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Since the first use of vaccine tell the last COVID-19 pandemic caused by spread of SARS-CoV-2 worldwide, the use of advanced biotechnological techniques has accelerated the development of different types and methods for immunization. The last pandemic showed that the nucleic acid-based vaccine, especially mRNA, has an advantage in terms of development time; however, it showed a very critical drawback namely, the higher costs when compared to other strategies, and its inability to protect against new variants. This showed the need of more improvement to reach a better delivery and efficacy. In this review we will describe different vaccine delivery systems including, the most used viral vector, and also variable strategies for delivering of nucleic acid-based vaccines especially lipid-based nanoparticles formulation, polymersomes, electroporation and also the new powerful tools for the delivery of mRNA, which is based on the use of cell-penetrating peptides (CPPs). Additionally, we will also discuss the main challenges associated with each system. Finlay, the efficacy and safety of the vaccines depends not only on the formulations and delivery systems, but also the dosage and route of administration are also important players, therefore we will see the different routes for the vaccine administration including traditionally routes (intramuscular, Transdermal, subcutaneous), oral inhalation or via nasal mucosa, and will describe the advantages and disadvantage of each administration route.
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Breast cancer is a wide-spread threat to the women's health. The drawbacks of conventional treatments necessitate the development of alternative strategies, where gene therapy has regained hope in achieving an efficient eradication of aggressive tumors. Monocarboxylate transporter 4 (MCT4) plays pivotal roles in the growth and survival of various tumors, which offers a promising target for treatment. In the present study, pH-responsive lipid nanoparticles (LNPs) based on the ionizable lipid,1,2-dioleoyl-3-dimethylammonium propane (DODAP), were designed for the delivery of siRNA targeting MCT4 gene to the breast cancer cells. Following multiple steps of characterization and optimization, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1, in comparison with a normal cell line, LX-2. The selection of the helper phospholipid to be incorporated into the LNPs had a dramatic impact on their gene delivery performance. The optimized LNPs enabled a powerful MCT4 silencing by â¼90â¯% at low siRNA concentrations, with a subsequent â¼80â¯% cytotoxicity to 4T1 cells. Meanwhile, the LNPs demonstrated a 5-fold higher affinity to the breast cancer cells versus the normal cells, in which they had a minimum effect. Moreover, the MCT4 knockdown by the treatment remodeled the cytokine profile in 4T1 cells, as evidenced by 90â¯% and â¼64â¯% reduction in the levels of TNF-α and IL-6; respectively. The findings of this study are promising for potential clinical applications. Furthermore, the simple and scalable delivery vector developed herein can serve as a breast cancer-targeting platform for the delivery of other RNA therapeutics.
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Neoplasias da Mama , Citocinas , Transportadores de Ácidos Monocarboxílicos , Proteínas Musculares , Nanopartículas , RNA Interferente Pequeno , Microambiente Tumoral , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Nanopartículas/química , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Feminino , Citocinas/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Animais , Camundongos , Técnicas de Silenciamento de Genes , Tamanho da Partícula , Concentração de Íons de HidrogênioRESUMO
[This corrects the article DOI: 10.1016/j.heliyon.2023.e13876.].
