Neuroprotective potential of Ayahuasca and untargeted metabolomics analyses: applicability to Parkinson's disease.

ETHNOPHARMACOLOGY RELEVANCE: Ayahuasca is a tea produced through decoction of Amazonian plants. It has been used for centuries by indigenous people of South America. The beverage is considered to be an ethnomedicine, and it is traditionally used for the treatment of a wide range of diseases, including neurological illness. Besides, some scientific evidence suggests it may be applicable to Parkinson's disease (PD) treatment. Thus, Ayahuasca deserves in depth studies to clarify its potential role in this disease. AIM OF THE STUDY: This study aimed to use an untargeted metabolomics approach to evaluate the neuroprotective potential of the Ayahuasca beverage, the extracts from its matrix plants (Banisteriopsis caapi and Psychotria viridis), its fractions and its main alkaloids on the viability of SH-SY5Y neuroblastoma cells in an in vitro PD model. MATERIAL AND METHODS: The cytotoxicity of Ayahuasca, crude extracts, and fractions of B. caapi and P. viridis, as well as neuroprotection promoted by these samples in a 6-hydroxydopamine (6-OHDA)-induced neurodegeneration model, were evaluated by the MTT assay at two time-points: 48 h (T1) and 72 h (T2). The main alkaloids from Ayahuasca matrix plants, harmine (HRE) and N,N-dimethyltryptamine (DMT), were also isolated and evaluated. An untargeted metabolomics approach was developed to explore the chemical composition of samples with neuroprotective activity. Ultra-Performance Liquid Chromatography coupled to Electrospray Ionisation and Time-of-Flight (UPLC-ESI-TOF) metabolome data was treated and further analysed using multivariate statistical analyses (MSA): principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The metabolites were dereplicated using the Dictionary of Natural Products and an in house database. The main alkaloids were also quantified by UPLC-MS/MS. RESULTS: The samples did not cause cytotoxicity in vitro and three of samples intensely increased cell viability at T1. The crude extracts, alkaloid fractions and HRE demonstrated remarkable neuroprotective effect at T2 while the hydroalcoholic fractions demonstrated this neuroprotective effect at T1 and T2. Several compounds from different classes, such as ß-carbolines and monoterpene indole alkaloids (MIAs) were revealed correlated with this property by MSA. Additionally, a total of 2419 compounds were detected in both ionisation modes. HRE showed potent neuroprotective action at 72 h, but it was not among the metabolites positively correlated with the most efficacious neuroprotective profile at either time (T1 and T2). Furthermore, DMT was statistically important to differentiate the dataset (VIP value > 1), although it did not exhibit sufficient neuroprotective activity by in vitro assay, neither a positive correlation with T1 and T2 neuroprotective profile, which corroborated the MSA results. CONCLUSION: The lower doses of the active samples stimulated neuronal cell proliferation and/or displayed the most efficacious neuroprotection profile, namely by preventing neuronal damage and improving cell viability against 6-OHDA-induced toxicity. Intriguingly, the hydroalcoholic fractions exhibited enhanced neuroprotective effects when compared to other samples and isolated alkaloids. This finding corroborates the significance of a holistic approach. The results demonstrate that Ayahuasca and its base plants have potential applicability for PD treatment and to prevent its progression differently from current drugs to treat PD.

Improved vascularisation but inefficient in vivo bone regeneration of adipose stem cells and poly-3-hydroxybutyrate-co-3-hydroxyvalerate scaffolds in xeno-free conditions.

