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1.
Med Biol Eng Comput ; 59(10): 2085-2114, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34453662

RESUMO

This proof of concept (PoC) assesses the ability of machine learning (ML) classifiers to predict the presence of a stenosis in a three vessel arterial system consisting of the abdominal aorta bifurcating into the two common iliacs. A virtual patient database (VPD) is created using one-dimensional pulse wave propagation model of haemodynamics. Four different machine learning (ML) methods are used to train and test a series of classifiers-both binary and multiclass-to distinguish between healthy and unhealthy virtual patients (VPs) using different combinations of pressure and flow-rate measurements. It is found that the ML classifiers achieve specificities larger than 80% and sensitivities ranging from 50 to 75%. The most balanced classifier also achieves an area under the receiver operative characteristic curve of 0.75, outperforming approximately 20 methods used in clinical practice, and thus placing the method as moderately accurate. Other important observations from this study are that (i) few measurements can provide similar classification accuracies compared to the case when more/all the measurements are used; (ii) some measurements are more informative than others for classification; and (iii) a modification of standard methods can result in detection of not only the presence of stenosis, but also the stenosed vessel. Graphical Abstract An overview of methodology fo the creation of virtual patients and their classification.


Assuntos
Diagnóstico por Imagem , Aprendizado de Máquina , Constrição Patológica , Humanos , Estudo de Prova de Conceito
2.
J Phys Chem Lett ; 12(32): 7859-7865, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34382803

RESUMO

Fluorescence-encoded vibrational spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoded vibrational spectroscopy methods lack sensitivity in the low-frequency region, which if addressed could further enhance their capabilities. Here, we present a method for highly sensitive low-frequency fluorescence-encoded vibrational spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS). By first exciting molecules into vibrationally excited states and then promoting the vibrating molecules to electronic states at varying times, the molecular vibrations can be encoded onto the emitted time-domain fluorescence intensity. We demonstrate the sensitive low-frequency detection capability of FLETCHERS by measuring vibrational spectra in the lower fingerprint region of rhodamine 800 solutions as dilute as 250 nM, which is ∼1000 times more sensitive than conventional vibrational spectroscopy. These results, along with further improvement of the method, open up the prospect of performing single-molecule vibrational spectroscopy in the low-frequency region.


Assuntos
Corantes Fluorescentes/química , Rodaminas/química , Análise Espectral Raman/métodos , Fluorescência , Limite de Detecção , Estudo de Prova de Conceito , Espectrometria de Fluorescência , Vibração
3.
Undersea Hyperb Med ; 48(3): 221-226, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34390626

RESUMO

Historically, electronic devices have been generally prohibited during hyperbaric oxygen (HBO2) therapy due to risk of fire in a pressurized, oxygen-rich environment. Point-of-care ultrasound (POCUS) however has emerged as a useful imaging modality in diverse clinical settings. Hyperbaric chambers treating critically ill patients would benefit from the application of POCUS at pressure to make real-time patient assessments. Thus far, POCUS during HBO2 therapy has been limited due to required equipment modifications to meet safety standards. Here we demonstrate proof of concept, safety, and successful performance of an off-the-shelf handheld POCUS system (SonoSite iViz) in a clinical hyperbaric environment without need for modification.


Assuntos
Oxigenação Hiperbárica , Testes Imediatos , Estudo de Prova de Conceito , Ultrassonografia/instrumentação , Cuidados Críticos , Estado Terminal , Fontes de Energia Elétrica , Desenho de Equipamento , Segurança de Equipamentos , Estudos de Viabilidade , Temperatura Alta , Humanos , Fatores de Tempo
4.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428374

RESUMO

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Assuntos
Testes Respiratórios/instrumentação , Teste para COVID-19/instrumentação , Máscaras , SARS-CoV-2/isolamento & purificação , Microbiologia do Ar , Anticorpos Antivirais/imunologia , Testes Respiratórios/métodos , Teste para COVID-19/métodos , Colódio/química , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Cimento de Policarboxilato/química , Porosidade , Estudo de Prova de Conceito , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/química , Proteínas Virais/análise , Proteínas Virais/imunologia
5.
Trials ; 22(1): 508, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34332638