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BACKGROUND: Familial Hypercholesterolemia (FH) is a hereditary condition that causes a rise in blood cholesterol throughout a person's life. FH can result in myocardial infarction and even sudden death if not treated. FH is thought to be caused mainly by variants in the gene for the low-density lipoprotein receptor (LDLR). This study aimed to investigate the genetic variants in FH patients, verify their pathogenicity, and comprehend the relationships between genotype and phenotype. Also, review studies assessed the relationship between the LDLR null variants and the reaction to lipid-lowering therapy. METHODS: The study utilised high-throughput next-generation sequencing for genetic screening of FH-associated genes and capillary sequencing for cascade screening. Furthermore, bioinformatic analysis was employed to describe the pathogenic effects of the revealed novel variant on the structural features of the corresponding RNA molecule. RESULTS: We studied the clinical signs of hypercholesterolemia in a Saudi family with three generations of FH. We discovered a novel frameshift variant (c.666_670dup, p.(Asp224Alafs*43) in the LDLR and a known single nucleotide variant (c.9835A > G, p.(Ser3279Gly) in the APOB gene. It is thought that the LDLR variant causes a protein to be prematurely truncated, likely through nonsense-mediated protein decay. The LDLR variant is strongly predicted to be pathogenic in accordance with ACMG guidelines and co-segregated with the FH clinical characteristics of the family. This LDLR variant exhibited severe clinical FH phenotypes and was restricted to the LDLR protein's ligand-binding domain. According to computational functional characterization, this LDLR variant was predicted to change the free energy dynamics of the RNA molecule, thereby affecting its stability. This frameshift variant is thought to eliminate important functional domains in LDLR that are required for receptor recycling and LDL particle binding. We provide insight into how FH patients with a null variant in the LDLR gene respond to lipid-lowering therapy. CONCLUSIONS: The findings expand the range of FH variants and assist coronary artery disease preventive efforts by improving diagnosis, understanding the genotype-phenotype relationship, prognosis, and personalised therapy for patients with FH.
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Lung cancer is one of the most aggressive and deadliest health threats. There has been an increasing interest in non-coding RNA (ncRNA) recently, especially in the areas of carcinogenesis and tumour progression. However, ncRNA-directed therapies are still encountering obstacles on their way to the clinic. In the present article, we provide an overview on the potential of targeting ncRNA in the treatment of lung cancer. Then, we discuss the delivery challenges and recent approaches enabling the delivery of ncRNA-directed therapies to the lung cancer cells, where we illuminate some advanced technologies including chemically-modified oligonucleotides, nuclear targeting, and three-dimensional in vitro models. Furthermore, advanced non-viral delivery systems recruiting nanoparticles, biomimetic delivery systems, and extracellular vesicles are also highlighted. Lastly, the challenges limiting the clinical trials on the therapeutic targeting of ncRNAs in lung cancer and future directions to tackle them are explored.
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Neoplasias Pulmonares , RNA não Traduzido , Humanos , RNA não Traduzido/genética , RNA não Traduzido/uso terapêutico , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/tratamento farmacológico , Carcinogênese , Terapia de Alvo Molecular/métodosRESUMO
Graft versus host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic stem cell transplantation, especially for intestinal GVHD, as steroid resistant GVHD results in high mortality. For this reason, new treatments of GVHD are needed. One approach is the reduction of pathogenic bacteria using anti-E. coli Immunoglobulin Yolk (IgY). In a haploidentical murine model, B6D2F1 mice conditioned with total body irradiation (TBI), received bone marrow cells (BM) and splenocytes (SC) from either syngeneic (Syn = B6D2F1) or allogeneic (Allo = C57BL/6) donors. Following this, animals received from day -2 until day +28 chow contained IgY or control chow. Thereafter the incidence and severity of aGVHD, the cytokines, chemokines, IDO1 and different pathogen-recognition receptors (PRR) were analyzed and compared to control animals (received chow without IgY). We found that animals receiving chow with IgY antibody showed reduced GVHD severity compared to control animals. On day28 after alloBMT, IDO, NOD2, TLR2, TLR4 and the inflammatory chemokine CCL3, were reduced in the colon and correlated with a significant decrease in E. coli bacteria. In summary chow containing chicken antibodies (IgY) improved GVHD via decrease in bacterial load of E coli conducting to reduction of pathogen receptors (NOD2, TLR2 and 4), IDO, chemokines and cytokines.