Bone defects are a common clinical situation. However, bone regeneration remains a challenge and faces the limitation of poor engraftment due to deficient vascularisation. Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and human adipose stem cells (hASC) are promising for vascularisation and bone regeneration. Therefore, we sought to investigate the bone regenerative capacity of hASCs cultured in allogeneic human serum (aHS) and PHB-HV scaffolds in a nude mouse model of the critical-sized calvarial defect. We evaluated bone healing for three treatment groups: empty (control), PHB-HV and PHB-HV + hASCs. The pre-implant analysis showed that hASCs colonised the PHB-HV scaffolds maintaining cell viability before implantation. Histological analysis revealed that PHB-HV scaffolds were tolerated in vivo; they integrated with adjacent tissue eliciting a response like a foreign body reaction, and tiny primary bone was observed only in the PHB-HV group. Also, the µ-CT analysis revealed only approximately 10% of new bone in the bone defect area in both the PHB-HV and PHB-HV + hASCs groups. The expression of BGLAP and its protein (osteocalcin) by PHB-HV + hASCs group and native bone was similar while the other bone markers RUNX2, ALPL and COL1A1 were upregulated, but this expression remained significantly lower compared to the native bone. Nevertheless, the PHB-HV group showed neovascularisation at 12 weeks post-implantation while PHB-HV + hASCs group also exhibited higher VEGFA expression as well as a higher number of vessels at 4 weeks post-implantation, and, consequently, earlier neovascularisation. This neovascularisation must be due to scaffold architecture, improved by hASCs, that survived for the long term in vivo in the PHB-HV + hASCs group. These results demonstrated that hASCs cultured in aHS combined with PHB-HV scaffolds were ineffective to promote bone regeneration, although the construct of hASCs + PHB-HV in xeno-free conditions improved scaffold vascularisation representing a strategy potentially promising for other tissue engineering applications.

Nanocellulose/bioactive glass cryogels as scaffolds for bone regeneration.

A major challenge exists in the preparation of scaffolds for bone regeneration, namely, achieving simultaneously bioactivity, biocompatibility, mechanical performance and simple manufacturing. Here, cellulose nanofibrils (CNF) are introduced for the preparation of scaffolds taking advantage of their biocompatibility and ability to form strong 3D porous networks from aqueous suspensions. CNF are made bioactive for bone formation through a simple and scalable strategy that achieves highly interconnected 3D networks. The resultant materials optimally combine morphological and mechanical features and facilitate hydroxyapatite formation while releasing essential ions for in vivo bone repair. The porosity and roughness of the scaffolds favor several cell functions while the ions act in the expression of genes associated with cell differentiation. Ion release is found critical to enhance the production of the bone morphogenetic protein 2 (BMP-2) from cells within the fractured area, thus accelerating the in vivo bone repair. Systemic biocompatibility indicates no negative effects on vital organs such as the liver and kidneys. The results pave the way towards a facile preparation of advanced, high performance CNF-based scaffolds for bone tissue engineering.

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis.

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.

Biochemical analysis and identification of linear B-cell epitopes from recombinant Sm21.7 antigen from Schistosoma mansoni.

Schistosoma mansoni tegument is a dynamic host-interactive layer that is an essential source of parasite antigens and a relevant field for schistosome vaccine research. Sm21.7 is a cytoskeleton antigen found in S. mansoni tegument that engenders protection in experimental challenge infection. Because of its crucial role in the parasite tegument and its promising protective capability, Sm21.7 is an exciting target for the development of therapeutic strategies. The present study describes Sm21.7 structural and biophysical features using circular dichroism spectroscopy and identifies linear B-cell epitopes of Sm21.7 using in-silico methods and immunoassay. The Sm21.7 gene was cloned into the pETDEST42 vector, and the recombinant protein was overexpressed in Escherichia coli DE3. The soluble protein was purified by affinity chromatography followed by ion-exchange chromatography. Purified recombinant Sm21.7 was analyzed by circular dichroism spectroscopy which demonstrated that the rSm21.7 structure was comprised of approximately 38% α-helices and its conformation remains stable at temperatures of up to 60 °C. Prediction of rSm21.7 B-cell epitopes was based on amino acid physicochemical properties. Sixteen peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by spot peptide array using pooled rSm21.7-immunized mice sera or patients' sera with different clinical forms of S. mansoni infection. Immunoassays revealed that sera from rSm21.7-immunized mice reacted predominantly with peptides located in the dynein-light chain domain (DLC) at the C-terminal region of rSm21.7. Comparative analysis of the antibody response of acute, intestinal and hepatosplenic patients' sera to the Sm21.7 peptides showed that a differential recognition pattern of Sm21.7-derived peptides by intestinal patients' sera might contribute to down-regulate the immune response in chronic intestinal patients. Together, the results may help the development of S. mansoni vaccine strategies based on the rSm21.7 antigen.

Dose-dependent effect of triiodothyronine on the chondrogenic differentiation of mesenchymal stem cells from the bone marrow of female rats.

