RESUMEN
OBJECTIVES: Analysis of breath, specifically the patterns of volatile organic compounds (VOCs), has shown the potential to distinguish between patients with lung cancer (LC) and healthy individuals (HC). However, the current technology relies on complex, expensive and low throughput analytical platforms, which provide an offline response, making it unsuitable for mass screening. A new portable device has been developed to enable fast and on-site LC diagnosis, and its reliability is being tested. METHODS: Breath samples were collected from patients with histologically proven non-small-cell lung cancer (NSCLC) and healthy controls using Tedlar bags and a Nafion filter attached to a one-way mouthpiece. These samples were then analysed using an automated micro portable gas chromatography device that was developed in-house. The device consisted of a thermal desorption tube, thermal injector, separation column, photoionization detector, as well as other accessories such as pumps, valves and a helium cartridge. The resulting chromatograms were analysed using both chemometrics and machine learning techniques. RESULTS: Thirty NSCLC patients and 30 HC entered the study. After a training set (20 NSCLC and 20 HC) and a testing set (10 NSCLC and 10 HC), an overall specificity of 83.3%, a sensitivity of 86.7% and an accuracy of 85.0% to identify NSCLC patients were found based on 3 VOCs. CONCLUSIONS: These results are a significant step towards creating a low-cost, user-friendly and accessible tool for rapid on-site LC screening. CLINICAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT06034730.
RESUMEN
Hepatotropic viruses are amongst the most ubiquitous pathogens worldwide, causing significant morbidity and mortality. As hepatocytes are among the primary targets of these viruses, their ability to mount early effective innate defence responses is of major research interest. Interferon lambda (IFNL) is produced early in response to viral stimulation in other cell types, but hepatocyte production of this interferon is little investigated. Due to the difficulty and significant costs in obtaining and culturing human primary hepatocytes, surrogate systems are widely sought. Here we used induced pluripotent stem (iPS)-derived hepatocyte-like cells (HLCs) to investigate hepatic IFNL expression in response to viral-like ligands. We demonstrate that hepatocytes rely on cytoplasmic pattern recognition receptors (PRRs) such as Protein Kinase RNA-dependent (PKR) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) for the detection of double stranded RNA. Stimulation of HLCs by viral-like RNA ligands activating cytosolic RNA sensors resulted in thousand fold increase of type III interferon gene expression. These results are in contrast with type I IFN expression, which was induced to a lower extent. Concomitant induction of interferon stimulated genes, such as interferon-stimulated gene 15 (ISG15) and CXCL10, indicated the ability of HLCs to activate interferon-dependent activity. These results demonstrate that HLCs mount an innate antiviral response upon stimulation with viral-like RNA characterized by the induction of type III IFN.
RESUMEN
Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.
Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Lisosomas , Degeneración Macular , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Sirtuinas , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuinas/metabolismo , Sirtuinas/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Lisosomas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
We have demonstrated a Pd(0)-catalyzed Heck/C(sp3)-H activation cascade for the synthesis of spirocyclopropyl oxindoles in high yields from easily accessible ortho-bromoacrylamides. The formation of spirocyclopropyl oxindole is guided by an unconventional four-membered palladacycle through C(sp3)-H activation. The reaction exhibits a wide range of substrate scope and operates efficiently with a mere 0.5 mol % of Pd-catalyst. In addition, the use of microwave conditions facilitates rapid completion of the reaction. Furthermore, this spirocyclopropanation strategy can be coupled with [3 + 2] cycloaddition to produce spiropyrrolidine oxindoles, offering a valuable approach for the preparation of alkaloids such as (±)-horsfiline and (±)-coerulescine.
RESUMEN
Screening methods available for colorectal cancer (CRC) to date are burdened by poor reliability and low patient adherence and compliance. An altered pattern of volatile organic compounds (VOCs) in exhaled breath has been proposed as a non-invasive potential diagnostic tool for distinguishing CRC patients from healthy controls (HC). The aim of this study was to evaluate the reliability of an innovative portable device containing a micro-gas chromatograph in enabling rapid, on-site CRC diagnosis through analysis of patients' exhaled breath. In this prospective trial, breath samples were collected in a tertiary referral center of colorectal surgery, and analysis of the chromatograms was performed by the Biomedical Engineering Department. The breath of patients with CRC and HC was collected into Tedlar bags through a Nafion filter and mouthpiece with a one-way valve. The breath samples were analyzed by an automated portable gas chromatography device. Relevant volatile biomarkers and discriminant chromatographic peaks were identified through machine learning, linear discriminant analysis and principal component analysis. A total of 68 subjects, 36 patients affected by histologically proven CRC with no evidence of metastases and 32 HC with negative colonoscopies, were enrolled. After testing a training set (18 CRC and 18 HC) and a testing set (18 CRC and 14 HC), an overall specificity of 87.5%, sensitivity of 94.4% and accuracy of 91.2% in identifying CRC patients was found based on three VOCs. Breath biopsy may represent a promising non-invasive method of discriminating CRC patients from HC.