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Tumor necrosis factor (TNF-α) and inflammatory cytokine (IL-6) play a vital role in various cellular incidents such as the proliferation and death of cells during carcinogenesis. Hence, regulation of these biomarkers could be a promising tool for controlling tumor progression using nanoformulations. Silver nanoparticles-poly (vinyl pyrrolidone) (AgNPs-PVP) were prepared using the reduction of silver nitrate and stabilized with PVP. They are characterized through yield percentage, UV-VIS, FT-IR, size, charge, and morphology. The obtained AgNPs were tested for anticancer activity against prostate cancer (PC 3) and human skin fibroblast (HFS) cell lines. Moreover, biomarker-based confirmations like TNF-α and IL-6 were estimated. The synthesized AgNPs-PVP were stable, spherical in shape, with particle sizes of 122.33 ± 17.61 nm, a polydispersity index of 0.49 ± 0.07, and a negative surface charge of -19.23 ± 0.61 mV. In vitro cytotoxicity testing showed the AgNPs-PVP exhibited antiproliferation properties in PC3 in a dose-dependent manner. In addition, when compared to control cells, AgNPs-PVP has lower TNF-α with a significant value ( ∗ p < 0.05); the value reached 16.84 ± 0.71 pg/ml versus 20.81 ± 0.44 pg/ml, respectively. In addition, HSF cells showed a high level of reduction ( ∗∗∗ p < 0.001) in IL-6 production. This study suggested that AgNPs-PVP could be a possible therapeutic agent for human prostate cancer and anti-IL-6 in cancerous and noncancerous cells. Further studies will be performed to investigate the effect of AgNPs-PVP in different types of cancer.
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BACKGROUND: Familial hypercholesterolemia (FH) is a common illness mainly caused by variants occurring in the low-density lipoprotein receptor (LDLR) gene. FH is a leading cause of coronary artery disease. OBJECTIVE: This study aims to determine genetic defect(s) in homozygous and heterozygous FH index patients and their first-degree blood relatives and understand the genotype-phenotype correlation. METHODS: This study employed the genetic screening of FH-related genes by next-generation sequencing and cascade screening by capillary sequencing. RESULTS: We identified the presence of a novel frameshift variant [c.335_336insCGAG, p.(F114Rfs*17)] and three known missense variants [c.622G>A, p.(E208K)], [c.1474G>A, p.(D492N)], [c.1429G>A, p.(D477N)] in the LDLR gene of four unrelated Saudi families with FH. In proband 1, a nonsense variant c.1421C>G, p.(S474*) was also detected at exon 9 of the lipoprotein lipase gene. The segregation arrangement of the identified variants corresponded with the clinical characteristics. In this study, all the detected variants were confined in the ligand-binding domain and epidermal growth factor (EGF)-precursor homology domain of the LDLR protein, which portrayed severe clinical phenotypes of FH. Moreover, these LDLR variants were in a highly conserved residue of the proteins. CONCLUSION: In addition to the finding of the novel variant in the LDLR gene that extends the spectrum of variants causing FH, the results of this study also support the need for diagnostic screening and cascade genetic testing of this high-risk condition and to understand the genotype-phenotype correlation, which could lead to better prevention of coronary artery disease.
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Doença da Artéria Coronariana , Hiperlipoproteinemia Tipo II , Humanos , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Arábia Saudita/epidemiologia , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Receptores de LDL/química , Homozigoto , Fenótipo , MutaçãoRESUMO
Cetuximab (CTX) is known to have cytotoxic effects on several human cancer cells in vitro; however, as CTX is poorly water soluble, there is a need for improved formulations can reach cancer cells at high concentrations with low side effects. We developed (PEG-4000) polymeric nanoparticles (PEGNPs) loaded with CTX and evaluated their in vitro cytotoxicity and anticancer properties against human lung (A549) and breast (MCF-7) cancer cells. CTX-PEGNPs were formulated using the solvent evaporation technique, and their morphological properties were evaluated. Further, the effects of CTX-PEGNPs on cell viability using the MTT assay and perform gene expression analysis, DNA fragmentation measurements, and the comet assay. CTX-PEGNP showed uniformly dispersed NPs of nano-size range (253.7 ± 0.3 nm), and low polydispersity index (0.16) indicating the stability and uniformity of NPs. Further, the zeta potential of the preparations was -17.0 ± 1.8 mv. DSC and FTIR confirmed the entrapping of CTX in NPs. The results showed IC50 values of 2.26 µg/mL and 1.83 µg/mL for free CTX and CTX-PEGNPs on the A549 cancer cell line, respectively. Moreover, CTX-PEGNPs had a lower IC50 of 1.12 µg/mL in MCF-7 cells than that of free CTX (2.28 µg/mL). The expression levels of p21 and stathmin-1 were significantly decreased in both cell lines treated with CTX-PEGNPs compared to CTX alone. The CTX-PEGNP-treated cells also showed increased DNA fragmentation rates in both cancer cell lines compared with CTX alone. The results indicated that CTX-PEGNP was an improved formulation than CTX alone to induce apoptosis and DNA damage and inhibit cell proliferation through the downregulation of P21 and stathmin-1 expression.
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Cetuximab/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Polietilenoglicóis/farmacologia , Células A549 , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/administração & dosagem , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Portadores de Fármacos/farmacologia , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Nanopartículas/química , Polímeros , Estatmina/efeitos dos fármacos , Estatmina/metabolismoRESUMO
BACKGROUND: Different common gene variants were related to coronary artery disease (CAD) in many studies. Yet, the relation of these loci to the severity of CAD is not completely elucidated. METHODS: We enrolled 520 subjects (315 CAD cases and 205 controls). CAD presence and extension were assessed by coronary angiography (CAG). Genotyping of five SNPs (namely, rs2230806 (1051G > A) in ABCA1 on chromosome 9, rs2075291 (553G > T) in ApoA5 on chromosome 11, rs320 in LPL on chromosome 8 intron (T â G at position 481), rs10757278 (c.22114477A > G), and rs2383206 (c.22115026 A > G) on chromosome 9p21 locus) was performed by allele-specific PCR. The degree and site of arterial lesions were used to classify patients, tested for association with CAD severity, and related to allele dosage. RESULTS: The polymorphisms rs2383206 and rs10757278 showed significant associations with 2- and 3-vessel coronary disease (p =0.003 and 0.006, respectively). The homozygous GG genotypes of rs10757278 was associated with higher frequency of left anterior descending (LAD), right coronary artery (RCA) and left circumflex (LCX) diseases (p =0.002, 0.016 and 0.002, respectively). The GG genotypes of rs2383206 were found in higher percentage in patients with left main (LM) trunk and left circumflex (LCX) diseases (p = 0.013 and 0.002, respectively). CONCLUSION: SNPs rs10757278 and rs2383206 allele dosage could predict CAD severity in the Saudi Arab population.
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Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Índice de Gravidade de Doença , Idoso , Estudos de Casos e Controles , Angiografia Coronária , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Systemic steroids are used to treat acute graft-versus-host disease (aGVHD) caused by allogenic bone marrow transplantation (allo-BMT); however, their prolonged use results in complications. Hence, new agents for treating aGVHD are required. Recently, a new compound A (CpdA), with anti-inflammatory activity and reduced side effects compared to steroids, has been identified. Here, we aimed to determine whether CpdA can improve the outcome of aGVHD when administered after transplantation in a mouse model (C57BL/6 in B6D2F1). After conditioning with 9Gy total body irradiation, mice were infused with bone marrow (BM) cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. The animals were subsequently treated (3 days/week) with 7.5 mg/kg CpdA from day +15 to day +28; the controls received 0.9% NaCl. Thereafter, the incidence and severity of aGVHD in aGVHD target organs were analyzed. Survival and clinical scores did not differ significantly; however, CpdA-treated animals showed high cell infiltration in the target organs. In bulk mixed lymphocyte reactions, CpdA treatment reduced the cell proliferation and expression of inflammatory cytokines and chemokines compared to controls, whereas levels of TNF, IL-23, chemokines, and chemokine receptors increased. CpdA significantly reduced proliferation in vitro but increased T cell infiltration in target organs.
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Anti-Inflamatórios/farmacologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Aguda , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacosRESUMO
Multiple genes have been implicated to have a role in asthma predisposition by association studies. Pediatric patients often manifest a more extensive form of this disease and a particularly severe disease course. It is likely that genetic predisposition could play a more substantial role in this group. This study is aimed at identifying the spectrum of rare and novel variation in known pediatric asthma susceptibility genes using whole exome sequencing analysis in nine individual cases of childhood onset allergic asthma. DNA samples from the nine children with a history of bronchial asthma diagnosis underwent whole exome sequencing on Ion Proton. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued. The analysis showed 21 variants in the subjects, 13 had been previously identified, and 8 were novel. Also, among of which, nineteen were nonsynonymous and 2 were nonsense. With regard to the novel variants, the 2 nonsynonymous variants in the PRKG1 gene (PRKG1: p.C519W and PRKG1: p.G520W) were presented in 4 cases, and a nonsynonymous variant in the MAVS gene (MAVS: p.A45V) was identified in 3 cases. The variants we found in this study will enrich the variant spectrum and build up the database in the Saudi population. Novel eight variants were identified in the study which provides more evidence in the genetic susceptibility in asthma among Saudi children, providing a genetic screening map for the molecular genetic determinants of allergic disease in Saudi children, with the goal of reducing the impact of chronic diseases on the health and the economy. We believe that the advanced specified statistical filtration/annotation programs used in this study succeeded to release such results in a preliminary study, exploring the genetic map of that disease in Saudi children.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Asma/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Predisposição Genética para Doença , Polimorfismo Genético , Adolescente , Idade de Início , Asma/diagnóstico , Asma/fisiopatologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Fenótipo , Arábia Saudita , Sequenciamento do ExomaRESUMO
The current COVID-19 pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has raised significant economic, social, and psychological concerns. The rapid spread of the virus, coupled with the absence of vaccines and antiviral treatments for SARS-CoV-2, has galvanized a major global endeavor to develop effective vaccines. Within a matter of just a few months of the initial outbreak, research teams worldwide, adopting a range of different strategies, embarked on a quest to develop effective vaccine that could be effectively used to suppress this virulent pathogen. In this review, we describe conventional approaches to vaccine development, including strategies employing proteins, peptides, and attenuated or inactivated pathogens in combination with adjuvants (including genetic adjuvants). We also present details of the novel strategies that were adopted by different research groups to successfully transfer recombinantly expressed antigens while using viral vectors (adenoviral and retroviral) and non-viral delivery systems, and how recently developed methods have been applied in order to produce vaccines that are based on mRNA, self-amplifying RNA (saRNA), and trans-amplifying RNA (taRNA). Moreover, we discuss the methods that are being used to enhance mRNA stability and protein production, the advantages and disadvantages of different methods, and the challenges that are encountered during the development of effective vaccines.
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BACKGROUND: Coronary artery disease (CAD) is one of the common genetic and clinical risk factors associated with cardiovascular and multifactorial disorder. ATP-binding cassette transporter A1 (ABCA1) gene plays an important role in lipid metabolism and in multiple studies associated with CAD. However, more studies are needed to identify the exact role of single nucleotide polymorphisms which may cause CAD. OBJECTIVES: The aim of this study is to investigate the genetic association of polymorphism g.1051G > A in the ABCA1 gene with CAD patients in the Saudi population. METHODS: We included 315 confirmed CAD cases, and 205 non-CAD or control subjects in this case-control study. DNA isolation was carried out for all registered participants and the polymorphism g.1051G > A was genotyped with Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism analysis with EcoNI restriction enzyme. RESULTS: Modifiable risk factors such as Body Mass Index, smoking and diabetes were strongly associated and non-modifiable risk factors such as hypertension (Systolic Blood Pressure and Diastolic Blood Pressure) and serum analysis such as Fasting Blood Glucose, Total cholesterol (TC), Triglyceride (TG) and LDL-c were significantly associated in CAD cases (p < 0.05). Allele (OR-1.73;95% CI:1.33-2.26; p = 0.0004), GA vs GG (OR-2.26; 95% CI: 1.53-3.35; p = 0.0003 and dominant inheritance pattern (OR-2.23; 95% CI:1.56-3.20; p = 0.00009 was strongly associated with CAD cases and control subjects. The frequency level of use of atorvastatin was significantly different among GG, GA and AA subjects. Additionally, TC and TG levels were influenced by the presence of g.1051G > A polymorphism. CONCLUSION: The polymorphism g.1051G > A in the gene ABCA1 is closely associated with the existence of the CAD subjects. This polymorphism could also affect the serum levels of the lipid profile, suggesting a possible occurrence of CAD in the Saudi population.