OBJECTIVES: Verify the in-vitro effect of triiodothyronine (T3) on the chondrogenic differentiation of female rat bone marrow mesenchymal stem cells (BMMSCs) over several time periods and at several doses. METHODS: CD54 + /CD73 + /CD90 +  BMMSCs from Wistar female rats were cultured in chondrogenic medium with or without T3 (0.01; 1; 100; 1000 nm). At seven, 14 and 21 days, the cell morphology, chondrogenic matrix formation and expression of Sox9 and collagen II were evaluated. KEY FINDINGS: The dose of 100 nm did not alter the parameters evaluated in any of the periods studied. However, the 0.01 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and expression of Sox9 and collagen II in at least one of the evaluated periods; the 1 nm T3 dose also improved the chondrogenic potential by increasing the chondrogenic matrix formation and the expression of collagen II in at least one of the evaluated periods. The 1000 nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and Sox9 expression in at least one of the evaluated periods. CONCLUSIONS: T3 has a dose-dependent effect on the differentiation of BMMSCs from female rats.

Upregulation of p38 pathway accelerates proliferation and migration of MDA-MB-231 breast cancer cells.

Tumor cells capture the signaling pathways used by normal tissue to promote their own survival and dissemination and among them, the NF-κB and MAPK pathways (ERK, JNK and p38). MAPK activation has ambiguous effects on tumor cell fate depending on cell type, cancer stage and the engaged MAPK isoforms. A synthetic peptide named LyeTx II, derived from the venom of the Brazilian spider Lycosa erythrognatha, was capable of increasing MDA-MB-231 aggressive breast cancer cell proliferation as indicated by MTT and BrdU (5-bromo-2'-deoxyuridine) incorporation assay and cell migration. A correlation has been established between the accelerated proliferation and migration observed in the presence of LyeTx II and the upregulation of p38 MAPK phosphorylation. The use of the selective inhibitor of p38α/ß (SB203580) abrogated the peptide effect in MDA-MB-231 cells. Besides, an augment of the canonical NF-κB pathway activation considered as crucial in cancer progression was noted after cell incubation with LyeTx II. Importantly, activation of p38 and NF-κB pathways was dependent on TAK1 activity. Together, these data suggest that TAK1-p38 pathway may represent an interesting target for treatment of aggressive breast cancers.

Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. OBJECTIVE: The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. METHODS: To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. RESULTS: The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. CONCLUSION: The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, ß-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and ß-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and ß-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and ß-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

Estudo comparativo da diferenciação osteogênica das células tronco mesenquimais da medula óssea e do tecido adiposo de cães adultos / Comparative study of the osteogenic differentiation of mesenchymal stem cells from bone marrow and adipose tissue of adult dogs

O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)

The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(AU)

Inner nuclear membrane localization of epidermal growth factor receptor (EGFR) in spontaneous canine model of invasive micropapillary carcinoma of the mammary gland.

The epidermal growth factor receptor (EGFR) has been described in the nucleus of primary tumors. Accumulation of EGFR at the nucleus is linked to DNA synthesis and cell proliferation, but the pathological significance of nuclear EGFR is not completely understood. The aim of this study was to investigate the nuclear localization of EGFR in invasive micropapillary carcinoma (IMPC) that is an aggressive neoplasm of canine mammary gland. Confocal immunofluorescence of formalin and paraffin-embedded tissue was used to access the subcellular localization of EGFR. Our results demonstrated that EGFR co-localizes with the inner nuclear envelope marker, Lamin B1 in IMPC. Furthermore, EGFR was not localized within the nucleus or at the inner nuclear envelope membrane in mammary carcinoma in mixed tumor (CMT) that is associated with a better prognosis than other malignant histological types. This finding could be useful as a predictive biomarker of therapeutic response for IMPC.

Anti-Toxoplasma activity and impact evaluation of lyophilization, hot molding process, and gamma-irradiation techniques on CLH-PLGA intravitreal implants.

Intraocular delivery systems have been developed to treat many eye diseases, especially those affecting the posterior segment of the eye. However, ocular toxoplasmosis, the leading cause of infectious posterior uveitis in the world, still lacks an effective treatment. Therefore, our group developed an intravitreal polymeric implant to release clindamycin, a potent anti-Toxoplasma antibiotic. In this work, we used different techniques such as differential scanning calorimetry, thermogravimetry, X-ray diffraction, scanning electron microscopy, and fourier-transform infrared spectroscopy to investigate drug/polymer properties while manufacturing the delivery system. We showed that the lyophilization, hot molding process, and sterilization by gamma irradiation did not change drug/polymer physical-chemistry properties. The drug was found to be homogeneously dispersed into the poly lactic-co-glycolic acid (PLGA) chains and the profile release was characterized by an initial burst followed by prolonged release. The drug profile release was not modified after gamma irradiation and non-covalent interaction was found between the drug and the PLGA. We also observed the preservation of the drug activity by showing the potent anti-Toxoplasma effect of the implant, after 24-72 h in contact with cells infected by the parasite, which highlights this system as an alternative to treat toxoplasmic retinochoroiditis.