RESUMO

BACKGROUND: Although Alzheimer's disease affects around 800,000 people in the UK and costs almost £23 billion per year, currently licenced treatments only offer modest benefit at best. Seizures, which are more common in patients with Alzheimer's disease than age matched controls, may contribute to the loss of nerve cells and abnormal brain discharges can disrupt cognition. This aberrant electrical activity may therefore present potentially important drug targets. The anti-seizure medication levetiracetam can reduce abnormal cortical discharges and reverse memory deficits in a mouse model of Alzheimer's disease. Levetiracetam has also been shown to improve memory difficulties in patients with mild cognitive impairment, a precursor to Alzheimer's disease. Clinical use of levetiracetam is well-established in treatment of epilepsy and extensive safety data are available. Levetiracetam thus has the potential to provide safe and efficacious treatment to help with memory difficulties in Alzheimer's disease. METHODS: The proposed project is a proof of concept study to test whether levetiracetam can help cognitive function in people with dementia. We plan to recruit thirty patients with mild to moderate Alzheimer's disease with no history of previous seizures or other significant co-morbidity. Participants will be allocated to a double-blind placebo-controlled crossover trial that tests levetiracetam against placebo. Standardised scales to assess cognition and a computer-based touchscreen test that we have developed to better detect subtle improvements in hippocampal function will be used to measure changes in memory. All participants will have an electroencephalogram (EEG) at baseline. The primary outcome measure is a change in the computer-based touchscreen cognitive task while secondary outcomes include the effect of levetiracetam on mood, quality of life and modelling of the EEG, including time series measures and feature-based analysis to see whether the effect of levetiracetam can be predicted. The effect of levetiracetam and placebo will be compared within a given patient using the paired t-test and the analysis of covariance adjusting for baseline values. DISCUSSION: This is the first study to evaluate if an anti-seizure medication can offer meaningful benefit to patients with Alzheimer's disease. If this study demonstrates at least stabilisation of memory function and/or good tolerability, the next step will be to rapidly progress to a larger study to establish whether levetiracetam may be a useful and cost-effective treatment for patients with Alzheimer's disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT03489044 . Registered on April 5, 2018.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/tratamento farmacológico , Animais , Estudos Cross-Over , Método Duplo-Cego , Humanos , Levetiracetam/efeitos adversos , Camundongos , Estudo de Prova de Conceito , Qualidade de Vida
6.
J Am Chem Soc ; 143(30): 11544-11553, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34288684

RESUMO

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/análise , Amarelo de Eosina-(YS)/análogos & derivados , Espectrometria de Fluorescência/métodos , 3,3'-Diaminobenzidina/química , Biomarcadores/química , Catálise/efeitos da radiação , Amarelo de Eosina-(YS)/síntese química , Amarelo de Eosina-(YS)/efeitos da radiação , Fluorescência , Luz , Limite de Detecção , Oxirredução/efeitos da radiação , Fosfoproteínas/análise , Polietilenoglicóis/química , Polimerização , Estudo de Prova de Conceito , SARS-CoV-2/química
7.
J Am Chem Soc ; 143(31): 12315-12327, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34324336

RESUMO

Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a one-component multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity in vitro and 31 (57%) in vivo. Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments in vitro and in vivo. Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.


Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Nanopartículas/química , RNA Mensageiro/metabolismo , Tensoativos/química , Animais , Dendrímeros/síntese química , Portadores de Fármacos/síntese química , Liberação Controlada de Fármacos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Estudo de Prova de Conceito , Tensoativos/síntese química
8.
Methods Mol Biol ; 2342: 825-841, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272719

RESUMO

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15 years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.


Assuntos
Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , RNA Interferente Pequeno/farmacologia , RNA/análise , Ativação Metabólica , Animais , Cromatografia Líquida , Hidrólise , Cinética , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Estudo de Prova de Conceito
9.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34234013

RESUMO

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.


Assuntos
Bacteriófagos/genética , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Programas de Imunização , Administração por Inalação , Animais , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Dependovirus/genética , Armazenamento de Medicamentos , Feminino , Programas de Imunização/métodos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Estudo de Prova de Conceito , Temperatura
10.
Sensors (Basel) ; 21(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34282770

