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1.
Science ; 367(6484): 1372-1376, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193327

RESUMO

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.


Assuntos
Compostos de Anilina/química , Ascorbato Peroxidases/genética , Engenharia Genética , Neurônios/fisiologia , Nitrocompostos/química , Fenilenodiaminas/química , Polímeros/química , Potenciais de Ação , Animais , Ascorbato Peroxidases/metabolismo , Caenorhabditis elegans , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Condutividade Elétrica , Células HEK293 , Hipocampo , Humanos , Potenciais da Membrana , Camundongos , Neurônios Motores/fisiologia , Células Musculares/fisiologia , Neurônios/enzimologia , Técnicas de Patch-Clamp , Polímeros/metabolismo , Ratos , Transdução Genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-31451496

RESUMO

Recurrent vulvovaginal candidiasis (RVVC) is a widespread chronic infection that has a substantial negative impact on work and quality of life. The development of antimicrobial resistance and biofilm formation are speculated to contribute to Candida pathogenicity and treatment ineffectiveness. Designed antimicrobial peptides (dAMPs) are chemically modified from endogenous antimicrobial peptides that provide the first line of defense against pathogens. The goal here is to identify a dAMP for the topical treatment of RVVC. The dAMP MICs were determined for 46 fluconazole-susceptible and fluconazole-resistant Candida spp. clinical isolates. The possibility of inducing dAMP drug resistance and comparison of dAMP and fluconazole activity against preformed Candida biofilm and biofilm formation were evaluated. Assessment of mammalian cell viability was determined using bioluminescent human keratinocytes. The dAMP effect on fungus was probed via scanning electron microscopy, and topically applied dAMP activity was evaluated in a rodent vulvovaginal candidiasis (VVC) infection model. dAMPs demonstrated broad-spectrum antimicrobial activity against common causative clinical Candida isolates, reduced preformed biofilm, and inhibited biofilm formation. An evaluated dAMP did not induce resistance after repeated exposure of Candida tropicalis The dAMPs were selective for Candida cells with limited mammalian cytotoxicity with substantial activity in a rodent VVC model. dAMPs are described as having potent antifungal and antibiofilm activity, likely direct membrane action with selectivity for Candida cells, with limited resistance development. Combined with activity in a rodent VVC model, the data support clinical evaluation of dAMPs for topical treatment of VCC and recurrent VVC infections.

3.
ACS Nano ; 13(7): 7985-7995, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31259527

RESUMO

By their nature, tumors pose a set of profound challenges to the immune system with respect to cellular recognition and response coordination. Recent research indicates that leukocyte subpopulations, especially tumor-associated macrophages (TAMs), can exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. To better study and understand the roles of TAMs in determining immunotherapeutic outcomes, significant technical challenges associated with dynamically monitoring single cells of interest in relevant live animal models of solid tumors must be overcome. However, imaging techniques with the requisite combination of spatiotemporal resolution, cell-specific contrast, and sufficient signal-to-noise at increasing depths in tissue are exceedingly limited. Here we describe a method to enable high-resolution, wide-field, longitudinal imaging of TAMs based on speckle-modulating optical coherence tomography (SM-OCT) and spectral scattering from an optimized contrast agent. The approach's improvements to OCT detection sensitivity and noise reduction enabled high-resolution OCT-based observation of individual cells of a specific host lineage in live animals. We found that large gold nanorods (LGNRs) that exhibit a narrow-band, enhanced scattering cross-section can selectively label TAMs and activate microglia in an in vivo orthotopic murine model of glioblastoma multiforme. We demonstrated near real-time tracking of the migration of cells within these myeloid subpopulations. The intrinsic spatiotemporal resolution, imaging depth, and contrast sensitivity reported herein may facilitate detailed studies of the fundamental behaviors of TAMs and other leukocytes at the single-cell level in vivo, including intratumoral distribution heterogeneity and roles in modulating cancer proliferation.

4.
Am J Trop Med Hyg ; 101(2): 383-391, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31219005

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evaluated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell metabolism in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.


Assuntos
Amiodarona/farmacologia , Reposicionamento de Medicamentos , Itraconazol/farmacologia , Miócitos Cardíacos/parasitologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , Células-Tronco/parasitologia , Tripanossomicidas/farmacologia , Células Vero
5.
Med Mycol ; 57(Supplement_2): S239-S244, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30816969