Effect of the ionic product of bioglass 60s on osteoblastic activity in canines.

BACKGROUND: The objective of the present study was to evaluate the effect of the ionic product (IP) of BG60S on osteoblastic activity. The following media groups were created: DMEM, which is formed by osteoblasts in basal medium; IP DMEM, which is formed by osteoblasts in IP with basal medium; OST, which is formed by osteoblasts in osteogenic medium; and IP OST, which is formed by osteoblasts in IP with osteogenic medium. The osteoblasts were cultivated in an incubator at 37 °C and 5 % CO2 for 7, 14 and 21 days. After each period, the alkaline phosphatase (AP) activity, mineralised area per field and expression of osterix (OSX), bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC) were evaluated by reverse transcription (RT)-PCR. RESULTS: The IP significantly increased the AP activity in the IP DMEM group at 7 and 14 days and reduced the AP activity in the IP OST group at 14 and 21 days relative to their respective controls (DMEM and OST). The groups that received the IP displayed a significant increase in the percentage of mineralised area per field and more advance maturation of the extracellular matrix relative to those that did not receive IP. The IP significantly increased the expression of OSX, BSP and ON in osteoblast cultures maintained in IP DMEM compared with the control (DMEM) for the majority of studied periods. In osteogenic medium, IP also significantly increased OSX, BSP, ON and OC expression compared with the control (OST) for the majority of studied periods. CONCLUSIONS: The IP of BG60S alters the gene expression of canine osteoblasts, favouring the synthesis and mineralisation of the extracellular matrix.

Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation.

INTRODUCTION: Human adipose tissue-derived stem cells (hASCs) are attractive cells for therapeutic applications and are currently being evaluated in multiple clinical trials. Prior to their clinical application, hASCs must be expanded ex vivo to obtain the required number of cells for transplantation. Fetal bovine serum is the supplement most widely used for cell culture, but it has disadvantages and it is not safe for cell therapy due to the risks of pathogen transmission and immune reaction. Furthermore, the cell expansion poses a risk of accumulating genetic abnormalities that could lead to malignant cell transformation. In this study, our aim was to evaluate the proliferation pattern as well as the resistance to spontaneous transformation of hASCs during expansion in a xeno-free culture condition. METHODS: hASCs were expanded in Dulbecco's modified Eagle's medium supplemented with pooled allogeneic human serum or fetal bovine serum to enable a side-by-side comparison. Cell viability and differentiation capacity toward the mesenchymal lineages were assessed, along with immunophenotype. Ki-67 expression and the proliferation kinetics were investigated. The expression of the transcription factors c-FOS and c-MYC was examined with Western blot, and MYC, CDKN2A, ERBB2 and TERT gene expression was assessed with quantitative PCR. Senescence was evaluated by ß-gal staining. Karyotype analysis was performed and tumorigenesis assay in vivo was also evaluated. RESULTS: The hASCs expanded in medium with pooled allogeneic human serum did not show remarkable differences in morphology, viability, differentiation capacity or immunophenotype. The main difference observed was a significantly higher proliferative effect on hASCs cultured in pooled allogeneic human serum. There was no significant difference in C-FOS expression; however, C-MYC protein expression was enhanced in pooled allogeneic human serum cultures compared to fetal bovine serum cultures. No difference was observed in MYC and TERT mRNA levels. Moreover, the hASCs presented normal karyotype undergoing senescence, and did not form in vivo tumors, eliminating the possibility that spontaneous immortalization of hASCs had occurred with pooled allogeneic human serum. CONCLUSIONS: This complete characterization of hASCs cultivated in pooled allogeneic human serum, a suitable xeno-free approach, shows that pooled allogeneic human serum provides a high proliferation rate, which can be attributed for the first time to C-MYC protein expression, and showed cell stability for safe clinical applications in compliance with good manufacturing practice.