RESUMO

Humans typically fixate on objects before moving their arm to grasp the object. Patients with ALS disorder can also select the object with their intact eye movement, but are unable to move their limb due to the loss of voluntary muscle control. Though several research works have already achieved success in generating the correct grasp type from their brain measurement, we are still searching for fine controll over an object with a grasp assistive device (orthosis/exoskeleton/robotic arm). Object orientation and object width are two important parameters for controlling the wrist angle and the grasp aperture of the assistive device to replicate a human-like stable grasp. Vision systems are already evolved to measure the geometrical attributes of the object to control the grasp with a prosthetic hand. However, most of the existing vision systems are integrated with electromyography and require some amount of voluntary muscle movement to control the vision system. Due to that reason, those systems are not beneficial for the users with brain-controlled assistive devices. Here, we implemented a vision system which can be controlled through the human gaze. We measured the vertical and horizontal electrooculogram signals and controlled the pan and tilt of a cap-mounted webcam to keep the object of interest in focus and at the centre of the picture. A simple 'signature' extraction procedure was also utilized to reduce the algorithmic complexity and system storage capacity. The developed device has been tested with ten healthy participants. We approximated the object orientation and the size of the object and determined an appropriate wrist orientation angle and the grasp aperture size within 22 ms. The combined accuracy exceeded 75%. The integration of the proposed system with the brain-controlled grasp assistive device and increasing the number of grasps can offer more natural manoeuvring in grasp for ALS patients.


Assuntos
Força da Mão , Equipamentos de Autoajuda , Eletroculografia , Mãos , Humanos , Movimento , Estudo de Prova de Conceito
11.
J Alzheimers Dis ; 82(2): 791-802, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34092628

RESUMO

BACKGROUND: Medical imaging methods such as PET and MRI aid clinical assessment of Alzheimer's disease (AD). Less expensive, less technically demanding, and more widely deployable technologies are needed to expand objective screening for diagnosis, treatment, and research. We previously reported brain tissue near-infrared optical spectroscopy (NIR) in vitro indicating the potential to meet this need. OBJECTIVE: To determine whether completely non-invasive, clinical, NIR in vivo can distinguish AD patients from age-matched controls and to show the potential of NIR as a clinical screen and monitor of therapeutic efficacy. METHODS: NIR spectra were acquired in vivo. Three groups were studied: autopsy-confirmed AD, control and mild cognitive impairment (MCI). A feature selection approach using the first derivative of the intensity normalized spectra was used to discover spectral regions that best distinguished "AD-alone" (i.e., without other significant neuropathology) from controls. The approach was then applied to other autopsy-confirmed AD cases and to clinically diagnosed MCI cases. RESULTS: Two regions about 860 and 895 nm completely separate AD patients from controls and differentiate MCI subjects according to the degree of impairment. The 895 nm feature is more important in separating MCI subjects from controls (ratio-of-weights: 1.3); the 860 nm feature is more important for distinguishing MCI from AD (ratio-of-weights: 8.2). CONCLUSION: These results form a proof of the concept that near-infrared spectroscopy can detect and classify diseased and normal human brain in vivo. A clinical trial is needed to determine whether the two features can track disease progression and monitor potential therapeutic interventions.


Assuntos
Doença de Alzheimer/diagnóstico , Encéfalo/diagnóstico por imagem , Disfunção Cognitiva/diagnóstico , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Idoso , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , Estudo de Prova de Conceito , Reprodutibilidade dos Testes
12.
Methods Mol Biol ; 2277: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080153

RESUMO

Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Esquizofrenia/metabolismo , Benzimidazóis/química , Carbocianinas/química , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Fibroblastos/patologia , Corantes Fluorescentes/química , Humanos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Mitocôndrias/patologia , Estudo de Prova de Conceito , Esquizofrenia/patologia , Xantenos/química
13.
ACS Appl Mater Interfaces ; 13(26): 30397-30403, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34161059

RESUMO

Engineering cell-derived nanovesicles with active-targeting ligands is an important strategy to enhance the targeting efficiency. However, the enhanced binding capability to targeting cells also leads to the binding with nontarget cells that share the same biomarkers. DNA-based logic gate is a kind of molecular system that responds to chemical inputs by generating output signals, and the relationship between the input and the output is based on a certain logic. Thus, the DNA-based logic gate could provide a new approach to improve the delivery efficiency of the nanovesicle. In this work, we developed a DNA logic-gated module that coupled two tumor cell-targeting factors (e.g., low pH and a tumor cell biomarker) in a Boolean manner. Immobilization of this module on the surface of the nanovesicle enables the nanovesicle to sense tumor cell-targeting factors and regard these cues as inputs AND logic gate. With the guide of DNA-based logic gate, gold carbon dots (GCDs) encapsulated within nanovesicles were delivered into target cells, and then the intracellular redox status variation was reflected by fluorescence change of GCDs. Overall, we developed DNA logic-gated nanovesicles that contract different targeting factors into a unique tag for target cells. This facile functionalization strategy can pave the way for constructing smart nanovesicles and would broaden their application in the field of precision medicine and personalized treatment.