RESUMO

The general ability and tendency of bacteria and fungi to assemble into bacterial communities, termed biofilms, poses unique challenges to the treatment of human infections. Fungal biofilms, in particular, are associated with enhanced virulence in vivo and decreased sensitivity to antifungals. Much attention has been given to the complex cell wall structures in fungal organisms, yet beyond the cell surface, Aspergillus fumigatus and other fungi assemble a self-secreted extracellular matrix that is the hallmark of the biofilm lifestyle, protecting and changing the environment of resident members. Elucidation of the chemical and molecular detail of the extracellular matrix is crucial to understanding how its structure contributes to persistence and antifungal resistance in the host. We present a summary of integrated analyses of A. fumigatus biofilm architecture, including hyphae and the extracellular matrix, by scanning electron microscopy and A. fumigatus matrix composition by new top-down solid-state NMR approaches coupled with biochemical analysis. This combined methodology will be invaluable in formulating quantitative and chemical comparisons of A. fumigatus isolates that differ in virulence and are more or less resistant to antifungals. Ultimately, knowledge of the chemical and molecular requirements for matrix formation and function will drive the identification and development of new strategies to interfere with biofilm formation and virulence.


Assuntos
Aspergillus fumigatus/química , Aspergillus fumigatus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Aspergillus fumigatus/ultraestrutura , Matriz Extracelular/química , Hifas/química , Hifas/crescimento & desenvolvimento , Hifas/ultraestrutura
6.
Nat Nanotechnol ; 14(5): 420-425, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833691

RESUMO

Electron microscopy has been instrumental in our understanding of complex biological systems. Although electron microscopy reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An electron-microscopy-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesize small lanthanide-doped nanoparticles and measure the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nanolabels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colours, so the imaging of biomolecules in a multicolour electron microscopy modality may be possible.


Assuntos
Corantes Fluorescentes/química , Microscopia Eletrônica de Transmissão , Nanopartículas/química
7.
J Ethnopharmacol ; 235: 122-132, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30738119

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Limitations of clinical antifungal treatments and drug-resistance are drivers of the search for novel antifungal strategies. Extracts prepared from the tubers of the medicinal plant, Pelargonium sidoides, are known for their antiviral and antibacterial activities and are used in ethnomedicine for the treatment of acute respiratory infections. Their impact on fungi has not been well characterised. Here, we provide a first report on the antifungal activity of a P. sidoides aerial tissue extract against Cryptococcus neoformans as well as the effects of both tuber and aerial tissue extracts on selected virulence factors. AIM OF THE STUDY: Novel antimicrobial strategies that target multiple cellular pathways or make use of anti-pathogenic compounds that inhibit virulence factors have been proposed. This work aimed to evaluate P. sidoides plant parts for their anticryptococcal activity and antipathogenic properties on selected virulence factors. MATERIALS AND METHODS: The antifungal activity of crude P. sidoides tuber and aerial tissue extracts (15% m/m ethanol) were compared using a modified colourimetric antifungal susceptibility test. Fungicidal activity of the extracts was confirmed by plate counts. To test yeast resistance to the extracts, it was conditioned by multiple passages in sub-lethal doses followed by antifungal susceptibility testing. Cytotoxicity of the extracts was tested with a blood agar haemolysis assay. Extracts were evaluated for the presence of multiple bioactive compounds by solid-phase fractionation and visualisation by thin-layer chromatography in combination with bioassays. The influence of extracts on the production of the polysaccharide capsule, ergosterol content as well as laccase and urease activities were also evaluated. Cell surface variations after extract exposure were visualised by scanning electron microscopy (SEM). RESULTS: Both tuber and aerial tissue extracts were fungicidal and contained multiple bioactive compounds which constrained the development of antifungal resistance. No haemolytic activity was observed, and the extracts did not appear to target ergosterol biosynthesis. However, the extracts displayed anti-pathogenic potential by significantly inhibiting laccase and urease activity while also significantly reducing capsule size. SEM revealed notable cell surface variations and provided support for the observed reduction in capsule size. CONCLUSIONS: Our results provide support to the exploration of medicinal plants as sources of alternative antifungal therapies and the potential use of multicomponent inhibition and or virulence attenuation for next-generation treatment strategies. Our data also provide relevant information that may support the further use of P. sidoides in traditional medicines as well as in commercialised phytopharmaceuticals.


Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Pelargonium/química , Extratos Vegetais/farmacologia , Animais , Antifúngicos/isolamento & purificação , Antifúngicos/toxicidade , Cromatografia em Camada Delgada , Cryptococcus neoformans/patogenicidade , Hemólise/efeitos dos fármacos , Cavalos , Medicina Tradicional/métodos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Componentes Aéreos da Planta , Extratos Vegetais/toxicidade , Ovinos
8.
Cell Death Discov ; 4: 113, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534421

