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1.
Biopolymers ; 107(10)2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28741310

RESUMEN

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly.


Asunto(s)
Biopolímeros/química , Queratinas Específicas del Pelo/química , Proteínas Recombinantes/química , Ingeniería de Tejidos , Biopolímeros/genética , Humanos , Queratinas Específicas del Pelo/genética , Nanoestructuras/química , Multimerización de Proteína , Proteínas Recombinantes/genética
2.
J R Army Med Corps ; 163(3): 158-163, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27625370

RESUMEN

BACKGROUND: Fatigue in military operations leads to safety and operational problems due to a decrease in alertness and performance. The primary method of counteracting the effects of sleep deprivation is to increase nightly sleep time, which in operational situations is not always feasible. History has taught us that surgeons and surgical teams are finite resources that cannot operate on patients indefinitely. METHODS: A systematic review was conducted using the search terms 'sleep' and 'deprivation' examining the impact of sleep deprivation on cognitive performance in military surgical teams. Studies examining outcomes on intensive care patients and subjects with comorbidities were not addressed in this review. RESULTS: Sleep deprivation in any 'out-of-hours' surgery has a significant impact on overall morbidity and mortality. Sleep deprivation in surgeons and surgical trainees negatively impacts cognitive performance and puts their own and patients' health at risk. All published research lacks consensus when defining 'sleep deprivation' and 'rested' states. It is recognised that it would be unethical to conduct a well-designed randomised controlled trial, to determine the effects of fatigue on performance in surgery; however, there is a paucity between surrogate markers and applying simulated results to actual clinical performance. This requires further research. Recommended methods of combating fatigue include: prophylactically 'sleep-banking' prior to known periods of sleep deprivation, napping, use of stimulant or alerting substances such as modafinil, coordinated work schedules to reduce circadian desynchronisation and regular breaks with enforced rest periods. CONCLUSIONS: A forward surgical team will become combat-ineffective after 48 hours of continuous operations. This systematic review recommends implementing on-call periods of no more than 12 hours in duration, with adequate rest periods every 24 hours. Drug therapies and sleep banking may, in the short term, prevent negative effects of acute sleep deprivation.


Asunto(s)
Competencia Clínica , Cognición , Fatiga/fisiopatología , Medicina Militar , Privación de Sueño/fisiopatología , Cirujanos , Análisis y Desempeño de Tareas , Compuestos de Bencidrilo/uso terapéutico , Fatiga/psicología , Personal de Salud , Humanos , Personal Militar , Modafinilo , Grupo de Atención al Paciente , Descanso , Sueño , Privación de Sueño/psicología , Promotores de la Vigilia/uso terapéutico
3.
Biochem Soc Trans ; 43(5): 856-60, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26517894

RESUMEN

Advances in protein engineering tools, both computational and experimental, has afforded many new protein structures and functions. Here, we present a snapshot of repeat-protein engineering efforts towards new, versatile, alternative binding scaffolds for use in analytical sensors and as imaging agents. Analytical assays, sensors and imaging agents based on the direct binding of analyte are increasingly important for research and diagnostics in medicine, food safety, and national security.


Asunto(s)
Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas , Secuencias Repetitivas de Aminoácido , Animales , Técnicas Biosensibles/tendencias , Humanos , Ligandos , Imagen Molecular/tendencias
5.
J Mech Behav Biomed Mater ; 125: 104921, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34758444

RESUMEN

Collagen is the most abundant structural protein in humans, with dozens of sequence variants accounting for over 30% of the protein in an animal body. The fibrillar and hierarchical arrangements of collagen are critical in providing mechanical properties with high strength and toughness. Due to this ubiquitous role in human tissues, collagen-based biomaterials are commonly used for tissue repairs and regeneration, requiring chemical and thermal stability over a range of temperatures during materials preparation ex vivo and subsequent utility in vivo. Collagen unfolds from a triple helix to a random coil structure during a temperature interval in which the midpoint or Tm is used as a measure to evaluate the thermal stability of the molecules. However, finding a robust framework to facilitate the design of a specific collagen sequence to yield a specific Tm remains a challenge, including using conventional molecular dynamics modeling. Here we propose a de novo framework to provide a model that outputs the Tm values of input collagen sequences by incorporating deep learning trained on a large data set of collagen sequences and corresponding Tm values. By using this framework, we are able to quickly evaluate how mutations and order in the primary sequence affect the stability of collagen triple helices. Specifically, we confirm that mutations to glycines, mutations in the middle of a sequence, and short sequence lengths cause the greatest drop in Tm values.