Asunto(s)
Pruebas Respiratorias , Neoplasias Colorrectales , Humanos , Datos Preliminares , Estudios Prospectivos , Reproducibilidad de los Resultados , Neoplasias Colorrectales/diagnósticoRESUMEN
Purpose: The aim of this study was to develop and validate a test to assess visual function in pigs using the visual psychophysics contrast sensitivity function. Methods: We utilized a touchscreen along with a pellet reward dispenser to train three Göttingen pigs on a visual psychophysics test and determined their contrast sensitivity function. Images with different contrast resolutions were used as visual stimuli and presented against a control image in a two-choice test. Following animals' acclimatization and the first phase of training, the system was arranged such that animals could self-run multiple consecutive trials without human intervention. Results: All animals were trained within a week and remembered the task with 1 day of reinforcement when tested 1 month after the last visual assessment. All trained animals performed well during the trial with minimal screen side bias, especially at contrast threshold above 40%. Conclusions: Göttingen pigs are trainable for a visual psychophysics test and able to self-run the trial without human intervention. Translational Relevance: Contrast sensitivity is one of the key parameters to assess visual function in humans. The possibility of measuring the same parameters in a large animal model allows for a better translation and understanding of drug safety and efficacy in preclinical ophthalmology.
Asunto(s)
Oftalmología , Humanos , Animales , Porcinos , Modelos Animales , PsicofísicaRESUMEN
A sesquiterpenic alkaloid dendrobine is the major bioactive compound extracted from Dendrobium nobile Lindl. This compound was structurally very similar to picrotoxinin (neurotoxin) containing bicyclic or tricyclic ring while dendrobine containing tetracyclic rings with up to 7 stereogenic centers showing numerous neuroprotective effects. Several sesquiterpenic alkaloids such as (+)-(1R,5R,6S,8R,9R)- 8,12-dihydroxy-copacamphan-3-en-2-one, dendrobine, denrine B, (+)- (1R,2S,3R,4S,5R,6S,9R)-3,11,12-trihydroxypicrotoxane-2(15)-lactone, dendroxine B, nobilonine, 13-Hydroxy-14-oxodendrobine, 6-Hydroxy-nobilonine and (-)-(1S,2R,3S,4R,5S,6R,9S,12R)- 3,11,13-trihydroxypicrotoxane-2(15)-lactone were isolated from D. nobile This comprehensive review summarizes the necessary information on the morphology, biochemistry and pharmacology of dendrobine. The phytochemical profile had potent in-vivo and in-vitro efficacy in neuroprotection, nerve function, memory enhancement, cognitive disorders, anxiety and depression, anti-oxidant, psychological conditions, increasing serotonin concentration in synapse anxiolytic, tranquilizing, anti-stress, neurodegenerative (Alzheimer's disease and Parkinson), anti-inflammatory and analgesic. The compound dendrobine activates neuro synapses, serotonergic synapse and signaling pathways during neurotransmission playing important role in Alzheimer's disease, Parkinson's disease, anxiety and long-term depression.
Asunto(s)
Alcaloides , Fármacos Neuroprotectores , Sesquiterpenos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Humanos , Sesquiterpenos/química , Sesquiterpenos/farmacología , Sesquiterpenos/aislamiento & purificación , Alcaloides/química , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Animales , Enfermedades Neurodegenerativas/tratamiento farmacológicoRESUMEN
A 1D-guided differential rescaling algorithm for a contour plot is developed based on our recently proposed comprehensive two-dimensional gas chromatography (GC × GC) system with a first-dimensional (1D) detector added. Chromatograms obtained from 1D and second-dimensional (2D) detectors are both incorporated during the data processing. As compared to the conventional contour plot methods using only 2D data, our algorithm can significantly improve precision and consistency of GC × GC results in terms of retention times, peak widths, and peak areas or volumes, regardless of modulation time selection, modulation phase shift fluctuations, and modulation duty cycle. The peak identification, quantification, and capacity can therefore be enhanced. Furthermore, the 1D-guided differential rescaling method is shown to better handle the coelution and missing peak issues often encountered in the conventional methods. Finally, the new method exhibits high versatility in 1D and 2D detector selection, which greatly broadens GC × GC utility. Our method can easily be adapted to other two-dimensional chromatography systems that have direct access to 1D chromatograms.