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BACKGROUND: Anthocyanins are antioxidant compounds constitute the primary dyes of the pomegranate arils. Anthocyanins could protect the aged skin induced by oxidant exposure as a major role in aging processing and skin degeneration. PURPOSE: The study aimed to evaluate the anti-aging activity of anthocyanins rich pomegranate (Punica granatum) after formulated into a topical cream. Also, its effect on human dermal fibroblast function and epidermal keratinocyte were evaluated. METHOD: Anthocyanins were extracted from fresh pomegranate arils using acidified methanol and were purified by Sephadex LH-20 gel-column chromatography. Further, the fusion method was used to prepare cold cream containing pomegranate anthocyanins. The formulated cream was evaluated for their compatibility study, irritation, homogeneity, drug content, drug release, and stability tests. Furthermore, permeation study through abdominal rabbits, as well as Human application was performed. RESULTS: Compatibility study showed the absence of any interaction between anthocyanins and the used polymers. The formulated cream was nonirritant, homogenous and potentially reduced skin aging when applied to Human volunteers' skin. Furthermore, the skin permeation displayed a good permeation of 43.16% after 210 min. CONCLUSION: Pomegranate anthocyanins could be used as a safe, stable, homogeneous, nonirritant and effective topical anti-aging drug formulation for aged human people.
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Lythraceae , Punica granatum , Envelhecimento , Animais , Antocianinas/farmacologia , Frutas , CoelhosRESUMO
BACKGROUND: Leukaemia is the most prevalent form of cancer-causing death in a large number of populations and needs prompt and effective treatment. Chemotherapeutics can be used to treat leukaemia, but their pronounced killing effects to other living cells is still an issue. Active targeting to certain specific receptors in leukaemic cells is the best way to avoid damage to other living cells. Leukaemic cells can be targeted using novel nanoparticles (NPs) coated with a specific ligand, such as octreotide (OCD), to target somatostatin receptor type 2 (SSTR2), which is expressed in leukaemic cells. METHODS: Amino-PEGylated quantum dots (QDs) were chosen as model NPs. The QDs were first succinylated using succinic anhydride and then coated with OCD. The reactivity and selectivity of the formulated QDs-OCD were studied in cell lines with well-expressed SSTR2, while fluorescence was detected using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). Conclusively, QD-OCD targeting to blood cells was studied in vivo in mice and detected using inductively coupled plasma mass spectrometry and CLSM in tissues. RESULTS: Highly stable QDs coated with OCD were prepared. FACS and CLSM showed highly definite interactions with overexpressed SSTR2 in the investigated cell lines. Moreover, the in vivo results revealed a higher concentration of QDs-OCD in blood cells. The fluorescence intensity of the QDs-OCD was highly accumulated in blood cells, while the unmodified QDs did not accumulate significantly in blood cells. CONCLUSION: The formulated novel QDs-OCD can target SSTR2 overexpressed in blood cells with great potential for treating blood cancer.
