RESUMEN
Diabetes mellitus is a complicated metabolic disease that has become one of the fastest-growing health crises in modern society. Diabetic patients may suffer from various complications, and diabetic foot is one of them. It can lead to increased rates of lower-extremity amputation and mortality, even seriously threatening the life and health of patients. Because its healing process is affected by various factors, its management and treatment are very challenging. To address these problems, smart biomaterials have been developed to expedite diabetic wound closure and improve treatment outcomes. This review begins with a discussion of the basic mechanisms of wound recovery and the limitations of current dressings used for diabetic wound healing. Then, the categories and characteristics of the smart biomaterial scaffolds, which can be utilized as a delivery system for drugs with anti-inflammatory activity, bioactive agency, and antibacterial nanoparticles for diabetic wound treatment were described. In addition, it can act as a responsive system to the stimulus of the pH, reactive oxygen species, and glucose concentration from the wound microenvironment. These results show that smart biomaterials have an enormous perspective for the treatment of diabetic wounds in all stages of healing. Finally, the advantages of the construction of smart biomaterials are summarized, and possible new strategies for the clinical management of diabetic wounds are proposed.
Asunto(s)
Diabetes Mellitus , Pie Diabético , Humanos , Materiales Biocompatibles/uso terapéutico , Pie Diabético/tratamiento farmacológico , Cicatrización de Heridas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Vendajes , Diabetes Mellitus/terapiaRESUMEN
The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor-targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor-mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non-targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide-dependent indicating integrin-mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN-amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor-targeting, with minimal clearance by the liver and so enable delivery of tumor-targeted siRNA therapeutics.
RESUMEN
Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.
Asunto(s)
Activación de Complemento , Nanopartículas , PolietilenglicolesRESUMEN
Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its ß-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of ß-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100â¯mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Ingeniería Metabólica/métodos , Polihidroxialcanoatos/metabolismo , Polímeros/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos Volátiles/metabolismo , Técnicas de Inactivación de Genes , Peso Molecular , Oxidación-Reducción , Plásmidos/genéticaRESUMEN
With increasing demand of environmentally friendly materials, development on biobased polymers such as polyhydroxyalkanoate (PHA) is indispensable. An unsaturated PHA, namely, poly(3-hydroxydodecanoate- co-3-hydroxy-9-decenoate), short as P(3HDD- co-3H9D), provides possibilities for functionalization. Two different strategies are explored for synthesis of PHA- graft-graphene nanocomposites with graphene content ranging from 0.2 to 1.5 wt %. Chemical structures of intermediates and products were confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Uniform dispersion of graphene was observed in formed PHA nanocomposites under scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). PHA- graft-graphene nanocomposites exhibited higher thermal degradation temperature and enhanced electricity conductivity compared with that of neat PHA. Moreover, lower critical filling content and lower electrical resistivity at same graphene content demonstrated enhanced electrical conductivity of PHA- graft-graphene nanocomposites compared with previously reported blends. The lowest electrical resistivity was 2 Ω·m in sample PHA- graft-graphene nanocomposites with approximately 1.5 wt % graphene content.
Asunto(s)
Conductividad Eléctrica , Grafito/química , Nanocompuestos/química , Polihidroxialcanoatos/química , Nanocompuestos/ultraestructuraRESUMEN
The published online version contains mistake in the funding information. Instead of 30370096, it should have been 31370096.
RESUMEN
A thermoresponsive graft copolymer polyhydroxyalkanoate-g-poly(N-isopropylacrylamide) or short as PHA-g-PNIPAm, was successfully synthesized via a three-step reaction. First, PNIPAm oligomer with a trithiocarbonate-based chain transfer agent (CTA), short as PNIPAm-CTA, with designed polymerization degree was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Subsequently, the PNIPAm-CTA was treated with n-butylamine for aminolysis in order to obtain a pendant thiol group at the end of the chain (PNIPAm-SH). Finally, the PNIPAm-SH was grafted onto unsaturated P(3HDD-co-3H10U), a random copolymer of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-10-undecylenate (3H10U), via a thiol-ene click reaction. Enhanced hydrophilicity and thermoresponsive property of the resulted PHA-g-PNIPAm were confirmed by water contact angle studies. The biocompatibility of PHA-g-PNIPAm was comparable to poly-3-hydroxybutyrate (PHB). The graft copolymer PHA-g-PNIPAm based on biopolyester PHA could be a promising material for biomedical applications.
