocalizes from the septum region to the cytoplasm affecting cell division, specifically in the septum formation. These mechanisms are orchestrated by Dps1 and Dps2, which play a crucial role in metal homeostasis, and are associated with the D. radiodurans tolerance against reactive oxygen species.

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.

Thermoactivation of a cellobiohydrolase.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

Self-assembly PEGylation assists SLN-paclitaxel delivery inducing cancer cell apoptosis upon internalization.

In past years, a considerable progress has been made in the conversion of conventional chemotherapy into potent and safe nanomedicines. The ultimate goal is to improve the therapeutic window of current chemotherapeutics by reducing systemic toxicities and to deliver higher concentrations of the chemotherapeutic agents to malignant cells. In this work, we report that PEGylation of the nanocarriers increases drug intracellular bioavailability leading therefore to higher therapeutic efficacy. The surface of the already patented solid lipid nanoparticles (SLN) loaded with paclitaxel (SLN-PTX) was coated with a PEG layer (SLN-PTX_PEG) through an innovative process to provide stable and highly effective nanoparticles complying with the predefined pharmaceutical quality target product profile. We observed that PEGylation not only stabilizes the SLN, but also modulates their cellular uptake kinetics. As a consequence, the intracellular concentration of chemotherapeutics delivered by SLN-PTX_PEG increases. This leads to the increase of efficacy and thus it is expected to significantly circumvent cancer cell resistance and increase patient survival and cure.

Implications of Akt2/Twist crosstalk on breast cancer metastatic outcome.

Akt2 is a pivotal player in a complex web of signaling pathways controlling cell growth, proliferation, and survival. The deregulation or aberrations of Akt2 have been associated with tumor progression, metastatic spread, and, lastly, chemoresistance. The impairment of its activity has gained more attention because Akt2 is intertwined with a range of signaling paths, including the Phosphatidylinositol 3 kinase/Akt/Mammalian target of rapamycin (PI3K/mTOR) signaling axis, which are involved in macromolecules synthesis and metabolism. Here, we focus on Akt2 because of its involvement in the acquisition of stem cell-like properties, responsible for invasiveness and chemoresistance, also promoted by Twist. We also suggest therapeutic strategies targeting Akt2 to overcome the drawbacks of current cancer therapies.

Expression, purification and crystallization of MnSOD from Arabidopsis thaliana.

Manganese superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme. MnSOD plays an important role in plant tolerance to abiotic stress and is a target candidate for increasing stress tolerance in crop plants. Although the structure and kinetic parameters of MnSODs from several organisms have been determined, this information is still lacking for plant MnSODs. Here, recombinant MnSOD from Arabidopsis thaliana (AtMnSOD) was expressed, purified and crystallized. A nearly complete data set could only be obtained when a total rotation range of 180° was imposed during data collection, despite the seemingly tetragonal metric of the AtMnSOD crystal diffraction. The data set extended to 1.95 Å resolution and the crystal belonged to space group P1. Molecular-replacement calculations using an ensemble of homologous SOD structures as a search model gave a unique and unambiguous solution corresponding to eight molecules in the asymmetric unit. Structural and kinetic analysis of AtMnSOD is currently being undertaken.

Molecular mobility, composition and structure analysis in glycerol plasticised chitosan films.

This study was developed with the purpose to investigate the effect of polysaccharide/plasticiser concentration on the microstructure and molecular dynamics of polymeric film systems, using transmission electron microscope imaging (TEM) and nuclear magnetic resonance (NMR) techniques. Experiments were carried out in chitosan/glycerol films prepared with solutions of different composition. The films obtained after drying and equilibration were characterised in terms of composition, thickness and water activity. Results show that glycerol quantities used in film forming solutions were responsible for films composition; while polymer/total plasticiser ratio in the solution determined the thickness (and thus structure) of the films. These results were confirmed by TEM. NMR allowed understanding the films molecular rearrangement. Two different behaviours for the two components analysed, water and glycerol were observed: the first is predominantly moving free in the matrix, while glycerol is mainly bounded to the chitosan chain.

Experimental design towards an optimal lipid nanosystem: a new opportunity for paclitaxel-based therapeutics.