Asunto(s)
Aprendizaje Profundo , Animales , Materiales Biocompatibles , Colágeno , Humanos , Temperatura , Cicatrización de Heridas
6.
Bioact Mater ; 6(11): 3671-3677, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33898871

RESUMEN

Lipid nanoparticles are promising carriers for oral drug delivery. For bioactive cargos with intracellular targets, e.g. gene-editing proteins, it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells. However, limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier. Herein, we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle (carrier) and protein (cargo) nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells. We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase, penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer. After transporting across the intestinal epithelia, the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination. These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.

7.
ACS Appl Mater Interfaces ; 13(2): 3186-3198, 2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33398989

RESUMEN

Mesoscaled assemblies are organized in native collagen tissues to achieve remarkable and diverse performance and functions. In this work, a facile, low-cost, and controllable liquid exfoliation method was applied to directly extract these collagen mesostructures from bovine Achilles tendons using a sodium hydroxide (NaOH)/urea aqueous system with freeze-thaw cycles and sonication. A series of collagen fibrils with diameters of 26-230 nm were harvested using this process, and in situ observations under polarizing microscopy (POM) and using molecular dynamics simulations revealed the influence of the NaOH/urea system on the tendon collagen. FTIR and XRD results confirmed that these collagen fibrils preserved typical structural characteristics of type I collagen. These isolated collagen fibrils were then utilized as building blocks to fabricate free-standing collagen membranes, which exhibited good stability in solvents and outstanding mechanical properties and transparency, with potential for utility in optical and electronic sensors. Moreover, in vitro and vivo evaluations demonstrated that these new resulting collagen membranes had good cytocompatibility, biocompatibility, and degradability for potential applications in biomedicine. This work provides a new approach for collagen processing by liquid exfoliation with utility for the formation of robust collagen materials that consist of native collagen mesostructures as building blocks.


Asunto(s)
Tendón Calcáneo/química , Materiales Biocompatibles/química , Colágeno/química , Membranas Artificiales , Animales , Bovinos , Línea Celular , Colágeno/ultraestructura , Femenino , Congelación , Ratones , Ratas Sprague-Dawley , Hidróxido de Sodio/química , Sonicación , Resistencia a la Tracción , Urea/química
8.
Sci Justice ; 50(3): 141-9, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20709275

RESUMEN

Geophysics may assist scent dogs and divers in the search of water bodies for human and animal remains, contraband, weapons and explosives by surveying large areas rapidly and identifying targets or environmental hazards. The most commonly applied methods are described and evaluated for forensic searches. Seismic reflection or refraction and CHIRPS are useful for deep, open water bodies and identifying large targets, yet limited in streams and ponds. The use of ground penetrating radar (GPR) on water (WPR) is of limited use in deep waters (over 20 m) but is advantageous in the search for non-metallic targets in small ditches and ponds. Large metal or metal-bearing targets can be successfully imaged in deep waters by using towfish magnetometers: in shallow waters such a towfish cannot be used, so a non-metalliferous boat can carry a terrestrial magnetometer. Each device has its uses, depending on the target and location: unknown target make-up (e.g. a homicide victim with or without a metal object) may be best located using a range of methods (the multi-proxy approach), depending on water depth. Geophysics may not definitively find the target, but can provide areas for elimination and detailed search by dogs and divers, saving time and effort.