Asunto(s)
Resinas Acrílicas/química , Ensayo de Materiales , Polihidroxialcanoatos/química , Polímeros/química , Agua/química , Modelos Químicos , Conformación Molecular , Polimerizacion , Temperatura , TermodinámicaRESUMEN
Novel poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) copolymers produced by haloarchaea are excellent candidate biomaterials. However, there is no report hitherto focusing on the biodegradation of PHBHV synthesized by haloarchaea. In this study, an environmental biodegradation of haloarchaea-produced PHBHV films, with 10~60 mol% 3-hydroxyvalerate (3HV) composition and different microchemical structures, was studied in nutrition-depleted activated sludge. The changes in mass, molar mass, chemical composition, thermal properties, and surface morphology were monitored. The mass and molar mass of each film decreased significantly, while the PHA monomer composition remained unchanged with time. Interestingly, the sample of random copolymer PHBHV-2 (R-PHBHV-2) (3HV, 30 mol%) had the lowest crystallinity and was degraded faster than R-PHBHV-3 containing the highest 3HV content or the higher-order copolymer PHBHV-1 (O-PHBHV-1) possessing the highest surface roughness. The order of biodegradation rate was in the opposite trend to the degree of crystallizability of the films. Meanwhile, thermal degradation temperature of most films decreased after biodegradation. Additionally, the surface erosion of films was confirmed by scanning electron microscopy. The dominant bacteria probably responsible for the degradation process were identified in the activated sludge. It was inferred that the degradation rate of haloarchaea-produced PHBHV films mainly depended on sample crystallinity, which was determined by monomer composition and microchemical structure and in turn strongly influenced surface morphology.
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Archaea/metabolismo , Biodegradación Ambiental , Ácidos Pentanoicos/metabolismo , Polímeros/metabolismo , Aguas del Alcantarillado/microbiología , Cristalización , Microscopía Electrónica de Rastreo , Plásticos/metabolismo , Poliésteres/metabolismoRESUMEN
Poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial polyhydroxyalkanoates (PHA) synthesized by bacteria grown on 1,3-propandiol or glycerol. In this study synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli was constructed harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyzes formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pdup) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP. When phaA and phaB from Ralstonia eutropha respectively encoding ß-ketothiolase and acetoacetate reductase, were introduced into the above P3HP producing recombinant E. coli, copolymers poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) were synthesized from glucose as a sole carbon source. The above E. coli recombinants grown on glucose LB medium successfully produced 5g/L cell dry weight containing 18% P3HP and 42% P(3HB-co-84mol% 3HP), respectively, in 48h shake flask studies.
Asunto(s)
Proteínas Bacterianas , Cupriavidus necator/genética , Escherichia coli , Glucosa/metabolismo , Hidroxibutiratos/metabolismo , Ingeniería Metabólica , Poliésteres/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cupriavidus necator/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Dendrimers are three-dimensional macromolecular structures originating from a central core molecule and surrounded by successive addition of branching layers (generation). These structures exhibit a high degree of molecular uniformity, narrow molecular weight distribution, tunable size and shape characteristics, as well as multivalency. Collectively, these physicochemical characteristics together with advancements in design of biodegradable backbones have conferred many applications to dendrimers in formulation science and nanopharmaceutical developments. These have included the use of dendrimers as pro-drugs and vehicles for solubilization, encapsulation, complexation, delivery, and site-specific targeting of small-molecule drugs, biopharmaceuticals, and contrast agents. We briefly review these advances, paying particular attention to attributes that make dendrimers versatile for drug formulation as well as challenging issues surrounding the future development of dendrimer-based medicines.