RESUMO

Glioblastoma is the most common yet most lethal of primary brain cancers with a one-year post-diagnosis survival rate of 65% and a five-year survival rate of barely 5%. Recently the U.S. Food and Drug Administration approved a novel fourth approach (in addition to surgery, radiation therapy, and chemotherapy) to treating glioblastoma; namely, tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but its mechanisms of action are not fully understood. Current theories involve TTFields disrupting mitosis due to interference with proper mitotic spindle assembly. We show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. Increased membrane permeability through the imposition of TTFields was shown by several approaches. For example, increased permeability was indicated through increased bioluminescence with TTFields exposure or with the increased binding and ingress of membrane-associating reagents such as Dextran-FITC or ethidium D or with the demonstration by scanning electron microscopy of augmented number and sizes of holes on the cellular membrane. Further investigations showed that increases in bioluminescence and membrane hole production with TTFields exposure disappeared by 24 h after cessation of alternating electric fields thus demonstrating that this phenomenom is reversible. Preliminary investigations showed that TTFields did not induce membrane holes in normal human fibroblasts thus suggesting that the phenomenom was specific to cancer cells. With TTFields, we present evidence showing augmented membrane accessibility by compounds such as 5-aminolevulinic acid, a reagent used intraoperatively to delineate tumor from normal tissue in glioblastoma patients. In addition, this mechanism helps to explain previous reports of additive and synergistic effects between TTFields and other chemotherapies. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies for these diseases.

9.
Sci Transl Med ; 10(469)2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487249

RESUMO

Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1α) that preceded adhesion development. Inhibition of HIF1α with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.


Assuntos
Anticorpos/farmacologia , Biomarcadores/metabolismo , Epitélio/patologia , Aderências Teciduais/patologia , Animais , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peritônio/efeitos dos fármacos , Peritônio/lesões , Peritônio/patologia , Aderências Teciduais/genética , Transcrição Genética
10.
Mol Psychiatry ; 23(12): 2302-2313, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30254235

RESUMO

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.


Assuntos
Hipocampo/metabolismo , Memória de Curto Prazo/fisiologia , Dor/metabolismo , Animais , Plasticidade Celular/fisiologia , Cognição , Disfunção Cognitiva/fisiopatologia , Matriz Extracelular/metabolismo , Interneurônios , Potenciação de Longa Duração/fisiologia , Masculino , Metaloproteinase 8 da Matriz/metabolismo , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Plasticidade Neuronal/fisiologia , Lobo Temporal
11.
Nat Photonics ; 12(9): 548-553, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31258619

RESUMO

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising single-molecule probes given their non-blinking, photobleach-resistant luminescence upon infrared excitation. However, the weak luminescence of sub-50 nm UCNPs limits their single-particle detection to above 10 kWcm-2 that is impractical for live cell imaging. Here, we systematically characterize single-particle luminescence for UCNPs with various formulations over a 106 variation in incident power, down to 8 Wcm-2. A core-shell-shell (CSS) structure (NaYF4@NaYb1-xF4:Erx@NaYF4) is shown to be significantly brighter than the commonly used NaY0.78F4:Yb0.2Er0.02. At 8 Wcm-2, the 8% Er3+ CSS particles exhibit a 150-fold enhancement given their high sensitizer Yb3+ content and the presence of an inert shell to prevent energy migration to defects. Moreover, we reveal power-dependent luminescence enhancement from the inert shell, which explains the discrepancy in enhancement factors reported by ensemble and previous single-particle measurements. These brighter probes open the possibility of cellular and single-molecule tracking at low irradiance.

12.
Microbiology ; 163(11): 1568-1577, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28982395

RESUMO

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/virologia , Ferro/metabolismo , Fagos de Pseudomonas/fisiologia , Birrefringência , Candida albicans/fisiologia , Humanos , Interações Microbianas , Modelos Biológicos , Pseudomonas aeruginosa/virologia
13.
Angew Chem Int Ed Engl ; 56(51): 16357-16362, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29080292

RESUMO

Template-free fabrication of non-spherical polymeric nanoparticles is desirable for various applications, but has had limited success owing to thermodynamic favorability of sphere formation. Herein we present a simple way to prepare cubic nanoparticles of block copolymers by self-assembly from aqueous solutions at room temperature. Nanocubes with edges of 40-200 nm are formed spontaneously on different surfaces upon water evaporation from micellar solutions of triblock copolymers containing a central poly(ethylene oxide) block and terminal trimethylene carbonate/dithiolane blocks. These polymers self-assemble into 28±5 nm micelles in water. Upon drying, micelle aggregation and a kinetically controlled crystallization of central blocks evidently induce solid cubic particle formation. An approach for preserving the structures of these cubes in water by thiol- or photo-induced crosslinking was developed. The ability to solubilize a model hydrophobic drug, curcumin, was also explored.

14.
Science ; 358(6362): 506-510, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-29074771

RESUMO

Whereas standard transmission electron microscopy studies are unable to preserve the native state of chemically reactive and beam-sensitive battery materials after operation, such materials remain pristine at cryogenic conditions. It is then possible to atomically resolve individual lithium metal atoms and their interface with the solid electrolyte interphase (SEI). We observe that dendrites in carbonate-based electrolytes grow along the <111> (preferred), <110>, or <211> directions as faceted, single-crystalline nanowires. These growth directions can change at kinks with no observable crystallographic defect. Furthermore, we reveal distinct SEI nanostructures formed in different electrolytes.