Lipid based nanoparticles represent a class of nanocarriers that have caused great expectation, particularly due to their suitability to incorporate BCS class II and IV drugs. The use of solid lipid nanoparticles (SLNs) as a nanocarrier for antineoplastic agents has been underexplored when compared to the encapsulation of the same agents in polymeric particles. The preparation and efficacy assessment of a SLN platform as drug delivery carrier for anticancer agents, herein proposed as a strategy to find innovative formulations, could dramatically improve the outcome of cancer therapy. Considering these lipid nanoparticles, despite the great amount of insights described in the literature, it seems that improving their manufacturability could be the missing step to convert this system into a drug product. A way to circumvent that problem would be to select a preparation method that could take advantage of the pharmaceutical industry installed capabilities, thus speeding-up the scale-up translational steps while maintaining both regulatory compliance and flexibility. The High Pressure Homogenization (HPH) has proved to be a reliable process for SLN preparation. However, the use of the high-shear mixer, a well established process to manufacture coarse dispersions at industrial scale, has still not been fully explored to prepare SLN. In this study, we explore the possibility of using the hot emulsification/solidification method to prepare SLN's that complies with the current pharmaceutical quality requirements. Thus, a high-shear based process that consistently accomplishes performance requirements was optimized in order to standardize the nanocarrier production following the identification of some process and formulation critical parameters. A hydrophobic drug, Paclitaxel (Ptx) was successfully incorporated using the proposed developed method. The particles physicochemical characteristics changes caused by the drug entrapment as well as the particles stability were also evaluated. In addition the ability of SLN to travel across biological barriers due to its matrix lipid nature was explored upon comparing the efficacy of the drug loaded SLN with the conventional marketed drug product (Taxol®). The cellular uptake studies showed that the developed Ptx loaded SLN were in fact internalized and demonstrated higher efficacy in the cancer cells death process than Taxol. The experimental data demonstrated that the hot homogenization technique using a high-shear mechanical homogenizer allows the preparation of suitable size (around 150 nm) SLN. Overall, the results obtained can be particularly impactful in the forthcoming SLN research.

Antioxidant potential of two red seaweeds from the Brazilian coasts.

In this work, in vitro antioxidant activity of two Brazilian red seaweeds, Gracilaria birdiae and Gracilaria cornea, was characterized. The total phenolic content, the radical-scavenging activity and the antioxidant activity were determined in two solvent extracts of the algae. Liquid chromatography-mass spectrometry (LC-MS/MS) allowed identification of important antioxidant compounds. The ethanol extract of G. birdiae was found to have the highest value of total phenolic content: 1.13 mg of gallic acid equiv (GAE)/g of extract. The radical-scavenging activity of G. birdiae and G. cornea extracts has been evaluated at different extract concentrations; the IC(50) values of ethanolic extracts of G. cornea and G. birdiae were 0.77 and 0.76 mg mL(-1), respectively, while for methanolic extracts, the IC(50) values of G. cornea and G. birdiae were 0.86 and 0.76 mg mL(-1), respectively. The antioxidant activities of these two seaweeds' extracts as assessed by the ß-carotene-linoleic acid assay were equally high, achieving values of ß-carotene oxidation inhibition of up to 40%. Finally, in the methanolic extracts, LC-MS/MS allowed identification in both algae of two important antioxidants: apigenin and gallic acid.

Utilização de medicamentos e risco de interações medicamentosas em idosos atendidos pelo Programa de Atenção ao Idoso da Unijuí / Use of medications and risk of drug interactions in the elderly population at the Elderly Care Program in Unijuí

Idosos utilizam elevado número de medicamentos, apresentando alto potencial para desenvolver interações medicamentosas. Objetivou-se verificar o perfil de utilização de medicamentos e conhecer possíveis interações medicamentosas em idosos acompanhados pelo Programa de Atenção ao Idoso (PAI) da Unijuí. Os dados foram coletados em julho de 2009. Classificaramse os medicamentos no sistema Anatomical Therapeutic Chemical e possibilidades de interações segundo Drug Interaction Facts. De março/2008 até julho/2009 o PAI atendeu 31 idosos, estando 16 em acompanhamento, os quais apresentaram média de 78,4 ± 6,8 anos e utilizaram 83 medicamentos, com média de 5,2 ± 3,7/ idoso. Os fármacos mais prevalentes foram os que atuam no aparelho cardiovascular, no sistema nervoso e para o trato alimentar e metabolismo. Verificaram-se 36 possíveis interações entre 9 pacientes, com média de 4/idoso. O fármaco mais envolvido foi digoxina. Quanto a classificação, 4 apresentaram nível de significância 1, dez nível 2, cinco nível 3, sete nível 4 e dez nível 5. O número de possíveis interações verificadas foi expressivo, entretanto nem todos os idosos apresentarão reações relacionadas às interações, mas apresentaram o risco. Portanto, é importante que prescritores conheçam a influência de um fármaco sobre outro. Seria necessário treinamento intensivo para intervenção farmacêutica, sendo o relacionamento com o médico indispensável.