Asunto(s)
Agua Dulce , Fenómenos Geológicos , Animales , Ahogamiento , Medicina Legal/métodos , Humanos , Magnetismo , Radar
9.
Adv Drug Deliv Rev ; 160: 186-198, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33080258

RESUMEN

Advances in medical science have led to diverse new therapeutic modalities, as well as enhanced understanding of the progression of various disease states. These findings facilitate the design and development of more customized and exquisite drug delivery systems that aim to improve therapeutic indices of drugs to treat a variety of conditions. Synthetic polymer-based drug carriers have often been the focus of such research. However, these structures suffer from challenges with heterogeneity of the starting material, limited chemical features, complex functionalization methods, and in some cases a lack of biocompatibility. Consequently, protein-based polymers have garnered much attention in recent years due to their monodisperse features, ease of production and functionalization, and biocompatibility. Genetic engineering techniques enable the advancement of protein-based drug delivery systems with finely tuned physicochemical properties, and thus an expanded level of customization unavailable with synthetic polymers. Of these genetically engineered proteins, elastin-like proteins (ELP), silk-like proteins (SLP), and silk-elastin-like proteins (SELP) provide a unique set of alternatives for designing drug delivery systems due to their inherent chemical and physical properties and ease of engineering afforded by recombinant DNA technologies. In this review we examine the advantages of genetically engineered drug delivery systems with emphasis on ELP and SLP constructions. Methods for fabrication and relevant biomedical applications will also be discussed.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Elastina/química , Ingeniería de Proteínas/métodos , Seda/química , Materiales Biocompatibles/química , Técnicas de Transferencia de Gen , Humanos , Hidrogeles/química , Nanopartículas , Tamaño de la Partícula , Proteínas Recombinantes/química
10.
Adv Healthc Mater ; 9(11): e2000266, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32338463

RESUMEN

Three-dimensional organoid tissue culture models are a promising approach for the study of biological processes including diseases. Advances in these tissue culture technologies improve in vitro analysis compared to standard 2D cellular approaches and are more representative of the physiological environment. However, a major challenge associated with organoid systems stems from the laborious processing involved in the analysis of large numbers of organoids. Here the design, characterization, and application of silk-elastin-like protein-based smart carrier arrays for processing organoids is presented. Fabrication of hydrogel-based carrier systems at room temperature result in organized arrays of organoids that maintain tissue culture plate orientation and could be processed simultaneously for histology. The system works by transfer of the organoids to the hydrogel arrays after which the material is subjected to 65 °C to induce hydrogel contraction to secure the organoids, resulting in multisample constructs and allowing for placement on a microscope slide. Histological processing and immunostaining of these arrayed cerebral organoids analyzed within the contracted silk-elastin-like proteins (SELP) show retention of native organoid features compared to controls without the hydrogel carrier system, thus avoiding any artifacts. These SELP carriers present a useful approach for improving efficiency of scaled organoid screening and processing.


Asunto(s)
Fenómenos Biológicos , Materiales Inteligentes , Elastina , Hidrogeles , Organoides , Seda
11.
Sci Adv ; 6(19): eaay8828, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32494701

RESUMEN

Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive decline, memory loss, and inability to perform everyday functions. Hallmark features of AD-including generation of amyloid plaques, neurofibrillary tangles, gliosis, and inflammation in the brain-are well defined; however, the cause of the disease remains elusive. Growing evidence implicates pathogens in AD development, with herpes simplex virus type I (HSV-1) gaining increasing attention as a potential causative agent. Here, we describe a multidisciplinary approach to produce physiologically relevant human tissues to study AD using human-induced neural stem cells (hiNSCs) and HSV-1 infection in a 3D bioengineered brain model. We report a herpes-induced tissue model of AD that mimics human disease with multicellular amyloid plaque-like formations, gliosis, neuroinflammation, and decreased functionality, completely in the absence of any exogenous mediators of AD. This model will allow for future studies to identify potential downstream drug targets for treating this devastating disease.