Assuntos
Computadores Moleculares , DNA/química , Lipossomos/química , Lógica , Nanoestruturas/química , Motivos de Aminoácidos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Carbono/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , DNA/metabolismo , Corantes Fluorescentes/química , Ouro/química , Humanos , Concentração de Íons de Hidrogênio , Estudo de Prova de Conceito , Pontos Quânticos/química , Receptores Proteína Tirosina Quinases/metabolismo
14.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165969

RESUMO

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Assuntos
Hidrogéis/química , Reação em Cadeia da Polimerase Multiplex/métodos , Nanotubos de Carbono/química , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Compostos de Boro/química , Coronavirus/química , Primers do DNA/química , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Grafite/química , Vírus da Influenza A/química , Vírus da Doença de Newcastle/química , Estudo de Prova de Conceito , RNA Viral/química , Viroses/diagnóstico
15.
Biomolecules ; 11(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069580

RESUMO

IRDs are one of the leading causes of visual loss in children and young adults. Mutations in over 271 genes lead to retinal dysfunction, degeneration and sight loss. Though no cure exists, gene augmentation therapy has brought hope to the field. This systematic review sought to assess the efficacy of available gene therapy treatments for IRDs. Databases and public resources were searched for randomised controlled trials (RCTs) and non-randomised studies of interventions (NRSIs). Standard methodological procedures were used, including a risk-of-bias assessment. One RCT and five NRSIs were assessed, all for adeno-associated virus two (AAV2)-mediated treatment of RPE-specific 65 kDa (RPE65)-associated LCA (Leber congenital amaurosis). Five outcomes were reported for meta-analyses. Modest improvements in visual acuity, ambulatory navigation/mobility testing or central retinal thickness was observed. There was significant improvement in red and blue light full-field stimulus testing (FST) (red light risk ratio of 1.89, treated v control, p = 0.04; and blue light risk ratio of 2.01, treated v control, p = 0.001). Study design assessment using a ROBIN-I tool (Cochrane Library) showed risk-of-bias judgement to be "low/moderate", whilst there were "some concerns" for the RCT using a RoB-2 tool (Cochrane Library). Although comparison by meta-analysis is compromised by, amongst other issues, a variable amount of vector delivered in each trial, FST improvements demonstrate a proof-of-principle for treating IRDs with gene therapy.


Assuntos
Terapia Genética/métodos , Amaurose Congênita de Leber/terapia , Retinite Pigmentosa/terapia , Ensaios Clínicos como Assunto , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Estudo de Prova de Conceito , Retinite Pigmentosa/genética , Retinite Pigmentosa/fisiopatologia , Resultado do Tratamento , Acuidade Visual
16.
Cell Death Dis ; 12(7): 624, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135313

RESUMO

Proteasomal activity is compromised in diabetic hearts that contributes to proteotoxic stresses and cardiac dysfunction. Osteocrin (OSTN) acts as a novel exercise-responsive myokine and is implicated in various cardiac diseases. Herein, we aim to investigate the role and underlying molecular basis of OSTN in diabetic cardiomyopathy (DCM). Mice received a single intravenous injection of the cardiotrophic adeno-associated virus serotype 9 to overexpress OSTN in the heart and then were exposed to intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for consecutive 5 days to generate diabetic models. Neonatal rat cardiomyocytes were isolated and stimulated with high glucose to verify the role of OSTN in vitro. OSTN expression was reduced by protein kinase B/forkhead box O1 dephosphorylation in diabetic hearts, while its overexpression significantly attenuated cardiac injury and dysfunction in mice with STZ treatment. Besides, OSTN incubation prevented, whereas OSTN silence aggravated cardiomyocyte apoptosis and injury upon hyperglycemic stimulation in vitro. Mechanistically, OSTN treatment restored protein kinase G (PKG)-dependent proteasomal function, and PKG or proteasome inhibition abrogated the protective effects of OSTN in vivo and in vitro. Furthermore, OSTN replenishment was sufficient to prevent the progression of pre-established DCM and had synergistic cardioprotection with sildenafil. OSTN protects against DCM via restoring PKG-dependent proteasomal activity and it is a promising therapeutic target to treat DCM.