RESUMEN
Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. "monocyte factory." We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. For complete details on the use and execution of this protocol, please refer to Karlson et al.1 and Panicker et al.2.
Asunto(s)
Células Madre Pluripotentes Inducidas , Monocitos , Humanos , Macrófagos , Diferenciación Celular , Cuerpos EmbrioidesRESUMEN
Parkinson's disease (PD) and Alzheimer's disease (AD) are neurodegenerative disorders caused by the interaction of genetic, environmental, and familial factors. These diseases have distinct pathologies and symptoms that are linked to specific cell populations in the brain. Notably, the immune system has been implicated in both diseases, with a particular focus on the dysfunction of microglia, the brain's resident immune cells, contributing to neuronal loss and exacerbating symptoms. Researchers use models of the neuroimmune system to gain a deeper understanding of the physiological and biological aspects of these neurodegenerative diseases and how they progress. Several in vitro and in vivo models, including 2D cultures and animal models, have been utilized. Recently, advancements have been made in optimizing these existing models and developing 3D models and organ-on-a-chip systems, holding tremendous promise in accurately mimicking the intricate intracellular environment. As a result, these models represent a crucial breakthrough in the transformation of current treatments for PD and AD by offering potential for conducting long-term disease-based modeling for therapeutic testing, reducing reliance on animal models, and significantly improving cell viability compared to conventional 2D models. The application of 3D and organ-on-a-chip models in neurodegenerative disease research marks a prosperous step forward, providing a more realistic representation of the complex interactions within the neuroimmune system. Ultimately, these refined models of the neuroimmune system aim to aid in the quest to combat and mitigate the impact of debilitating neuroimmune diseases on patients and their families.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Humanos , Sistema Inmunológico , MicroglíaRESUMEN
Cell-replacement therapies are a new class of treatments, which include induced pluripotent stem cell (iPSC)-derived tissues that aim to replace degenerated cells. iPSCs can potentially be used to generate any cell type of the body, making them a powerful tool for treating degenerative diseases. Cell replacement for retinal degenerative diseases is at the forefront of cell therapies, given the accessibility of the eye for surgical procedures and a huge unmet medical need for retinal degenerative diseases with no current treatment options. Clinical trials are ongoing in different parts of the world using stem cell-derived retinal pigment epithelium (RPE). This review focuses on scientific and regulatory considerations when developing an iPSC-derived RPE cell therapy from the development of a robust and efficient differentiation protocol to critical quality control assays for cell validation, the choice of an appropriate animal model for preclinical testing, and the regulatory aspects that dictate the final approval for proceeding to a first-in-human clinical trial.
Asunto(s)
Células Madre Pluripotentes Inducidas , Animales , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Diferenciación Celular , Modelos AnimalesRESUMEN
OBJECTIVES: Dysregulation of hepcidin-iron axis is presumed to account for abnormal iron status in patients with chronic liver disease (CLD). Our aim is to determine the effect of specific etiologies of CLD and of cirrhosis on serum hepcidin levels. METHODS: PubMed, Embase, Web of Science were searched for studies comparing serum hepcidin levels in patients with CLD to that in controls using enzyme-linked immunosorbent assay. The study was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Guidelines. Statistical analysis was carried out with STATA using random effects model to calculate the mean difference (MD) between two groups. RESULTS: Hepcidin levels were significantly lower in subjects with hepatitis C virus (16 studies) [MD -1.6 (95â¯% CI: -2.66 to -0.54), p<0.01] and alcoholic liver disease (3 studies) [MD -0.84 (95â¯% CI: -1.6 to -0.07), p=0.03] than controls. Serum hepcidin was significantly higher in subjects with non-alcoholic fatty liver disease (12 studies) [MD 0.62 (95â¯% CI: 0.21 to 1.03), p<0.01], but did not differ in subjects with hepatitis B and controls (eight studies) [MD -0.65 (95â¯% CI: -1.47 to 0.16), p=0.12]. Hepcidin levels were significantly lower in patients with cirrhosis of any etiology (four studies) [MD -1.02 (CI: -1.59 to -0.45), p<0.01] vs. controls (CI: confidence interval). CONCLUSIONS: Serum hepcidin levels are altered in common forms of CLD albeit not in a consistent direction. Additional study is needed to determine how changes in hepcidin levels are related to dysregulation of iron metabolism in CLD.
Asunto(s)
Hepcidinas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ferritinas , Cirrosis Hepática , Hierro/metabolismoRESUMEN
The clinical significance of volatile organic compounds (VOC) in detecting diseases has been established over the past decades. Gas chromatography (GC) devices enable the measurement of these VOCs. Chromatographic peak alignment is one of the important yet challenging steps in analyzing chromatogram signals. Traditional semi-automated alignment algorithms require manual intervention by an operator which is slow, expensive and inconsistent. A pipeline is proposed to train a deep-learning model from artificial chromatograms simulated from a small, annotated dataset, and a postprocessing step based on greedy optimization to align the signals.Clinical Relevance- Breath VOCs have been shown to have a significant diagnostic power for various diseases including asthma, acute respiratory distress syndrome and COVID-19. Automatic analysis of chromatograms can lead to improvements in the diagnosis and management of such diseases.
Asunto(s)
Aprendizaje Profundo , Compuestos Orgánicos Volátiles , Cromatografía de Gases/métodos , Algoritmos , Simulación por Computador , Compuestos Orgánicos Volátiles/análisisRESUMEN
Spices are a rich source of vitamins, polyphenols, proteins, dietary fiber, and minerals such as calcium, magnesium, iron, and zinc, all of which play an important role in biological functions. Since ancient times, spices have been used in our kitchen as a food coloring agent. Spices like cinnamon and turmeric allegedly contain various functional ingredients, such as phenolic and volatile compounds. Therefore, this review aims to summarize the current knowledge about the nutritional profiles of cinnamon and turmeric, as well as to analyze the clinical studies on their extracts and essential oils in animals and humans. Furthermore, their enrichment applications for food products and animal feed have also been investigated in terms of safety and toxicity. Numerous studies have shown that cinnamon and turmeric have various health benefits, including the reduction of insulin resistance and insulin signaling pathways in diabetic patients, the reduction of inflammatory biomarkers, and the maintenance of gut microflora in both animals and humans. The food and animal feed industries have taken notice of these health benefits and have begun to promote cinnamon and turmeric as healthy foods. This has resulted in the development of new food products and animal feeds that contain cinnamon and turmeric as primary ingredients, which have been deemed an effective means of promoting cinnamon and turmeric's health benefits.
RESUMEN
Traditional and scientific evidence attribute numerous bioactivities of Licorice (Glycyrrhiza glabra Linn.) in aging-related disorders. In this state-of-art review, an extensive search in several databases was conducted to collect all relevant literature and comprehensively analyze Licorice's pharmacological attributes, neuroprotective properties, safety, and its mechanistic role in treating various neurological conditions. Network pharmacology was employed for the first time exploring the mechanistic role of Licorice in neurological disorders. Its neuroprotective role is attributed to phytoconstituents, including liquiritin, glycyrrhizic acid, liquiritigenin, glabridin, 18ß-glycyrrhetinic acid, quercetin, isoliquiritigenin, paratocarpin B, glycyglabrone, and hispaglabridin B, as evident from in vitro and in vivo studies. Network pharmacology analysis reveals that these compounds protect against long-term depression, aging-associated diseases, Alzheimer's disease, and other addictions through interactions with cholinergic, dopaminergic, and serotonergic proteins, validated in animal studies only. Future clinical trials are warranted as Licorice administration has a limiting factor of mild hypertension and hypokalemia. Hopefully, scientific updates on Licorice will propagate a paradigm shift in medicine, research propagation, and development of the central nervous system phytopharmaceuticals.