Polyhydroxybutyrate-co-hydroxyvalerate structures loaded with adipose stem cells promote skin healing with reduced scarring.

Currently available skin substitutes are still associated with a range of problems including poor engraftment resulting from deficient vascularization, and excessive scar formation, among others. Trying to overcome these issues, this work proposes the combination of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) structures with adipose-derived stem cells (ASCs) to offer biomechanical and biochemical signaling cues necessary to improve wound healing in a full-thickness model. PHBV scaffold maintained the wound moisture and demonstrated enough mechanical properties to withstand wound contraction. Also, exudate and inflammatory cell infiltration enhanced the degradation of the structure, and thus healing progression. After 28 days all the wounds were closed and the PHBV scaffold was completely degraded. The transplanted ASCs were detected in the wound area only at day 7, correlating with an up-regulation of VEGF and bFGF at this time point that consequently led to a significant higher vessel density in the group that received the PHBV loaded with ASCs. Subsequently, the dermis formed in the presence of the PHBV loaded with ASCs possesses a more complex collagen structure. Additionally, an anti-scarring effect was observed in the presence of the PHBV scaffold indicated by a down-regulation of TGF-ß1 and α-SMA together with an increase of TGF-ß3, when associated with ASCs. These results indicate that although PHBV scaffold was able to guide the wound healing process with reduced scarring, the presence of ASCs was crucial to enhance vascularization and provide a better quality neo-skin. Therefore, we can conclude that PHBV loaded with ASCs possesses the necessary bioactive cues to improve wound healing with reduced scarring.

Dragging human mesenchymal stem cells with the aid of supramolecular assemblies of single-walled carbon nanotubes, molecular magnets, and peptides in a magnetic field.

Human adipose-derived stem cells (hASCs) are an attractive cell source for therapeutic applicability in diverse fields for the repair and regeneration of damaged or malfunctioning tissues and organs. There is a growing number of cell therapies using stem cells due to their characteristics of modulation of immune system and reduction of acute rejection. So a challenge in stem cells therapy is the delivery of cells to the organ of interest, a specific site. The aim of this paper was to investigate the effects of a supramolecular assembly composed of single-walled carbon nanotubes (SWCNT), molecular magnets (lawsone-Co-phenanthroline), and a synthetic peptide (FWYANHYWFHNAFWYANHYWFHNA) in the hASCs cultures. The hASCs were isolated, characterized, expanded, and cultured with the SWCNT supramolecular assembly (SWCNT-MA). The assembly developed did not impair the cell characteristics, viability, or proliferation. During growth, the cells were strongly attached to the assembly and they could be dragged by an applied magnetic field of less than 0.3 T. These assemblies were narrower than their related allotropic forms, that is, multiwalled carbon nanotubes, and they could therefore be used to guide cells through thin blood capillaries within the human body. This strategy seems to be useful as noninvasive and nontoxic stem cells delivery/guidance and tracking during cell therapy.

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization.

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.

BACKGROUND: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach. CONCLUSIONS/SIGNIFICANCE: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Comparison of the osteogenic potential of mesenchymal stem cells from the bone marrow and adipose tissue of young dogs.

BACKGROUND: The aim of the present study was to compare the osteogenic potential of mesenchymal stem cells extracted from the bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) of young dogs. The following parameters were assessed: dimethyl thiazolyl diphenyl tetrazolium (MTT) conversion, alkaline phosphatase (ALP) activity, collagen and mineralised matrix synthesis, and the expressions of osterix, bone sialoprotein (BSP), and osteocalcin (OC). RESULTS: MTT conversion was greater in BM-MSCs compared to AD-MSCs after 14 and 21 days of differentiation; ALP activity was greater in differentiated AD-MSCs on day 7; collagen synthesis was greater in BM-MSCs on days 14 and 21; the percentage of mineralized area per field was greater in BM-MSCs compared to AD-MSCs; osterix expression was greater in BM-MSCs in days 14 and 21, and BSP and OC expression levels were greater in BM-MSCs at all the investigation time-points. CONCLUSIONS: It was concluded that the osteogenic potential was greater in BM-MSCs than AD-MSCs when extracted from young dogs.

Synaptic plasticity and sensory-motor improvement following fibrin sealant dorsal root reimplantation and mononuclear cell therapy.

Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6-8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy.