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1.
J Food Sci Technol ; 60(9): 2486-2496, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37424575

RESUMO

Two types of mayonnaise viz. eggless and egg based were prepared by substituting refined soybean oil by tomato seed oil (TSO) at varying concentrations (0-30%). Aim of the study was to utilise the potential of TSO in replaced of refined oil. With respect to oil particle distribution pattern in both types of mayonnaise, higher specific surface area (Dmean ~ 11.49 µm) and homogenous distribution of oil droplets was noted in egg-based mayonnaise. Rheological behavior depicted shear thinning properties in all types of mayonnaise with low viscosity (1.08 Pa s and 2.29 Pa s) being exhibited by tomato seed oil (TSO) incorporated mayonnaise. Nutritionally, significant increase of 65.5 and 26% in lycopene content while 29 and 34% increase in carotenoid content was noted upon incorporation of TSO in eggless and egg-based mayonnaise. Good storage and oxidative stability were demonstrated by TSO egg-based and eggless mayonnaise in terms of acid value and free fatty acids, also the peroxide value was also found lower than their respective control at the end of storage. Overall, tomato seed oil could be used as a non-conventional source of oil for such food applications owing to its close proximity with other vegetable oils and better nutritional profile like dominance of linoleic fatty acid i.e., 54.23% assayed via gas chromatography. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05771-7.

2.
J Food Sci Technol ; 59(12): 4956-4968, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36276515

RESUMO

Burfi, an Indian traditional dairy dessert is highly popular, however, its low nutritive value and poor shelf life limits commercial viability. Kinnow juice was utilized to improve the phytonutritional profile and shelf stability of burfi. To further improve the quality, pectin was added at different concentrations (1-4%). Compared to control, inclusion of kinnow juice significantly (p < 0.05) improved minerals and phytochemical constituents as confirmed using FTIR analysis. Developed burfi presented a softer texture which was consistent with SEM results. Increasing the pectin levels, increased textural properties of kinnow burfi while decreased moisture content and water activity, without affecting color and nutritional properties. Based on desirable sensory and textural properties, kinnow burfi prepared with pectin (4% of added sugar) was selected for product development and evaluation of shelf life under room (25 ± 2 °C) and refrigerated (4 ± 2 °C) conditions. Irrespective of storage temperatures, moisture content and bioactive constituents decreased while titratable acidity, total and reducing sugars, free fatty acids, hardness of texture, and yeast and mould count increased significantly. Except moisture, the extent of quality changes was rapid at room temperature. The product showed high sensory acceptability as well as microbial safety up to 21 days at room temperature and 28 days under refrigeration.

3.
Adv Exp Med Biol ; 1167: 175-190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31520355

RESUMO

Accumulative studies suggest that a fraction of cells within a tumor, known as cancer stem cells (CSCs) that initiate tumors, show resistance to most of the therapies, and causes tumor recurrence and metastasis. CSCs could be either transformed normal stem cells or reprogrammed differentiated cells. The eventual goal of CSC research is to identify pathways that selectively regulate CSCs and then target these pathways to eradicate CSCs. CSCs and normal stem cells share some common features, such as self-renewal, the production of differentiated progeny, and the expression of stem-cell markers, however, CSCs vary from normal stem cells in forming tumors. Specifically, CSCs are normally resistant to standard therapies. In addition, CSCs and non-CSCs can be mutually convertible in response to different signals or microenvironments. Even though CSCs are involved in human cancers, the biology of CSCs, is still not well understood, there are urgent needs to study CSCs in model organisms. In the last several years, discoveries in Drosophila have greatly contributed to our understanding of human cancer. Stem-cell tumors in Drosophila share various properties with human CSCs and maybe used to understand the biology of CSCs. In this chapter, we first briefly review CSCs in mammalian systems, then discuss stem-cell tumors in the Drosophila posterior midgut and Malpighian tubules (kidney) and their unique properties as revealed by studying oncogenic Ras protein (RasV12)-transformed stem-cell tumors in the Drosophila kidney and dominant-negative Notch (NDN)-transformed stem-cell tumors in the Drosophila intestine. At the end, we will discuss potential approaches to eliminate CSCs and achieve tumor regression. In future, by screening adult Drosophila neoplastic stem-cell tumor models, we hope to identify novel and efficacious compounds for the treatment of human cancers.


Assuntos
Drosophila , Neoplasias/patologia , Células-Tronco Neoplásicas/citologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Microambiente Tumoral
4.
J Food Sci Technol ; 53(4): 1899-908, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27413216

RESUMO

The present study was carried out to develop waris from potato and legume blends and to analyze them for organoleptic, physicochemical, phytochemical and shelf life quality. Wari is a partially fermented legume based savoury, used as adjunct in vegetable curries. In this study, potato (boiled and dehydrated) was used to supplement black gram or urad dhal waris. Two processing cultivars (Kufri Chipsona-1, Kufri Chandramukhi) and one commonly grown cultivar (Kufri Pukhraj) were evaluated for processing into waris. Based on preliminary sensory trails, waris with potato (70 %) and urad dhal (30 %) level of supplementation were found to be most acceptable and these waris were subjected to nutritional evaluation. Storage stability of the waris was assessed by storing the product at room temperature for a period of 12 months. Results were compared with dhal waris (control). Protein content was significantly higher in control waris compared to potato supplemented waris. Bioactive compounds including ascorbic acid, total phenolics and total antioxidant activity measured as DPPH radical scavenging activity increased significantly on incorporation of potato. Between the cultivars, waris enriched with Kufri Pukhraj, a table variety which is considered unfit for processing, displayed the highest phytochemical content and total antioxidant activity. Sensory evaluation indicated higher overall acceptability scores of potato enriched waris compared to control waris. Between the treatments i.e. boiled mash and dehydrated flour, waris supplemented with boiled potato mash showed a significantly higher content of phytochemicals and total antioxidant activity compared to potato flour waris. However no significant difference was observed in sensory quality of the product prepared either with fresh potato mash or potato flour. Storage studies showed that the potato waris can be stored safely for 12 months with its nutrient constituents intact.

5.
J Food Sci Technol ; 52(9): 5882-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26345004

RESUMO

Texturized defatted meals from sunflower, soybean and flaxseed prepared using extrusion technology were incorporated in noodles to improve the protein content of noodles. Noodles were also evaluated for chemical composition, cooking quality, color, functional, textural and sensory properties. Sensory, color and cooking characteristics of noodles were negatively affected with increasing level as texturized flour compared with the control. Noodles with 10 % texturized sunflower and flaxseed flour received the highest sensory scores. Overall acceptability scores were maximum for control and noodles with 10 % texturized defatted meal of sunflower and flaxseed. Further incorporation of 20 % texturized defatted flour from soybean in noodle making gave satisfactory results in terms of overall acceptability. It was concluded that texturized defatted meal serve as good substitute to wheat flour with increased protein content in noodles production and utilization.

6.
Front Nutr ; 10: 1178797, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37215207

RESUMO

Introduction: Colored potatoes comprise many bioactive compounds that potentially support human health. Polyphenols present in them have associated therapeutic benefits like antimutagenic and anticarcinogenic properties. Method: The current study aimed to explore the effects of different blanching methods (steam blanching, hot water blanching, and microwave-assisted blanching) on the phytochemical and structural aspects of PP-1901 and Lady Rosetta (LR) potato varieties. Changes in the antioxidant activity, color, total ascorbic acid, phenolic, and flavonoid content were based on the variations in parameters including temperature (blanching using hot water and steam) and capacity 100- 900 W (blanching using microwave). Results: For both PP-1901 and LR varieties, all the blanching methods led to a significant reduction in residual peroxidase activity, as well as affecting their color. The preservation of bioactive substances exhibited a microwave steam>hot water blanching trend. Blanching significantly increased the antioxidant activity of all the samples. Additionally, Fourier-transform infrared spectroscopy revealed that phytocompounds were retained to their maximum in microwave-blanched samples, especially at 300 W. The type of blanching method significantly affected the thermal properties of potatoes by disrupting the ordered structure of the matrix. Discussion: Microwaves at 300 W can be used as a novel and suitable alternative technique for blanching potatoes, which successfully retained the original quality of it in comparison to steam and hot water blanching.

7.
Pharmaceuticals (Basel) ; 16(2)2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-37259445

RESUMO

In parallel with a steady rise in cancer incidence worldwide, the scientific community is increasingly focused on finding novel, safer and more efficient modalities for managing this disease. Over the past decades, natural products have been described as a significant source of new structural leads for novel drug candidates. Scutellaria root is one of the most studied natural products because of its anticancer potential. Besides just describing the cytotoxic properties of plant constituents, their molecular mechanisms of action in different cancer types are equally important. Therefore, this review article focuses on the role of the Scutellaria flavones wogonin, baicalein, baicalin, scutellarein and scutellarin in regulating the autophagic machinery in diverse cancer models, highlighting these molecules as potential lead compounds for the fight against malignant neoplasms. The knowledge that autophagy can function as a dual-edged sword, acting in both a pro- and antitumorigenic manner, further complicates the issue, revealing an amazing property of flavonoids that behave either as anti- or proautophagic agents.

8.
J Food Sci Technol ; 49(1): 110-4, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23572833

RESUMO

Studies were carried out to replace bakery shortening with refined rice bran oil in bread preparation. Physico-chemical properties of bakery shortening and rice bran oil were studied. Rice bran oil was found to have a higher content of essential fatty acid linoleic acid (34.98%) as compared to that of bakery shortening (5.14%). Chemical composition of wheat flour used was also evaluation. Bread samples were prepared by replacing bakery shortening with rice bran oil at 0, 25, 50, 75 and 100% levels. Breads were examined for bread making quality i.e. loaf weight, loaf height, loaf volume and specific volume and organoleptic quality i.e. appearance, crust colour, crumb colour, aroma, texture, taste and overall acceptability on a 9 point hedonic scale. Statistical analysis regarding bread making quality and organoleptic quality of bread revealed that bread making and organoleptic quality of breads prepared after replacing bakery shortening with rice bran oil at 50% level varied significantly from that of control. Statistically significant variations were observed in the texture of breads prepared with shortening from that prepared after replacing bakery shortening with rice bran oil at 50% level.

9.
Nat Rev Cancer ; 22(12): 703-722, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36253536

RESUMO

Growing evidence suggests that the gut microbiota modulates the efficacy and toxicity of cancer therapy, most notably immunotherapy and its immune-related adverse effects. The poor response to immunotherapy in patients treated with antibiotics supports this influential role of the microbiota. Until recently, results pertaining to the identification of the microbial species responsible for these effects were incongruent, and relatively few studies analysed the underlying mechanisms. A better understanding of the taxonomy of the species involved and of the mechanisms of action has since been achieved. Defined bacterial species have been shown to promote an improved response to immune-checkpoint inhibitors by producing different products or metabolites. However, a suppressive effect of Gram-negative bacteria may be dominant in some unresponsive patients. Machine learning approaches trained on the microbiota composition of patients can predict the ability of patients to respond to immunotherapy with some accuracy. Thus, interest in modulating the microbiota composition to improve patient responsiveness to therapy has been mounting. Clinical proof-of-concept studies have demonstrated that faecal microbiota transplantation or dietary interventions might be utilized clinically to improve the success rate of immunotherapy in patients with cancer. Here, we review recent advances and discuss emerging strategies for microbiota-based cancer therapies.


Assuntos
Microbioma Gastrointestinal , Microbiota , Neoplasias , Humanos , Microbioma Gastrointestinal/fisiologia , Imunoterapia/métodos , Transplante de Microbiota Fecal/métodos , Neoplasias/tratamento farmacológico
10.
Cell Rep ; 39(12): 110958, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35732115

RESUMO

We previously showed that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. Here we show that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. We identify an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity.


Assuntos
Drosophila , Lipólise , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular , Drosophila/metabolismo , Células-Tronco Neoplásicas/metabolismo
11.
Curr Res Food Sci ; 4: 503-520, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34401747

RESUMO

For many years, conventional plastics are manufactured and used for packaging applications in different sectors. As the food industries are increasing, the demand for packaging material is also increasing. Plastics have transformed the food industry to higher levels; however, conventional petroleum-based plastics are non-degradable which has created severe ecological problems to the environment like a threat to aquatic life and degrading air quality. Biodegradable polymers or biopolymers emerged as an alternative approach for many industrial applications to control the risk caused by non-biodegradable plastic. According to the type of starting material, they have been categorized as polymers extracted from biomass, synthesized from monomers, and produced from microorganisms. The quality of biopolymers depends on the physical, mechanical, thermal, and barrier properties. The present review highlights the characteristics of various biopolymers and their blends, comparison of properties between non-biodegradable and biopolymers, the market potential for food packaging applications. The review also emphasizes different commercial forms like films, trays, bags, coatings, and foamed products for application as modified atmosphere packaging, active packaging, and edible packaging. Different issues affecting market growth like harmful products formed during production and consumer perception have also been discussed. Information on biopolymers is widely scattered over many sources, this article aims to provide an overview of biodegradable polymer packages for food applications.

12.
Front Cell Infect Microbiol ; 11: 763687, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34970505

RESUMO

Within almost the last 2 years, the world has been shaken by the coronavirus disease 2019 (COVID-19) pandemic, which has affected the lives of all people. With nearly 4.92 million deaths by October 19, 2021, and serious health damages in millions of people, COVID-19 has been the most serious global challenge after the Second World War. Besides lost lives and long-term health problems, devastating impact on economics, education, and culture will probably leave a lasting impression on the future. Therefore, the actual extent of losses will become obvious only after years. Moreover, despite the availability of different vaccines and vaccination programs, it is still impossible to forecast what the next steps of the virus are or how near we are to the end of the pandemic. In this article, the route of molecular evolution of the coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thoroughly compiled, highlighting the changes that the virus has undergone during the last 2 years and discussing the approaches that the medical community has undertaken in the fight against virus-induced damages.


Assuntos
COVID-19 , Preparações Farmacêuticas , Vacinas , Evolução Molecular , Humanos , Pandemias , SARS-CoV-2
13.
Xenobiotica ; 40(8): 586-92, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20509748

RESUMO

Osteoarthritis (OA) is a degenerative joint disease that has no FDA-approved treatment. The current standard of care does not address the regeneration of the damaged cartilage. Human growth hormone (hGH) is part of the insulin-like growth factor (IGF)-1 axis. There has been preclinical data that suggest its potential regenerative property in the joint. However, unformulated recombinant hGH (rhGH) is short-lived in the joint, and does not provide a desirable pharmacokinetic (PK) profile to support a clinical treatment paradigm. Polyethylene glycol (PEG)ylation is a potential method to extend the half-life of rhGH in the joint. The purpose of this study was to delineate the PK/PD profile of PEG-rhGH in the knee joint in a rat preclinical model of OA. After intra-articular (IA) injection of 100 microg into a rat knee joint that underwent medial meniscectomy, PEG-rhGH exhibits 2-fold longer half-lives in joint than native hGH. However, PEG-rhGH has a much longer systemic exposure. IA injections of PEG-rhGH also resulted in higher levels of IGF-1 in the joint and serum when compared with native rhGH. In order to develop PEG-rhGH as an IA therapeutic treatment for OA, careful dose selection is necessary to avoid systemic effects while retaining its anabolic efficacy in the joint.


Assuntos
Hormônio do Crescimento Humano/análogos & derivados , Hormônio do Crescimento Humano/farmacocinética , Osteoartrite/metabolismo , Polietilenoglicóis/farmacocinética , Proteínas Recombinantes/farmacocinética , Animais , Modelos Animais de Doenças , Meia-Vida , Masculino , Osteoartrite/tratamento farmacológico , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley
14.
JNMA J Nepal Med Assoc ; 58(232): 1080-1082, 2020 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-34506397

RESUMO

Congenital external canal atresia is one of the congenital ear anomalies that can occur in patients. Similarly, congenital cholesteatoma is also another congenital disease that is often diagnosed in early adulthood. Both the above-mentioned diseases can occur independently but the presence of both these entities is a rare occurrence and needs a high degree of suspicion aided by a computed tomography scan to make the diagnosis. We are presenting a case of a sixteen-year-old patient who presented with unilateral ear anomaly, earache, facial palsy, and postaural swelling and was diagnosed as a right sided congenital aural atresia with congenital cholesteatoma. He was surgically managed with right-sided modified radical mastoidectomy with canaloplasty and closure of mastoid fistula under general anesthesia.


Assuntos
Colesteatoma , Otopatias , Adolescente , Adulto , Colesteatoma/diagnóstico por imagem , Colesteatoma/cirurgia , Orelha , Meato Acústico Externo , Humanos , Masculino , Processo Mastoide
15.
J Food Sci ; 85(8): 2340-2349, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32645217

RESUMO

The aim of this study was to optimize the drying conditions (pretreatments and drying temperatures) of red bell pepper to obtain red bell pepper powder (RBP) with maximum bioactive retention and assess its potential use as a functional ingredient in bread. The RBP pretreated (blanching + dipping in ascorbic and citric acid solution) and dried at 60 °C retained the highest bioactive compounds. Wheat flour blends were prepared with RBP at 0%, 2%, 4%, 6%, 8%, and 10% level of incorporation and among hydration properties, water absorption increased with increasing levels. The prepared breads were assayed for physical, nutritional, bioactive, and sensory characteristics. Bread supplementation with RBP improves its color, mineral, fiber, and bioactive properties. However, the texture of bread becomes hard with the increased level of RBP due to a decrease in specific volume. The significant increment was recorded for bioactive compounds, such as total phenols, antioxidant activity, flavonoids, and carotenoids as the level of powder increased. Bread enriched with 6% RBP showed highest sensory scores (8.45) and index of acceptability (87.83%) as compared to other breads. Thus, RBP acts as a valuable supplement for developing bread with improved nutritional and bioactive constituents. PRACTICAL APPLICATION: This study describes the effect of different pretreatments and drying temperatures for processing of RBP as a functional ingredient in bread, being a staple food around the world. RBP powder is a novel ingredient that improved the nutritional, bioactive, and appearance of bread. Hence, it will be helpful in the utilization of perishable crops like bell pepper and will demonstrate its commercial viability to improve the nutritive value of bakery products.


Assuntos
Pão/análise , Capsicum/química , Dessecação/métodos , Manipulação de Alimentos/métodos , Antioxidantes/análise , Carotenoides/análise , Comportamento do Consumidor , Fibras na Dieta , Flavonoides/análise , Farinha/análise , Alimento Funcional/análise , Valor Nutritivo , Fenóis/análise , Sensação
16.
Nitric Oxide ; 20(3): 150-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19146971

RESUMO

The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.


Assuntos
Óxido Nítrico Sintase Tipo II/fisiologia , Tirosina/análogos & derivados , Animais , Artrite Experimental , Artrite Reumatoide , Biomarcadores/sangue , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inflamação , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite , Ratos , Índice de Gravidade de Doença , Tirosina/sangue
17.
Anal Biochem ; 380(1): 68-76, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18539125

RESUMO

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.


Assuntos
Biomarcadores/análise , Biomarcadores/metabolismo , Líquidos Corporais/química , Cromatografia de Afinidade/métodos , Espectrometria de Massas em Tandem/métodos , Tirosina/análogos & derivados , Anticorpos/imunologia , Modelos Lineares , Pronase/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Líquido Sinovial/química , Tirosina/análise , Tirosina/imunologia , Tirosina/metabolismo
18.
Methods Mol Biol ; 1842: 123-137, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196406

RESUMO

Stem cells have emerged as a promising cell source to heal, replace or regenerate tissue and organs damaged by aging, injury or diseases. The intestinal epithelium is the most rapidly renewing tissue in our body, which is maintained by intestinal stem cells (ISCs), located at the bottom of the crypts. ISCs continuously replace lost or injured intestinal epithelial cells in organisms ranging from Drosophila to humans. The adult Drosophila midgut provides an excellent in vivo model system to study ISC behavior during stress, regeneration, aging and infection. There are several signaling pathways/genes have been identified to regulate ISCs self-renewal and differentiation during normal and pathological conditions. A significant number of genetic tools and markers have been developed in the last one decade to study Drosophila ISCs behavior. Here, we describe some of the markers and methods used to study ISCs behavior in adult midgut of Drosophila.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores , Imunofluorescência , Fenótipo , Animais , Diferenciação Celular , Linhagem da Célula/genética , Drosophila , Técnicas de Silenciamento de Genes , Camundongos Transgênicos , Interferência de RNA
19.
Genetics ; 207(1): 197-213, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28696218

RESUMO

One of the pertinent issues associated with cellular plasticity is to understand how the delicate balance between the determined state of cells and the extent to which they can transdetermine is maintained. Employing the well-established model of generating ectopic eyes in developing wing discs of Drosophila by ectopic eyeless expression, we provide evidence for the genetic basis of this mechanism. By both loss-of-function and gain-of-function genetic analyses, we demonstrate that Matrix metalloproteinase 1 (Mmp1) plays an important role in regulating the extent of ectopic ommatidial differentiation. Transcriptional activation of ectopic Mmp1 by the morphogen Decapentaplegic (Dpp) is not triggered by its canonical signaling pathway which involves Mad. Rather, Dpp activates an alternate cascade involving dTak1 and JNK, to induce ectopic Mmp1 expression. Mutational analyses reveal that Mmp1 negatively regulates ectopic eye differentiation by restricting the rate of proliferation and the levels of expression of retinal-determining genes dachshund and eyes absent This is primarily achieved by restricting the range of Hedgehog (Hh) signaling. Importantly, the increase in proliferation and upregulation of target retinal-determining genes, as observed upon attenuating Mmp1 activity, gets significantly rescued when ectopic eyes are generated in wing discs of hh heterozygous mutants. In conjunction with the previously established instructive and permissive roles of Dpp in facilitating ectopic eye differentiation in wing discs, the outcome of this study sheds light on a mechanism by which Dpp plays a dual role in modulating the delicate balance between the determined state of cells and the extent they can transdetermine.


Assuntos
Olho Composto de Artrópodes/embriologia , Proteínas de Drosophila/genética , Proteínas Hedgehog/genética , Metaloproteinase 1 da Matriz/genética , Transdução de Sinais , Animais , Proliferação de Células , Olho Composto de Artrópodes/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Mutação com Ganho de Função , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Mutação com Perda de Função , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
J Immunol Methods ; 441: 56-66, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27939300

RESUMO

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of several therapeutic antibody drugs and evaluation in ADCC bioassays is important in antibody drug development and maintenance. Three types of effector cells now routinely used in bioassay evaluation of ADCC are natural killer cells from human donors (FcγRIIIA+primary NK), FcγRIIIA engineered NK-92 cells and FcγRIIIA/NFAT-RE/luc2 engineered Jurkat T cells. Engineered effector cells were developed to address need for improved precision and accuracy of classic NK cell ADCC bioassays. The main purpose of our study was to rationalize which of these ADCC effector cells best simulate the expected response in human subjects and to identify which effector cells and assays best fit ADCC bioassay needs during antibody drug development. We characterized differences between the effector cells and compared ADCC biological activities using the well-known humanized IgG1 antibody drug, trastuzumab. The three effector cell types studied expressed either V-158 or F-158 allotype of FcγRIIIA, hence six cell preparations were compared. Our results demonstrate highest surface expression of FcγRIIIA in primary NK and engineered NK-92 (V-158) cells with nearly all expressed on the cell surface. In contrast, expression in engineered Jurkat T cells was low with only a small percentage expressed on the cell surface. Studies evaluating binding of trastuzumab to effector cells demonstrated the highest affinity of FcγRIIIA in primary NK and NK-92 (V-158) cells. ADCC cytotoxicity studies showed greatest trastuzumab potency in primary NK and engineered NK-92 (V-158) cells and negligible cell lysis obtained using engineered Jurkat T cells. In contrast, the engineered Jurkat T (V-158) cells responded as effectively as primary NK (V/V) cells to nuclear factor of activated T cells (NFAT2) activation upon binding of trastuzumab to FcγRIIIA, demonstrating similar ADCC pathway activation in these cells despite the low surface expression of FcγRIIIA and its low affinity for trastuzumab. Dose-response range of trastuzumab in activation of NFAT2 (measured as pNFAT2 dephosphorylation) was very similar to response in classic ADCC assay for primary and NK-92 cells and to response in ADCC reporter assay for Jurkat T effector cells, bridging the assays. Trastuzumab potency in ADCC reporter assay using the engineered Jurkat T cells was close to that seen using either primary NK or engineered NK-92 cells in classic ADCC assay. In summary, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, yet only primary NK and engineered NK-92 cells are capable of inducing ADCC-mediated cell lysis. Engineered Jurkat T effector cells have value to assure antibody manufacturing consistency and in other applications where accuracy and precision are important. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells may behave more reproducibly than primary NK, but this must be balanced with the objective for biological relevance in decisions on which NK cells to use in assay.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Bioensaio/métodos , Engenharia Celular , Células Matadoras Naturais/imunologia , Receptores de IgG/isolamento & purificação , Linhagem Celular Tumoral , Genótipo , Humanos , Células Jurkat , Células Matadoras Naturais/metabolismo , Cultura Primária de Células , Receptores de IgG/imunologia
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