Asunto(s)
Enfermedad de Alzheimer , Herpes Simple , Herpesvirus Humano 1 , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Gliosis/complicaciones , Herpesvirus Humano 1/fisiología , Humanos , Placa Amiloide
12.
ACS Appl Bio Mater ; 3(4): 2466-2472, 2020 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-35025296

RESUMEN

Sustained, local delivery of the antibiotic ciprofloxacin under different formats from porous silk protein-based memory foam systems was studied. Similarly, protease XIV was incorporated during processing to provide control of the degradation kinetics of the silk materials. In vitro antibiotic release studies combined with degradation assessments were utilized to assess the mechanisms and kinetics of release from the silk materials. The sequestered protease XIV affected the degradation profiles of the silk foams yet did not impact the release kinetics of the ciprofloxacin, which was controlled by solubility and diffusion of the drug. The ability to tune the release of ciprofloxacin between 1 and 200 days, combined with the option to modulate the degradation rate up to 80% in 2 weeks via incorporation of a protease, suggests utility for drug release devices. Further, we anticipate that this approach could also be extended to other medical implant needs and other drugs.

13.
Biomed Mater ; 15(6): 065006, 2020 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-32485704

RESUMEN

Natural biopolymers have found success in tissue engineering and regenerative medicine applications. Their intrinsic biocompatibility and biological activity make them well suited for biomaterials development. Specifically, keratin-based biomaterials have demonstrated utility in regenerative medicine applications including bone regeneration, wound healing, and nerve regeneration. However, studies of structure-function relationships in keratin biomaterials have been hindered by the lack of homogeneous preparations of materials extracted and isolated from natural sources such as wool and hair fibers. Here we present a side-by-side comparison of natural and recombinant human hair keratin proteins K31 and K81. When combined, the recombinant proteins (i.e. rhK31 and rhK81) assemble into characteristic intermediate filament-like fibers. Coatings made from natural and recombinant dimers were compared side-by-side and investigated for coating characteristics and cell adhesion. In comparison to control substrates, the recombinant keratin materials show a higher propensity for inducing involucrin and hence, maturation in terms of potential skin cell differentiation.


Asunto(s)
Biopolímeros/química , Regeneración Ósea , Cabello/metabolismo , Queratinas Específicas del Pelo/química , Proteínas Recombinantes/química , Ingeniería de Tejidos/métodos , Actinas/metabolismo , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular , Cromatografía , Escherichia coli , Fibroblastos/metabolismo , Humanos , Queratinas/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Músculo Liso/metabolismo , Medicina Regenerativa/métodos , Silanos/química , Piel/patología , Cicatrización de Heridas/efectos de los fármacos
14.
J Funct Biomater ; 10(4)2019 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-31726786

RESUMEN

Transmucosal drug delivery is a promising avenue to improve therapeutic efficacy through localized therapeutic administration. Drug delivery systems that increase retention in the mucosal layer are needed to improve efficiency of such transmucosal platforms. However, the applicability of such systems is often limited by the range of chemistries and properties that can be achieved. Here we present the design and implementation of silk-elastin-like proteins (SELPs) with mucoadhesive properties. SELP-based micellar-like nanoparticles provide a system to tailor chemical and physical properties through genetic engineering of the SELP sequence, which enables the fabrication of nanoparticles with specific chemical and physical features. Analysis of the adhesion of four different SELP-based nanoparticle systems in an artificial mucus system, as well as in in vitro cellular assays indicates that addition of mucoadhesive chemical features on the SELP systems increases retention of the particles in mucosal environments. The results indicated that SELP-based nanoparticles provide a useful approach to study and develop transmucosal protein drug delivery system with unique mucoadhesive properties. Future studies will serve to further expand the range of achievable properties, as well as the utilization of SELPs to fabricate mucoadhesive materials for in vivo testing.