Assuntos
Apoptose/efeitos dos fármacos , Cardiomiopatias Diabéticas/prevenção & controle , Proteínas Musculares/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/farmacologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Proteína Forkhead Box O1/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosforilação , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Neurology ; 97(8): e755-e764, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34144995

RESUMO

OBJECTIVE: Hematoma expansion (HE) is commonly analyzed as a dichotomous outcome in intracerebral hemorrhage (ICH) trials. In this proof-of-concept study, we propose an HE shift analysis model as a method to improve the evaluation of candidate ICH therapies. METHODS: Using data from the Antihypertensive Treatment of Acute Cerebral Hemorrhage II (ATACH-2) trial, we performed HE shift analysis in response to intensive blood pressure lowering by generating polychotomous strata based on previously established HE definitions, percentile/absolute quartiles of hematoma volume change, and quartiles of 24-hour follow-up hematoma volumes. The relationship between blood pressure treatment and HE shift was explored with proportional odds models. RESULTS: The primary analysis population included 863 patients. In both treatment groups, approximately one-third of patients exhibited no HE. With the use of a trichotomous HE stratification, the highest strata of ≥33% revealed a 5.8% reduction in hematoma growth for those randomized to intensive therapy (adjusted odds ratio [aOR] 0.77, 95% confidence interval [CI] 0.60-0.99). Using percentile quartiles of hematoma volume change, we observed a favorable shift to reduce growth in patients treated with intensive therapy (aOR 0.73, 95% CI 0.57-0.93). Similarly, in a tetrachotomous analysis of 24-hour follow-up hematoma volumes, shifts in the highest stratum (>21.9 mL) were most notable. CONCLUSIONS: Our findings suggest that intensive blood pressure reduction may preferentially mitigate growth in patients at risk of high volume HE. A shift analysis model of HE provides additional insights into the biological effects of a given therapy and may be an additional way to assess hemostatic agents in future studies. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier:NCT01176565.


Assuntos
Anti-Hipertensivos/administração & dosagem , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/patologia , Hematoma/tratamento farmacológico , Hematoma/patologia , Nicardipino/administração & dosagem , Avaliação de Processos e Resultados em Cuidados de Saúde , Doença Aguda , Idoso , Hemorragia Cerebral/diagnóstico por imagem , Hemorragia Cerebral Intraventricular/diagnóstico por imagem , Hemorragia Cerebral Intraventricular/patologia , Progressão da Doença , Feminino , Hematoma/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Estudos Prospectivos
18.
Front Immunol ; 12: 624821, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34149688

RESUMO

Kidney disease affects 10% of the world population and is associated with increased mortality. Steroid-resistant nephrotic syndrome (SRNS) is a leading cause of end-stage kidney disease in children, often failing standard immunosuppression. Here, we report the results of a prospective study to investigate the immunological impact and safety of a gluten-free and dairy-free (GF/DF) diet in children with SRNS. The study was organized as a four-week summer camp implementing a strict GF/DF diet with prospective collection of blood, urine and stool in addition to whole exome sequencing WES of DNA of participants. Using flow cytometry, proteomic assays and microbiome metagenomics, we show that GF/DF diet had a major anti-inflammatory effect in all participants both at the protein and cellular level with 4-fold increase in T regulatory/T helper 17 cells ratio and the promotion of a favorable regulatory gut microbiota. Overall, GF/DF can have a significant anti-inflammatory effect in children with SRNS and further trials are warranted to investigate this potential dietary intervention in children with SRNS.


Assuntos
Laticínios/efeitos adversos , Dieta Livre de Glúten , Síndrome Nefrótica/congênito , Adolescente , Biomarcadores/sangue , Biomarcadores/urina , Criança , Pré-Escolar , Citocinas/sangue , Dieta Livre de Glúten/efeitos adversos , Estudos de Viabilidade , Feminino , Microbioma Gastrointestinal , Humanos , Lactente , Mediadores da Inflamação/sangue , Intestinos/microbiologia , Masculino , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/dietoterapia , Síndrome Nefrótica/imunologia , Síndrome Nefrótica/microbiologia , Projetos Piloto , Estudo de Prova de Conceito , Estudos Prospectivos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
19.
N Engl J Med ; 385(1): 35-45, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34192430

