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Zika virus (ZIKV) and dengue virus (DENV) share a lot of similarities being both phylogenetically closely related, share the same insect vector passage for reaching the host, affinity for the same carbohydrate receptor domains (CRDs), indicating feasible competition between them on the natural field. Here, we prospected interactions of both envelope proteins with a DC-SIGN, a transmembrane c-type lectine receptor with the most implicated CRD with the Flavivirus infection presents on dendritic cells involved in viruses replication processes into the host, and among rares CRD receptors susceptible to interacting with a broad of subtypes of DENV. Protein-protein docking procedures produced structures for molecular dynamics experiments, suggesting the most energetically favorable complex. The difference found in the deltaG results prompted the experimentation with molecular dynamics. To investigate further specific residues involved with such interactions we produced a decomposition analysis using molecular dynamics of the docked proteins evaluated afterward with the Generalized Born Surface Area method. Solvent-accessible surface area (SASA) analysis for both showed very similar but with a slight reduction for ZIKV_E, which agreed with residues SASA analysis highlighting regions more exposed in the ZIVK protein than in DENV. Despite residues PHE313 is reponsible for most of the interactions with the envelope of these arboviruses, ZIKV interacted with this residue in DC-SIGN with lower energies and using more interactions with not expexted residues GLU241 and ARG386. Taken together these results suggest better competitive interaction of ZIKV with the DC-SIGN receptor, particularly in the CRD portion.

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Introduction: Three of the main requirements that remain major challenges in tissue engineering of the knee meniscus are to engineer scaffolds with compatible anatomical shape, good mechanical properties, and microstructure able to mimic the architecture of the extracellular matrix (ECM). In this context, we presented a new biofabrication strategy to develop a three-dimensional (3D) meniscus-regenerative scaffold with custom-made macroscopic size and microarchitecture bioinspired by the organization of structural fibers of native tissue ECM. Methods: The concept was based on the combination of bioprinted cell-laden hydrogel (type 1 collagen) reinforced by multilayers of biomimetically aligned electrospun nanofibrous mats (polycaprolactone/carbon nanotubes, PCL/CNT), using a patient-specific 3D digital meniscus model reconstructed from MRI data by free and open-source software. Results: The results showed that the incorporation of aligned nanofibers sheets between the hydrogel layers enhanced the scaffold's structural integrity and shape fidelity compared to the nanofiber-free collagen hydrogel. Furthermore, mechanical compression tests demonstrated that the presence of nanofiber layers significantly improved the mechanical properties of the bioprinted construct. Importantly, the introduction of PCL/CNT nanofibrous mats between the layers of the bioprinted collagen hydrogel did not negatively affect cell viability, in which mesenchymal stem cells remained viable even after 7 days of culture within the scaffold. Conclusion: Overall, these findings evidence that this bioengineering approach offers a promising strategy for fabricating biomimetic meniscus scaffolds for tissue engineering.

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Clinically, bone tissue replacements and/or bone repair are challenging. Strategies based on well-defined combinations of osteoconductive materials and osteogenic cells are promising to improve bone regeneration but still require improvement. Herein, we combined polycaprolactone (PCL) fibers, carbon nanotubes (CNT), and hydroxyapatite (nHap) nanoparticles to develop the next generation of bone regeneration material. Fibers formed by rotary jet spinning (RJS) instead of traditional electrospinning (ES) with embedded bone marrow mesenchymal stem cells (BMMSCs) showed the best outcomes to repair rat calvarial defects after 6 weeks. To understand this, it was observed that different morphologies were formed depending on the manufacturing method used. RJS fibers presented a particular topography with rough fibers, which allowed for better cellular growth and cell spreading in vitro around and into a three-dimensional (3D) mesh, while fibers made by ES were more smooth and cellular growth was only measured on the 3D mesh surface. The fibers with incorporated nHap/CNT nanoparticles enhanced in vitro cell performance as indicated by more cellular proliferation, alkaline phosphatase activity, proliferation, and deposition of calcium. Greater bone neoformation occurred by combining three characteristics: the presence of nHap and CNT nanoparticles, the topography of the RJS fibers, and the addition of BMMSCs. RJS fibers with nanoparticles and seeded with BMMSCs showed 10 136 mm3 of bone neoformation, meaning a 10-fold increase compared to using RJS only and BMMSCs (0.853 mm3) and a 5-fold increase from using ES only (2054 mm3) after 6 weeks of implantation. Conversely, none of these approaches used individually showed any significant difference for in vivo bone neoformation, suggesting that their combination is essential for optimizing bone formation. In summary, our work generated a potential material composed of well-defined combinations of suitable scaffolds seeded with BMMSCs for enhancing numerous orthopedic tissue engineering applications.