Elderly people take a large number of medicines, entailing a high risk of developing drug interactions. The aim was to study the profile of medicines prescribed and the possible drug interactions in the public Elderly Care Program (PAI) run at Unijuí, a university in south Brazil. The data were collected in July 2009. The Anatomical Therapeutic Chemical (ATC) Classification System was used to classify medicines, while the potential interactions were classified with the aid of Drug Interaction Facts. Between March 2008 and July 2008, the PAI attended 31 elderly patients, of whom 16 were monitored, with a mean age of 78.4 ± 6.8 years. This group received 83 medicines, making an average of 5.2 ± 3.7 medicines/patient. The most prevalent drugs were those acting on the cardiovascular system, nervous system, digestive tract and metabolism. There were 36 possible interactions in 9 patients, an average of 4 interactions per patient. The drug most frequently involved was digoxin. The interactions were classified as follows: 4 at level 1, 10 at level 2, 5 at level 3, 7 at level 4 and 10 at level 5. This number of possible interactions is considerable; although not all elderly patients show the effects of the drug interactions, they do run the risk. Therefore, it is highly important that prescribers are familiar with the drug interactions. For the pharmacist to help the patient, intensive training for pharmaceutical intervention and a good relationship with the doctor are indispensable.

The peopling of Madeira Archipelago (Portugal) according to HLA genes.

The Madeira-Porto Santo Archipelago was officially colonized in 1420 by Portuguese settlers. Its importance in Columbus' information for the American discovery and for slave traffic across the Atlantic is unquestionable. Thus, a complex peopling may have given rise to a present-day high admixture of ethnicities according to HLA genes. A sample of 173 healthy unrelated Madeirans was analysed and compared with 6986 HLA chromosomes from other worldwide populations. Genetic distances, neighbour-joining dendrograms and correspondence analyses were used for comparisons. Southern European, North African (including Canary Islands), Jewish and Mediterranean typical HLA alleles were found and genetic distances from Madeirans to these populations were the closest ones. In addition A*24-B*65-DRB1*0102-DQB1*0501 and A*68-B*08-DRB1*0301-DQB1*0201 haplotypes were newly found in Madeira and not found in any other population. Jewish-Armenian-Middle East haplotype (A*33-B*65-DRB1*0102-DQB1*0501) is one of the most common haplotypes; this haplotype is also present in Spaniards and North Africans. Quantitatively, Portuguese, North Africans (Algerians), Spaniards and Canary Islanders (in this order) are the most important parental populations to Madeirans. Results are discussed on the basis of the recorded historical peopling which does not show a noticeable African gene input in present-day Madeiran population according to our data; one of the closest related populations found is the Canary Islanders, suggesting that Guanche (Canary Islands first inhabitants) slaves gene flow is still noticed at present, both in Madeira and in Canary Islands populations.

Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles.

UNLABELLED: Lymphatic drainage plays an important role in the uptake of particulates in the respiratory system, being also associated to the spreading of lung cancer through metastasis development. In recent years solid lipid nanoparticles (SLN) have been proposed as carriers of anti-tumoural drugs, for their low toxicity and surface characteristics make them suitable for either imaging (gamma-scintigraphy) or therapy upon encapsulation of cytotoxic drugs. Assessment of inhaled radiolabelled SLN biodistribution is described in the present work. METHODS: Nanoparticles (200 nm) were radiolabelled with 99mTc using the lipophilic chelator D,L-hexamehylpropyleneamine oxime (HMPAO). Biodistribution studies were carried out following aerosolisation and administration of a 99mTc-HMPAO-SLN suspension to a group of adult male Wistar rats. A 60 min dynamic image acquisition was performed in a gamma-camera, followed by static image collection at 30 min intervals up to 4 h postinhalation. Radiation counting was performed in organ samples, collected after the animals were sacrificed. RESULTS: The data show an important and significant uptake of the radiolabelled SLN into the lymphatics after inhalation, and a high rate of distribution in periaortic, axillar and inguinal lymph nodes. CONCLUSION: Results indicate that SLN could be effective colloidal carriers for lymphoscintigraphy or therapy upon pulmonary delivery.