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Antineoplásicos/metabolismo , Corantes Fluorescentes/química , Leucemia/metabolismo , Monócitos/metabolismo , Octreotida/metabolismo , Pontos Quânticos , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Composição de Medicamentos , Citometria de Fluxo , Células HeLa , Humanos , Leucemia/tratamento farmacológico , Leucemia/patologia , Masculino , Camundongos , Microscopia Confocal , Octreotida/química , Octreotida/farmacologiaRESUMO
Background: Familial hypercholesterolemia (FH) is an autosomal dominant inherited genetic disorder and results in the development of coronary artery disease (CAD). Clinical diagnosis of homozygous HH patients is usually straightforward because persistent hypercholesterolemia can produce xanthoma and corneal arcus. However, xanthoma may also be misdiagnosed as skin lesions and could therefore be mistreated. The aim of this case study report is to highlight the plight of patients with FH as means of raising awareness of the condition among dermatologists and health care practitioners, also to determine the genotype-phenotype correlation in severely affected homozygous FH proband patients. Methods: Genetic screening of FH associated genes was performed by Ion Torrent next-generation sequencing and cascade screening by capillary sequencing. Results: We present two clinical cases with prominent skin lesions seen in a dermatology clinic that were referred to plastic surgery for excision. Genetic testing was performed later, and confirmed common single nucleotide deletion variant (c.2027delG) in the LDLR alleles consequent to a frameshift mutation p.(G676Afs*33). In addition to the LDLR variant, two possibly damaging APOB variants p.(L3313I) and p.(L1212M) and three damaging variants p.(R19*), p.(G83Q) and p.(S474*) in APOC3, PON2 and LPL genes respectively were identified. The PON2 gene variant p.(G83Q) was found to be novel, while others have been previously reported. Both patients were refractory to pharmacological therapies and are currently on lipoprotein apheresis (LA). Conclusions: The present report indicates the need for increased awareness of FH, among the public and healthcare practitioners and supports the need for diagnostic screening and cascade genetic testing of this high-risk condition, which could ultimately lead to better prevention of CHD in this lethal condition.
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Conscientização , Competência Clínica , Dermatologistas/psicologia , Testes Genéticos/métodos , Pessoal de Saúde/psicologia , Hiperlipoproteinemia Tipo II/diagnóstico , Xantomatose/diagnóstico , Adolescente , Adulto , Criança , Erros de Diagnóstico , Feminino , Variação Genética , Humanos , Hiperlipoproteinemia Tipo II/genética , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Xantomatose/genética , Adulto JovemRESUMO
BACKGROUND: Eczema is also known as atopic dermatitis is well-known for the skin disease globally. In Saudi Arabia, exome sequencing studies have not been documented. The purpose of this study was to scrutinize the disease causing mutations in children affected with eczema with exome sequencing in the Saudi population. METHODS: We recruited randomly three sporadic cases of children diagnosed with eczema and simultaneously, three more cases were adopted for control samples. Exome sequencing was carried out by applying a pipeline that captures all the variants of concern related to the samples by using the Ion torrent. RESULTS: In this study, we have documented 49 variants, among which 37 variants were confirmed through eczema children and remaining 30 variants through control children. However, from the analysis of the 6 samples, we have identified rs10192157 (1646C>T; Thr549Ile), rs2899642 (27C>G; Asn9Lys), chr1:152127950 (1625G>A; Gly542Asp) and chr1:152128041 (1534C>G; Gly512Arg) variants which are rarely linked to the disease eczema. In the rs10192157, we have documented these mutations in all three eczema children and one in the control; the rs2899642 mutation appeared in only a couple of eczema children, whereas the mutation in the chr1:152127950 regions appeared in only one eczema patient. However, the chr1:152128041 mutations appeared in only one case of eczema and also in two control children. CONCLUSION: Our study revealed four mutations which had not previously been connected with eczema within the database. However, the rs10192157 and rs2899642 mutations were documented with asthma disease. The remaining mutations such as chr1:152127950 and chr1:152128041 have not been reported anywhere else. This study recommends screening these 4 mutations in eczema cases and their relevant controls to confirm the prevalence in the Saudi population. It is recommended that future studies examine the 4 mutations in detail.
Assuntos
Eczema/genética , Sequenciamento do Exoma , Predisposição Genética para Doença , Mutação , Adolescente , Criança , Dermatite Atópica/genética , Eczema/epidemiologia , Feminino , Humanos , Masculino , Arábia Saudita/epidemiologia , Análise de Sequência de DNARESUMO
Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5'-LDLReGFP-3' fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3' and 5' ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD-eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein-protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.