15.
J Funct Biomater ; 10(3)2019 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-31540233

RESUMEN

Pseudomonas aeruginosa (P. aeruginosa) infections may lead to severe damage of the cornea, mucosa, and skin. The highly aggressive nature of P. aeruginosa and the rise in multi-drug resistance, particularly in nosocomial settings, lead to an increased risk for permanent tissue damage and potentially death. Thus, a growing need exists to develop alternative treatments to reduce both the occurrence of bacterial infection and biofilm development, as well as pathological progression post-infection. Silk derived from Bombyx mori silkworms serves as a unique biomaterial that is biocompatible with low immunogenicity and high versatility, and thereby ideal for stabilizing therapeutics. In this study, we assessed the cytotoxicity of P. aeruginosa on human corneal stromal stem cells and two mucosal cell lines (Caco-2 and HT29-MTX). To determine whether antibiotic-immobilized scaffolds can serve as alternative therapeutics to free, diffuse forms, we developed novel gentamicin-conjugated silk films as functional scaffolds and compared antimicrobial effects and free gentamicin. The advantages of generating a surface coating with a covalently-bound antibiotic may reduce potential side-effects associated with free gentamicin, as well as limit the diffusion of the drug. Our results suggest that gentamicin conjugated to native silk and carboxyl-enriched silk inhibits P. aeruginosa growth. Development of stabilized antibiotic treatments with surface toxicity selective against bacteria may serve as an alternative approach to treat active infections, as well as potential prophylactic use as coatings in high-risk cases, such as post-surgical complications or prolonged hospitalization.

16.
Curr Protoc Toxicol ; 81(1): e84, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31529796

RESUMEN

The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict corneal toxicity of chemicals or effective treatments for disease prior to ocular exposure, as well as to study the corneal effects of systemic, chronic conditions, such as diabetes, are needed to reduce use of animal testing and accelerate therapeutic development. We have previously bioengineered an innervated corneal tissue model using silk protein scaffolds to recapitulate the structural and mechanical elements of the anterior cornea and to model the functional aspects of corneal sensation with the inclusion of epithelial, stromal, and neural components. The purpose of this unit is to provide a step-by-step guide for preparation, assembly, and application of this three-dimensional corneal tissue system to enable the study of corneal tissue biology. © 2019 by John Wiley & Sons, Inc.


Asunto(s)
Córnea , Seda , Técnicas de Cultivo de Tejidos/instrumentación , Andamios del Tejido , Alternativas a las Pruebas en Animales , Dimetilpolisiloxanos , Humanos , Técnicas de Cultivo de Tejidos/métodos , Ingeniería de Tejidos/métodos , Pruebas de Toxicidad
18.
Protein Sci ; 23(6): 790-800, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24659515

RESUMEN

Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N- and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.


Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Pared Celular/química , Leucina/química , Proteínas/química , Proteínas/metabolismo , Peptidoglicano , Unión Proteica
19.
Patient Educ Couns ; 81(3): 332-7, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21094013

RESUMEN

OBJECTIVE: To develop and pilot a communication aid aimed at increasing the frequency with which sexual health issues are raised proactively with young people in primary care. METHODS: Group interviews among primary health care professionals to guide development of the tool, simulated consultations to pre-test it, and a pilot study to assess effectiveness. RESULTS: We developed an electronic consultation aid: Talking of Sex and piloted it in eight general practices across the UK. 188 patients and 58 practitioners completed questionnaires pre-intervention, and 92 patients and 45 practitioners post-intervention. There was a modest increase in the proportion of consultations in which sexual health was raised, from 28.1% pre-intervention to 32.6% post-intervention. In consultations with nurses the rise was more marked. More patients reported discussing preventive practices such as condom use post-intervention. Patients unanimously welcomed the opportunity to discuss sexual health matters with their practitioner. CONCLUSION: The tool has capacity to increase the frequency with which sexual health is raised in primary care, particularly by nurses, to influence the topics discussed, and to improve patient satisfaction. PRACTICE IMPLICATIONS: The tool has potential in increasing the proportion of young people whose sexual health needs are addressed in general practice.


Asunto(s)
Comunicación en Salud , Relaciones Médico-Paciente , Conducta Sexual , Sexualidad , Adolescente , Adulto , Femenino , Medicina General , Humanos , Masculino , Proyectos Piloto , Atención Primaria de Salud/organización & administración , Educación Sexual/métodos , Encuestas y Cuestionarios , Reino Unido , Adulto Joven
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