RESUMO

BACKGROUND: In celiac disease, small intestinal transglutaminase 2 causes deamidation of glutamine residues in gluten peptides, which enhances stimulation of T cells and leads to mucosal injury. Inhibition of transglutaminase 2 is a potential treatment for celiac disease. METHODS: In a proof-of-concept trial, we assessed the efficacy and safety of a 6-week treatment with ZED1227, a selective oral transglutaminase 2 inhibitor, at three dose levels as compared with placebo, in adults with well-controlled celiac disease who underwent a daily gluten challenge. The primary end point was the attenuation of gluten-induced mucosal damage, as measured by the ratio of villus height to crypt depth. Secondary end points included intraepithelial lymphocyte density, the Celiac Symptom Index score, and the Celiac Disease Questionnaire score (for assessment of health-related quality of life). RESULTS: Of the 41 patients assigned to the 10-mg ZED1227 group, the 41 assigned to the 50-mg group, the 41 assigned to the 100-mg group, and the 40 assigned to the placebo group, 35, 39, 38, and 30 patients, respectively, had adequate duodenal-biopsy samples for the assessment of the primary end point. Treatment with ZED1227 at all three dose levels attenuated gluten-induced duodenal mucosal injury. The estimated difference from placebo in the change in the mean ratio of villus height to crypt depth from baseline to week 6 was 0.44 (95% confidence interval [CI], 0.15 to 0.73) in the 10-mg group (P = 0.001), 0.49 (95% CI, 0.20 to 0.77) in the 50-mg group (P<0.001), and 0.48 (95% CI, 0.20 to 0.77) in the 100-mg group (P<0.001). The estimated differences from placebo in the change in intraepithelial lymphocyte density were -2.7 cells per 100 epithelial cells (95% CI, -7.6 to 2.2) in the 10-mg group, -4.2 cells per 100 epithelial cells (95% CI, -8.9 to 0.6) in the 50-mg group, and -9.6 cells per 100 epithelial cells (95% CI, -14.4 to -4.8) in the 100-mg group. Use of the 100-mg dose may have improved symptom and quality-of-life scores. The most common adverse events, the incidences of which were similar across all groups, were headache, nausea, diarrhea, vomiting, and abdominal pain. Rash developed in 3 of 40 patients (8%) in the 100-mg group. CONCLUSIONS: In this preliminary trial, treatment with ZED1227 attenuated gluten-induced duodenal mucosal damage in patients with celiac disease. (Funded by Dr. Falk Pharma; CEC-3 EudraCT number, 2017-002241-30.).


Assuntos
Doença Celíaca/tratamento farmacológico , Duodeno/patologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Imidazóis/administração & dosagem , Mucosa Intestinal/patologia , Piridinas/administração & dosagem , Transglutaminases/antagonistas & inibidores , Administração Oral , Adulto , Doença Celíaca/patologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Duodeno/imunologia , Feminino , Glutens/administração & dosagem , Glutens/efeitos adversos , Humanos , Imidazóis/efeitos adversos , Mucosa Intestinal/imunologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Estudo de Prova de Conceito , Piridinas/efeitos adversos , Qualidade de Vida , Índice de Gravidade de Doença
20.
Front Immunol ; 12: 628059, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122401

RESUMO

Artificial antigen-presenting cells (aAPCs) are synthetic versions of naturally occurring antigen-presenting cells (APCs) that, similar to natural APCs, promote efficient T effector cell responses in vitro. This report describes a method to produce acellular tolerogenic aAPCs made of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and encapsulating IL-2 and TGF-ß for a paracrine release to T cells. We document that these aAPCs can induce both human CD4+ and CD8+ T cells to become FoxP3+ T regulatory cells (Tregs). The aAPC NP-expanded human Tregs are functional in vitro and can modulate systemic autoimmunity in vivo in humanized NSG mice. These findings establish a proof-of-concept to use PLGA NPs as aAPCs for the induction of human Tregs in vitro and in vivo, highlighting the immunotherapeutic potential of this targeted approach to repair IL-2 and/or TGF-ß defects documented in certain autoimmune diseases such as systemic lupus erythematosus.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Doença Enxerto-Hospedeiro/prevenção & controle , Interleucina-2/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/transplante , Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Interleucina-2/química , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Estudo de Prova de Conceito , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/química
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