15.
ACS Nano ; 11(8): 8320-8328, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28682058

RESUMO

The interface between cells and nonbiological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influence cellular responses; for example, titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compared to a smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-material interface at the relevant nanometer length scale. Here, we present a method for in situ examination of the cell-to-material interface at any desired location, based on focused ion beam milling and scanning electron microscopy imaging to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary by more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future.

16.
Plast Reconstr Surg ; 139(6): 1305e-1314e, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28538572

RESUMO

BACKGROUND: Decellularized cadaveric tendons are a potential source for reconstruction. Reseeding to enhance healing is ideal; however, cells placed on the tendon surface result in inadequate delivery. The authors used an injection technique to evaluate intratendinous cell delivery. METHODS: Decellularized tendons were reseeded with adipose-derived stem cells in culture, and injected with fetal bovine serum or hydrogel. PKH26-stained cells in cross-section were quantified. To evaluate cell viability, the authors delivered luciferase-labeled cells and performed bioluminescent imaging. To evaluate synthetic ability, the authors performed immunohistochemistry of procollagen. Adipose-derived stem cells' ability to attract tenocytes was assessed using transwell inserts. Cell-to-cell interaction was assessed by co-culturing, measuring proliferation and collagen production, and quantifying synergy. Finally, tensile strength was tested. RESULTS: Both fetal bovine serum (p < 0.001) and hydrogel (p < 0.001) injection led to more cells inside the tendon compared with culturing. Hydrogel injection initially demonstrated greater bioluminescence than culturing (p < 0.005) and fetal bovine serum injection (p < 0.05). Injection groups demonstrated intratendinous procollagen staining correlating with the cells' location. Co-culture led to greater tenocyte migration (p < 0.05). Interaction index of proliferation and collagen production assays were greater than 1 for all co-culture ratios, demonstrating synergistic proliferation and collagen production compared with controls (p < 0.05). There were no differences in tensile strength. CONCLUSIONS: Hydrogel injection demonstrated the greatest intratendinous seeding efficiency and consistency, without compromising tensile strength. Intratendinous cells demonstrated synthetic capabilities and can potentially attract tenocytes inside the tendon, where synergy would promote intrinsic tendon healing. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.


Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Traumatismos dos Tendões/cirurgia , Tendões/efeitos dos fármacos , Tendões/transplante , Engenharia Tecidual/métodos , Adipócitos/citologia , Animais , Bovinos , Sobrevivência Celular , Técnicas de Cocultura , Humanos , Injeções Intralesionais , Ratos , Procedimentos Cirúrgicos Reconstrutivos/métodos , Sensibilidade e Especificidade , Células-Tronco/citologia , Tendões/citologia , Resistência à Tração , Tecidos Suporte
17.
J Microbiol Methods ; 132: 46-55, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27836634

RESUMO

Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components. Conventional and Variable Pressure Scanning Electron Microscopy were applied to analyze the structure of biofilms attached to plastic and formed at an air-liquid interface.


Assuntos
Aspergillus fumigatus/ultraestrutura , Biofilmes , Microscopia Eletrônica de Varredura , Fibrose Cística/microbiologia , Resistência Microbiana a Medicamentos , Matriz Extracelular/ultraestrutura , Interações Hospedeiro-Patógeno/imunologia , Humanos , Hifas/ultraestrutura , Pressão
18.
Nat Cell Biol ; 18(12): 1311-1323, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27842057

RESUMO

The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, 'cadherin fingers', which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular , Polaridade Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Fagocitose , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actomiosina/metabolismo , Cateninas/metabolismo , Contagem de Células , Membrana Celular/metabolismo , Células HEK293 , Humanos , Imageamento Tridimensional , Junções Intercelulares/metabolismo , Mitose , Modelos Biológicos , Optogenética , Polimerização , Pseudópodes/metabolismo
19.
Microbiology ; 162(9): 1583-1594, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27473221

RESUMO

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.


Assuntos
Aspergillus fumigatus/fisiologia , Bacteriófagos/fisiologia , Ferro/metabolismo , Pseudomonas aeruginosa/virologia , Aspergilose/microbiologia , Aspergillus fumigatus/virologia , Biofilmes , Humanos
20.
Cytotherapy ; 18(4): 510-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26971679

RESUMO

BACKGROUND: Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction. METHODS: Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified. RESULTS: Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%). CONCLUSIONS: Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.


Assuntos
Transdiferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Masculino , Músculo Liso/citologia , Músculo Liso/fisiologia , Ratos , Ratos Wistar
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