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Currently, oxygen supply for in vitro cell culture is one of the major challenges in tissue engineering, especially in three-dimensional (3D) structures, such as polymeric hydrogels, because oxygen is an essential element for cells survival. In this context, oxygen levels must be maintained in articular cartilage to promote the differentiation, viability, and proliferation of chondrocytes due to the low level of oxygen presence in this region. Although some technologies employ oxygen-generating materials to add sufficient oxygen levels, the limitations and challenges of current technologies include the lack of controlled, sustained, and prolonged release of the oxygen. Moreover, the fabrication methods may leave some impurities or residues resulting in toxicity to the cells. "Click" chemistry is a facile, versatile, and compatible chemical strategy to engineer hydrogels for tissue engineering applications. Herein, we disclose the engineering of oxygen-generating microparticles in chondrocytes-laden hydrogels through a versatile catalyst-free tetrazine and norbornene inverse electron demand Diels‒Alder (iEDDA) click reaction. The hydrogels combine chondroitin sulfate (CS) and poly(ethylene glycol) (PEG) crosslinked in situ, displaying tunable rheological and mechanical properties, for sustained and prolonged oxygen-release. Gene expression analysis of the chondrocytes by real-time reverse transcription polymerase chain reaction (RT-PCR) demonstrated promising cell response within the engineered hydrogel.

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Cartilage is one of our body's tissues which are not repaired automatically by itself. Problems associated with cartilage are very common worldwide and are considered the leading cause of pain and disability. Smart biomaterial or "Four dimensional" (4D) biomaterials has started emerging as a suitable candidate, which are principally three dimensional (3D) materials that change their morphology or generate a response measured at space and time to physiologic stimuli. In this context, the release of oxygen through hydrogels in contact with water is considered as 4D biomaterials. The objective of this study is to develop strategies to release oxygen in a sustainable and prolonged manner through hydrogels systems to promote chondrocytes survival in oxygen-free environment. The 4D biomaterials are engineered from gelatin methacryloyl (GelMA) loaded with calcium peroxide (CPO), which have the ability to generate oxygen in a controlled and sustained manner for up to 6 days. The incorporation of CPO into the hydrogel system provided materials with enhanced mechanical and porosity properties. Furthermore, the hydrogels promoted chondrocyte survival and reduced cell death under oxygen-free conditions.

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BACKGROUND: The facile preparation of oxygen-generating microparticles (M) consisting of Polycaprolactone (PCL), Pluronic F-127, and calcium peroxide (CPO) (PCL-F-CPO-M) fabricated through an electrospraying process is disclosed. The biological study confirmed the positive impact from the oxygen-generating microparticles on the cell growth with high viability. The presented technology could work as a prominent tool for various tissue engineering and biomedical applications. METHODS: The oxygen-generated microparticles fabricated through electrospraying processes were thoroughly characterization through various methods such as X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) analysis, and scanning electron microscopy (SEM)/SEM-Energy Dispersive Spectroscopy (EDS) analysis. RESULTS: The analyses confirmed the presence of the various components and the porous structure of the microparticles. Spherical shape with spongy characteristic microparticles were obtained with negative charge surface (ζ = -16.9) and a size of 17.00 ± 0.34 µm. Furthermore, the biological study performed on rat chondrocytes demonstrated good cell viability and the positive impact of increasing the amount of CPO in the PCL-F-CPO-M. CONCLUSION: This technological platform could work as an important tool for tissue engineering due to the ability of the microparticles to release oxygen in a sustained manner for up to 7 days with high cell viability.

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The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

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Murine norovirus (MNV) is a single-stranded positive-sense RNA virus of the Caliciviridae family. MNV has been reported to infect laboratory mice with the ability to cause lethal infections in strains lacking components of the innate immune response. Currently, MNV is considered the most prevalent infectious agent detected in laboratory mouse facilities. In this study, mice in 22 laboratory animal facilities within Brazil were analyzed for MNV infection. Using primers targeting a conserved region of the viral capsid, MNV was detected by RT-PCR in 137 of 359 mice from all 22 facilities. Nucleotide sequencing and phylogenetic analysis of the capsid region from the viral genome showed identity ranging from 87% to 99% when compared to reported MNV sequences. In addition, RAW264.7 cells inoculated with a mouse fecal suspension displayed cytopathic effect after the fifth passage. This study represents the first report of MNV in mouse colonies in Brazilian laboratory animal facilities, emphasizing the relevance of a health surveillance program in such environments.

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Protein restriction in the early stages of life can result in several changes in pancreatic function. These alterations include documented reductions in insulin secretion and in cytoplasmic calcium concentration [Ca(2+)]i. However, the mechanisms underlying these changes have not been completely elucidated and may result, in part, from alterations in signaling pathways that potentiate insulin secretion in the presence of glucose. Our findings suggest that protein restriction disrupts the insulin secretory synergism between Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca(2+)-dependent protein kinase C (PKC) in isolated islets. Western blot analysis demonstrated reduced levels of both phospho-cAMP response element-binding protein (phospho-CREB) at Ser-133 and substrates phosphorylated by PKCs (Phospho-(Ser) PKC substrate), suggesting that PKA and PKC activity was impaired in islets from rats fed a low-protein diet (LP). cAMP levels and global Ca(2+) entry were also reduced in LP islets. In summary, our findings showed that protein restriction altered the crosstalk between PKA and PKC signaling pathways, resulting in the alteration of secretory synergism in isolated islets.

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An association between in vitro and in vivo studies has been demonstrated for the first time, using a novel nanohydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube (nHAp/VAMWCNT-O2) nanocomposites. Human osteoblast cell culture and bone defects were used to evaluate the in vitro extracellular matrix (ECM) calcification process and bone regeneration, respectively. The in vitro ECM calcification process of nHAp/VAMWCNT-O2 nanocomposites were investigated using alkaline phosphatase assay. The in vivo biomineralization studies were carried out on bone defects of C57BL/6/JUnib mice. Scanning electron microscopy, micro-energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and X-ray difractometry analyses confirmed the presence of the nHAp crystals. nHAp/VAMWCNT-O2 nanocomposites induced in vitro calcification of the ECM of human osteoblast cells in culture after only 24 h. Bone regeneration with lamellar bone formation after 9 weeks was found in the in vivo studies. Our findings make these new nanocomposites very attractive for application in bone tissue regeneration.

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A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is presented. The formation of a thin homogeneous film with high crystallinity was observed without any thermal treatment and with bioactivity properties that accelerate the in vitro biomineralization process and osteoblast adhesion.

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As seqüelas neurológicas da cinomose canina são consideradas irreversíveis e têm sido tratadas sintomaticamente, quando não há indicação de eutanásia, o que ocorre na maioria dos casos de seqüelas graves. Visando desenvolver uma nova abordagem terapêutica, testamos a injeção de células mononucleares de medula óssea alogeneicas extraídas de 5 cães doadores saudáveis, em 11 cães com seqüelas neurológicas com pré-diagnóstico de cinomose, sendo 7 com manifestações clínicas recentes e 4 com sinais clínicos crônicos. Dos 7 animais com seqüelas agudas ou recentes, 5 apresentaram remissão completa dos sinais clínicos e 2 melhora parcial e momentânea. Dos animais com sinais crônicos, 3 apresentaram melhora visível na primeira semana após o transplante, contudo, 2 deles, após curto período de estabilidade, apresentaram novamente os mesmos sinais clínicos vistos antes do transplante. Este protocolo mostrou-se bastante promissor para o tratamento de seqüelas neurológicas de cinomose canina

The neurologic sequels of canine distemper are currently considered irreversible, and are usually treated with palliative therapies when euthanasia is not recommended, what happens in the majority of severe cases. Aiming to develop a new therapeutic approach, we tested the systemic injection of allogeneic bone marrow mononuclear cells extracted from 5 healthy dogs, in 11 dogs with severe neurologic sequels with distemper previous diagnosis. 7 dogs had recent clinical manifestations and 4 had chronic signs. In the group of 7 animals with acute or recent sequels, 5 had complete remission of clinical signs and 2 had partial recovery. In the group with chronic sequels, 3 had visible recovery in the first week after transplantation, but 2 of them, after a short period of stability, presented again the same clinical signs seen before the transplantation. This protocol has demonstrated to be very promising for the treatment of neurologic sequels of canine distemper

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As seqüelas neurológicas da cinomose canina são consideradas irreversíveis e têm sido tratadas sintomaticamente, quando não há indicação de eutanásia, o que ocorre na maioria dos casos de seqüelas graves. Visando desenvolver uma nova abordagem terapêutica, testamos a injeção de células mononucleares de medula óssea alogeneicas extraídas de 5 cães doadores saudáveis, em 11 cães com seqüelas neurológicas com pré-diagnóstico de cinomose, sendo 7 com manifestações clínicas recentes e 4 com sinais clínicos crônicos. Dos 7 animais com seqüelas agudas ou recentes, 5 apresentaram remissão completa dos sinais clínicos e 2 melhora parcial e momentânea. Dos animais com sinais crônicos, 3 apresentaram melhora visível na primeira semana após o transplante, contudo, 2 deles, após curto período de estabilidade, apresentaram novamente os mesmos sinais clínicos vistos antes do transplante. Este protocolo mostrou-se bastante promissor para o tratamento de seqüelas neurológicas de cinomose canina(AU)

The neurologic sequels of canine distemper are currently considered irreversible, and are usually treated with palliative therapies when euthanasia is not recommended, what happens in the majority of severe cases. Aiming to develop a new therapeutic approach, we tested the systemic injection of allogeneic bone marrow mononuclear cells extracted from 5 healthy dogs, in 11 dogs with severe neurologic sequels with distemper previous diagnosis. 7 dogs had recent clinical manifestations and 4 had chronic signs. In the group of 7 animals with acute or recent sequels, 5 had complete remission of clinical signs and 2 had partial recovery. In the group with chronic sequels, 3 had visible recovery in the first week after transplantation, but 2 of them, after a short period of stability, presented again the same clinical signs seen before the transplantation. This protocol has demonstrated to be very promising for the treatment of neurologic sequels of canine distemper(AU)

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Hereditary persistence of fetal hemoglobin (HPFH) is characterized by increased levels of Hb F during adult life. Nondeletional forms of HPFH are characterized by single base mutations in the (A)gamma and (G)gamma promoters, resulting in an increase of Hb F ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the (A)gamma -195 (C>G) mutation that causes the Brazilian type of HPFH (HPFH-B). To better understand this mechanism, we have developed HPFH-B transgenic mice. mRNA levels of human gamma-globin of -195 transgenic mice were clearly higher when compared with control transgenic mice bearing a wild type sequence of the gamma promoter. Thus, our data indicate that the -195 mutation is the unique cause of elevation of Hb F in Brazilian HPFH. These results could provide us with an opportunity to study the modifying effects of the Hb F in the phenotype of sickle cell disease and beta-thalassemia (beta-thal).