Asunto(s)
Ácido Glicirretínico , Glycyrrhiza , Enfermedades del Sistema Nervioso , Animales , Alimentos Funcionales , Ácido Glicirretínico/farmacología , Extractos Vegetales/farmacología , Ácido Glicirrínico/farmacología , Enfermedades del Sistema Nervioso/tratamiento farmacológicoRESUMEN
Early diagnosis of skin barrier dysfunction helps provide timely preventive care against diseases such as atopic dermatitis, psoriasis, food allergies, and other atopic skin disorders. Skin barrier function is commonly evaluated by measuring the transepidermal water loss (TEWL) through stratum corneum due to its noninvasive characteristics. However, existing commercial TEWL devices are significantly affected by many factors, such as ambient temperature, humidity, air flow, water accumulation, initial water contents on the skin surface, bulky sizes, high costs, and requirements for well-controlled environments. Here, we developed a wearable closed-chamber hygrometer-based TEWL device (Wearable Analytical Skin Probe, WASP) and the related algorithm for accurate and continuous monitoring of skin water vapor flux. The WASP uses short dry air purges to dry the skin surface and chamber before each water vapor flux measurement. Its design ensures a highly controlled local environment, such as consistent initial dry conditions for the skin surface and the chamber. We further applied WASP to measure the water vapor flux from six different locations of a small group of human participants. It is found that the WASP can not only measure and distinguish between insensible sweating (i.e., TEWL) and sensible sweating (i.e., thermal sweating) but also track skin dehydration-rehydration cycles. Comparisons with a commercial TEWL device, AquaFlux, show that the results obtained by both devices agree well. The WASP will be broadly applicable to clinical, cosmetic, and biomedical research.
Asunto(s)
Vapor , Pérdida Insensible de Agua , Humanos , Piel , Epidermis , HumedadRESUMEN
Nowadays, fruits are gaining high demand due to their promising advantages on human health. Astonishingly, their by-products, that is, seeds and peels, account for 10-35% of fruit weight and are usually thrown as waste after consumption or processing. But it is neglected that fruit seeds also have functional properties and nutritional value, and thus could be utilized for dietary and therapeutic purposes, ultimately reducing the waste burden on the environment. Owing to these benefits, researchers have started to assess the nutritional value of different fruits seeds, in addition to the chemical composition in various bioactive constituents, like carotenoids (lycopene), flavonoids, proteins (bioactive peptides), vitamins, etc., that have substantial health benefits and can be used in formulating different types of food products with noteworthy functional and nutraceutical potential. The current review aims to comprehend the known information of nutritional and phytochemical profiling of non-edible fruits seeds, viz. apple, apricot, avocado, cherry, date, jamun, litchi, longan, mango, and papaya. Additionally, clinical studies conducted on these selected non-edible fruit seed extracts, their safety issues and their enrichment in food products as well as animal feed has also been discussed. This review aims to highlight the potential applications of the non-edible fruit seeds in developing new food products and also provide a viable alternative to reduce the waste disposal issue faced by agro-based industries.
RESUMEN
Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances (n = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology.NEW & NOTEWORTHY This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.
Asunto(s)
Células Epiteliales , Epitelio Pigmentado de la Retina , Humanos , Epitelio/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Membrana Celular/metabolismo , Modelos TeóricosRESUMEN
The emergence of antibiotic resistance prompts exploration of viable antimicrobial peptides (AMPs) designs. The present study explores the antimicrobial prospects of Apoptin nuclear localization sequence (NLS2)-derived peptide ANLP (PRPRTAKRRIRL). Further, we examined the utility of the NLS dimerization strategy for improvement in antimicrobial activity and sustained bio-stability of AMPs. Initially, the antimicrobial potential of ANLP using antimicrobial peptide databases was analyzed. Then, ANLP along with its two homodimer variants namely ANLP-K1 and ANLP-K2 were synthesized and evaluated for antimicrobial activity against Escherichia coli and Salmonella. Among three AMPs, ANLP-K2 showed efficient antibacterial activity with 12 µM minimum inhibitory concentration (MIC). Slow degradation of ANLP-K1 (26.48%) and ANLP-K2 (13.21%) compared with linear ANLP (52.33%) at 480 min in serum stability assay indicates improved bio-stability of dimeric peptides. The AMPs presented no cytotoxicity in Vero cells. Dye penetration assays confirmed the membrane interacting nature of AMPs. The zeta potential analysis reveals effective charge neutralization of both lipopolysaccharide (LPS) and bacterial cells by dimeric AMPs. The dimeric AMPs on scanning electron microscopy studies showed multiple pore formations on the bacterial surface. Collectively, proposed Lysine scaffold dimerization of Apoptin NLS2 strategy resulted in enhancing antibacterial activity, bio-stability, and could be effective in neutralizing the off-target effect of LPS. In conclusion, these results suggest that nuclear localization sequence with a modified dimeric approach could represent a rich source of template for designing future antimicrobial peptides.
Asunto(s)
Antiinfecciosos , Lipopolisacáridos , Animales , Chlorocebus aethiops , Lipopolisacáridos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Dimerización , Células Vero , Antibacterianos/farmacología , Antibacterianos/química , Péptidos Antimicrobianos , Pruebas de Sensibilidad MicrobianaRESUMEN
The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.