Asunto(s)
Dendrímeros/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Transfección , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacocinética , Antiinfecciosos/farmacología , Antiinfecciosos/toxicidad , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Dendrímeros/farmacocinética , Dendrímeros/farmacología , Dendrímeros/toxicidad , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/toxicidad , Sistemas de Liberación de Medicamentos/métodos , Humanos , Ingeniería de Tejidos/métodos , Transfección/métodos , Virosis/tratamiento farmacológico , Virus/efectos de los fármacosRESUMEN
Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Polilisina/síntesis química , Polilisina/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Medicare Part A , Metabolómica , Peso Molecular , Fosforilación Oxidativa/efectos de los fármacos , Polilisina/química , Estados UnidosRESUMEN
We report the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymers (R-PHBV) or higher-order copolymers (O-PHBV) in Haloferax mediterranei, with adjustable 3-hydroxyvalerate (3HV) incorporation by cofeeding valerate with glucose. Their microchemical structure, molecular weight and its distribution, and thermal and mechanical properties were characterized by NMR, GPC, DSC, TGA, and universal testing machine, respectively. (13)C NMR studies showed that O-PHBV copolymers consisted of short segments of PHB and PHV covalently linked together with random PHBV segments. Consistently, two Tg were observed in the DSC curves of O-PHBV. The "blocky" feature of O-PHBV enhanced crystallinity percentages and improved Young's modulus. Notably, the film of one O-PHBV copolymer, O-PHBV-1, showed unique foveolar cluster-like surface morphology with high hydrophobicity and roughness, as characterized using static contact angle and SEM and AFM analyses. It also exhibited increased platelet adhesion and accelerated blood clotting. The excellent hemostatic properties endow this copolymer with great potential in wound healing.
Asunto(s)
Ácido 3-Hidroxibutírico/síntesis química , Haloferax mediterranei , Hemostáticos/síntesis química , Ácidos Pentanoicos/síntesis química , Ácido 3-Hidroxibutírico/farmacología , Haloferax mediterranei/metabolismo , Hemostáticos/farmacología , Humanos , Ácidos Pentanoicos/farmacología , Adhesividad Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/fisiología , ProhibitinasRESUMEN
Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Respiración de la Célula/efectos de los fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Neoplasias Pulmonares/metabolismo , Mitocondrias Hepáticas/metabolismo , Polietileneimina/farmacología , Protones , Adenosina Trifosfato/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Transporte de Electrón/efectos de los fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético , Femenino , Humanos , Neoplasias Pulmonares/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacosRESUMEN
Poly(4-hydroxybutyrate) (P4HB) is a highly elastic polymer, whereas poly(3-hydroxypropionate) (P3HP) is a polymer with enormous tensile strength. This study aimed to biosynthesize a block copolymer consisting of soft P4HB block with a strong P3HP block to gain unique and excellent material properties. A recombinant Escherichia coli strain that produces homopolymers of P3HP and P4HB was employed for the block copolymer synthesis. When the strain was grown in the presence of 1,4-butanediol (BDO) as a 4HB precursor, P4HB block was formed. Sequential supplementation of 1,3-propanediol (PDO) as a 3HP precursor allowed the strain to produce P3HP block. Thermal, NMR, fractionation, and mechanical characterizations confirmed the resulting polymer as a block copolymer of P3HP-b-P4HB. Two block copolymers were formed from this study, including the P3HP-b-29% P4HB and P3HP-b-37% P4HB, they showed superior properties over random copolymers P(3HP-co-4HB). The block copolymers had two glass transition temperatures (Tg) and two melting temperatures (Tm). In comparison to the homopolymers P3HP and P4HB, incorporation of block microstructure resulted in the lowering of Tm, block copolymers were revealed with higher Young's modulus, yield strengths, and tension strengths much better than the previously reported random copolymers of similar compositions. Block copolymerization of P3HP and P4HB adds a new vision on PHA polymerization by generation of new polymers with superior properties.
Asunto(s)
Escherichia coli/metabolismo , Poliésteres/metabolismo , Polímeros/metabolismo , Butileno Glicoles/química , Espectroscopía de Resonancia Magnética , Peso Molecular , Poliésteres/aislamiento & purificación , Glicoles de Propileno/química , TemperaturaRESUMEN
Blood vessels not only transport oxygen and nutrients to each organ, but also play an important role in the regulation of tissue regeneration. Impaired or occluded vessels can result in ischemia, tissue necrosis, or even life-threatening events. Bioengineered vascular grafts have become a promising alternative treatment for damaged or occlusive vessels. Large-scale tubular grafts, which can match arteries, arterioles, and venules, as well as meso- and microscale vasculature to alleviate ischemia or prevascularized engineered tissues, have been developed. In this review, materials and techniques for engineering tubular scaffolds and vasculature at all levels are discussed. Examples of vascularized tissue engineering in bone, peripheral nerves, and the heart are also provided. Finally, the current challenges are discussed and the perspectives on future developments in biofunctional engineered vessels are delineated.