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INTRODUCTION: Priapism is defined as prolonged and persistent penile erection, unassociated with sexual interest or stimulation, and is one of the many serious complications associated with sickle cell disease (SCD). AIM: The aim of this study was to evaluate the role of the NO-cGMP signaling pathway in priapism in Berkeley murine model of SCD (SS). METHODS: SS mice and C57BL/6 mice (control) penile tissues were removed and the erectile tissue within the corpus cavernosum (CC) was surgically dissected free. The strips were mounted in 10 mL organ baths containing Krebs solution at 37 degrees C (95% O(2), 5% CO(2), pH 7.4), and vertically suspended between two metal hooks. MAIN OUTCOME MEASURES: Cumulative concentration-response curves were constructed for acetylcholine (ACh; endothelium-dependent responses), sodium nitroprusside (SNP; endothelium-independent relaxations) and BAY 41-2272 (a potent activator of NO-independent site of soluble guanylate cyclase) in CC precontracted with phenylephrine. Cavernosal responses induced by frequency-dependent electrical field stimulation (EFS) were also carried out to evaluate the nitrergic cavernosal relaxations. RESULTS: In SS mice, ACh-induced cavernosal relaxations were leftward shifted by 2.6-fold (P < 0.01) that was accompanied by increases in the maximal responses (78 +/- 5% and 60 +/- 3% in SS and C57B6/6J mice, respectively). Similarly, SNP- and BAY 41-2272-induced CC relaxations were leftward shifted by approximately 3.3- and 2.2-fold (P < 0.01) in SS mice, respectively. A significant increase in maximal responses to SNP and BAY 41-2272 in SS mice was also observed (113 +/- 6% and 124 +/- 5%, respectively) compared with C57B6/6J mice (83 +/- 4% and 99 +/- 2%, respectively). The EFS-induced cavernosal relaxations were also significantly higher SS mice. CONCLUSION: These results showed that SS mice exhibit amplified corpus carvenosum relaxation response mediated by NO-cGMP signaling pathway. Intervention in this signaling pathway may be a potential therapeutic target to treat SCD priapism.

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OBJECTIVE: We have previously reported that myocyte enhancer factor-2 (MEF2) transcription factors and c-Jun are rapidly activated by pressure overload and that these events are involved in the early activation of the myocardial hypertrophic genetic program. In this study, we investigated whether focal adhesion kinase (FAK) mediates the activation of MEF2 and c-Jun by mechanical stress in isolated neonatal rat ventricular myocytes (NRVMs). METHODS: NRVMs were subjected to cyclic stretch up to 4 h and studied by immunoblotting, reverse transcriptase-polymerase chain reaction, laser confocal analysis, and reporter gene and electrophoretic mobility shift assays. Analysis was extended to NRVMs transfected with FAK-antisense oligodeoxynucleotide, treated with FAK/Src inhibitor PP2 or JNK/c-Jun inhibitor SP600125. RESULTS: Cyclic stretch increased c-Jun expression, JNK/c-Jun phosphorylation, and MEF2-DNA binding activity in NRVMs. Reporter gene assays indicated that the MEF2 site is critical to c-jun transcription in stretched cells. FAK-antisense transfection abolished MEF2 and c-jun promoter activation, while either FAK-antisense or PP2 treatment inhibited the stretch-induced c-Jun expression and JNK/c-Jun phosphorylation. Finally, treatment of NRVMs with the specific JNK/c-Jun inhibitor SP600125 significantly reduced the stretch-induced increase of atrial natriuretic factor promoter activity. CONCLUSION: The present data indicate that FAK regulates the activation of MEF2 and JNK/c-Jun pathways, which in turn have a key role in the early activation of the hypertrophic genetic program by mechanical stress in cardiac myocytes.

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This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C(2)C(12) myoblasts into myotubes. Differentiation of C(2)C(12) myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to approximately 50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, whereas the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well-organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild-type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum-starved C(2)C(12) cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C(2)C(12) cells to terminal differentiation. Transient inhibition of FAK phosphorylation at Tyr-397 contributes to trigger the myogenic genetic program, but its later activation is also central to terminal differentiation into myotubes.

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