RESUMEN
Rheumatoid arthritis (RA) is an inflammatory disease that leads to disability; however, existing therapies are still unsatisfactory. Activated fibroblast-like synoviocytes (FLSs) play an essential role in synovitis formation and joint destruction in RA. The Hedgehog signaling pathway is aberrantly activated and contributes to the aggressive phenotype of RA-FLSs. However, it remains uncertain whether inhibiting Smoothened (SMO), a critical component of the Hedgehog signaling pathway, is an effective treatment for RA. Here, we design a series of small interfering RNAs (siRNAs) that specifically target the SMO gene. With precise chemical modifications, siRNAs' efficacy and stability are significantly improved, and the off-target effects are minimized. The optimized chemically modified siRNA (si-S1A3-Chol) decreases RA-FLS proliferation and invasiveness without the transfection reagent. Furthermore, si-S1A3-Chol injected intra-articularly effectively alleviates joint destruction and improves motor function in collagen-induced arthritis mouse models. Consequently, our results demonstrate that chemically modified siRNA targeting the Hedgehog signaling pathway may be a potential therapy for RA.
RESUMEN
In this paper, an efficient synthesis of 2-iminothiazolidin-4-ones through a copper-catalyzed tandem annulation reaction of alkyl amines, isothiocyanates and diazo acetates is presented. Notable advantages of this [2 + 1 + 2] cyclization methodology include readily accessible starting materials, simple operation, mild reaction conditions, high yields, step-economy and diverse functional group tolerance. In addition, the reaction is applicable to the gram scale synthesis and the preparation of bioactive molecules.
RESUMEN
Both exogenous transcriptional factors and chemical-defined medium can transdifferentiate astrocytes into functional neurons. However, the regional preference for such transdifferentiation has not been fully studied. A previously reported 5C medium was infused into the mouse cortex and striatum to determine the regional preference for transdifferentiation from astrocytes to neurons. The numbers of NeuN+ GFAP+ EdU+ cells (intermediates) and NeuN+ EdU+ cells (end products) were determined by immunofluorescence to explore the regional preference of transdifferentiation. In addition, to optimize the delivery of the transdifferentiation medium, three key growth factors, insulin, bFGF and transferrin, were loaded onto chitosan nanoparticles, mixed with gelatin methacryloyl and tested in animals with motor cortex injury. A higher transdifferentiation efficiency was identified in the mouse cortex. Differences in cellular respiration and the balance between glutaminase (Gls) and glutamine synthetase were confirmed to be key regulators. In addition, the sustained drug release system induced transdifferentiation of cortex astrocytes both in vivo and in vitro, and partially facilitated the behaviour recovery of mice with motor cortex injury. We also applied this method in pigs and obtained consistent results. In summary, low Gls and glycolysis can be used to predict high transdifferentiation efficiency, which may be useful to identify better indications for the current transdifferentiation system. In addition, the current drug delivery system has the potential to treat diseases related to cortex injuries.
Asunto(s)
Transdiferenciación Celular , Glutaminasa , Ratones , Animales , Porcinos , Transdiferenciación Celular/fisiología , Glutaminasa/metabolismo , Células Cultivadas , Astrocitos/metabolismo , GlucólisisRESUMEN
Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the 13C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.
Asunto(s)
Técnicas Biosensibles , Infecciones por Helicobacter , Helicobacter pylori , Úlcera Péptica , Neoplasias Gástricas , Humanos , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Sustancias Reductoras , Sistemas CRISPR-Cas , Detección Precoz del Cáncer , Úlcera Péptica/diagnóstico , Úlcera Péptica/genética , Genotipo , Citotoxinas/genética , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismoRESUMEN
Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs' encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB. Their mechanical and thermal properties showed obvious changes depending on the monomer ratios. Morphologically, P(3HP-co-4HB) films only became fully transparent when monomer 4HB content was around 67 mol%. For the first time, P(3HP-co-4HB) with adjustable monomer ratios were produced and characterized.