RESUMO
Background: Evidence suggests cheese has a favourable or neutral effect on cardiometabolic health, compared to butter. To date, studies have only considered the cheese matrix in its unmelted form, while the effect of melted cheese remains unknown. Objective: To test the effect of 6-week daily consumption of â¼40 g dairy fat, eaten in either as unmelted cheese, melted cheese, or in a fully deconstructed form, on markers of metabolic health in overweight adults aged ≥50 years of age. Design: A 6-week randomised parallel intervention, where 162 participants (43.3% male) received â¼40 g of dairy fat per day, in 1 of 3 treatments: (A) 120 g full-fat Irish grass-fed cheddar cheese, eaten in unmelted form (n 58); (B) 120 g full-fat Irish grass-fed cheddar cheese eaten in melted form (n 53); or (C) the equivalent components; butter (49 g), calcium caseinate powder (30 g), and Ca supplement (CaCO3; 500 mg) (n 51). Results: There was no difference in weight, fasting glucose, or insulin between the groups post-intervention. Melted cheese, compared to unmelted cheese, increased total cholesterol (0.23 ± 0.79 mmol L-1vs. 0.02 ± 0.67 mmol L-1, P = 0.008) and triglyceride concentrations (0.17 ± 0.39 mmol L-1vs. 0.00 ± 0.42 mmol L-1, P = 0.016). Melted cheese increased total cholesterol concentrations by 0.20 ± 0.15 mmol L-1 and triglyceride concentrations by 0.17 ± 0.08 mmol L-1 compared to unmelted cheese. No significant differences were observed between the cheese forms for change in HDL, LDL or VLDL cholesterol. Conclusion: Compared to unmelted cheese, melted cheese was found to increase total cholesterol and triglyceride concentrations in middle-aged, overweight adults with no effect on weight or glycaemic control.
Assuntos
Queijo , Metabolismo dos Lipídeos , Triglicerídeos , Humanos , Queijo/análise , Pessoa de Meia-Idade , Masculino , Feminino , Idoso , Triglicerídeos/metabolismo , Glicemia/metabolismo , Glicemia/análise , Colesterol/sangue , Colesterol/metabolismo , Manteiga/análise , Insulina/metabolismo , Caseínas , Sobrepeso/metabolismo , Gorduras na Dieta/análise , Gorduras na Dieta/metabolismoRESUMO
Ex vivo cellular system that accurately replicates sickle cell disease and ß-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and ß-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.
Assuntos
Anemia Falciforme , Talassemia beta , Humanos , Talassemia beta/genética , Talassemia beta/terapia , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Células-Tronco Hematopoéticas/metabolismo , Genótipo , Sistemas CRISPR-CasRESUMO
ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.
RESUMO
BACKGROUND: Research on dairy consumption in China is lacking, however, some evidence has demonstrated significant changes in recent years, with a reported increase in the overall consumption of dairy products. To fully understand these changes, a systematic review was conducted to examine reported dairy intakes and differences between dairy consumption in different population groups in China. METHODS: Web of Science, Embase, and PubMed databases were searched for studies published from January 2000 to September 2022. The China National Knowledge Infrastructure (CNKI) was used to retrieve papers available in Chinese. Papers reporting dietary intakes of dairy consumption across age, sex, and geographical location sub-groups were considered for inclusion in this review. In addition, this review includes the consumption of different types of dairy foods and changes in dairy intake over time. RESULTS: Forty-seven papers were included in the present study. Twelve papers examined dairy consumption across age groups, showing that middle-aged adults tend to consume less dairy than other age groups. Studies comparing across location-specific cohorts reported dairy intakes among urban populations were higher than rural, as well as being higher than the national average. Coastal, Northern and Eastern residents consumed more dairy products than those living in other regions of China, and people in larger cities had higher reported intakes than smaller cities. Milk was the primary dairy product reportedly consumed by Chinese population, followed by yogurt. Concerning sex, evidence showed that females generally reported a greater daily dairy intake than males. CONCLUSIONS: This review shows that, in China, several different population groups displayed significant differences in the amount and type of dairy consumed. When considering the incorporation of dairy products into healthy eating guidelines or positioning specific dairy products on the market, it is important to consider the differences and variations in consumption patterns within population groups.
RESUMO
Elevated intakes of saturated fatty acids (SFA) can adversely affect serum cholesterol levels. Dairy fat contains ~60% SFA, prompting healthy eating guidelines to recommend low-fat dairy. Physiological, and environmental factors influence inter-individual variance in response to food consumption. Evidence exploring the dairy matrix has differing effects of dairy fat consumption on serum cholesterol levels when consumed in the form of cheese. The extent of this variability and determinants of response to dairy fat are currently unknown. The objective of this study was to determine factors associated with lipid metabolism response to a dairy fat intervention, with a focus on serum cholesterol. A 6-week randomized parallel intervention trial was carried out in healthy volunteers (≥50 years, BMI ≥25 kg/m2). Participants (n = 104) consumed ~40 g dairy fat daily in addition to their usual diet, in 1 of 3 forms: butter, cheese, or reduced-fat cheese and butter. For this analysis, "response" was based on the percentage (%) change in serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) from pre- to post-intervention. Participants were divided into tertiles for each lipid response. The upper and lower tertiles were used to categorize participants as "responders" and "non-responders." For TC and LDL-c, response was classified as a decrease, whereas "response" was defined as an increase for HDL-c. Clinical response was also considered, by calculating pre- and post-intervention prevalence of those meeting target levels of cholesterol recommendations. Participants demonstrating the largest % decrease (Tertile 1; "responders") in TC had significantly higher levels of TC and HDL-c, at baseline, and lower levels of triglycerides (TAGs) compared to those in tertile 3 (i.e., TC non-responders). Those with the largest % decrease in LDL-c (Tertile 1: LDL-c responders) had higher baseline levels of LDL-c and lower levels of TAGs. Multiple regression analysis revealed that the % change in TC and LDL-c was associated with baseline TC, TAG, body weight and high-sensitivity C-reactive protein (hsCRP; P < 0.05). Previous work has demonstrated the dairy food matrix affects lipid response to dairy consumption. This study suggests that phenotypic differences may also influence response to dairy fat in overweight individuals.
RESUMO
Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting.
RESUMO
BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.
Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Clonagem Molecular , Edição de Genes/métodos , Genoma Humano , HumanosRESUMO
ß-thalassemia (BT) is a hereditary blood disorder caused by mutations in the ß-globin (HBB) gene leading to severely reduced or no synthesis of the ß-chain of adult hemoglobin. IVS1-5 (G > C) is the most common BT mutation in Indian population and yet no patient-specific cellular models have been generated. Here, we have established an induced pluripotent stem cell (iPSC) line, IGIBi002-A from a thalassemia patient with a homozygous IVS1-5(G > C) mutation. Characterization of IGIBi002-A demonstrated that these iPSCs are free of exogenous reprogramming genes and expressed pluripotent stem cell markers, exhibited a normal karyotype and were potential of three germ layer differentiation.
RESUMO
Milk and dairy foods are naturally rich sources of a wide range of nutrients, and when consumed according to recommended intakes, contribute essential nutrients across all stages of the life cycle. Seminal studies recommendations with respect to intake of saturated fat have been consistent and clear: limit total fat intake to 30% or less of total dietary energy, with a specific recommendation for intake of saturated fat to less than 10% of total dietary energy. However, recent work has re-opened the debate on intake of saturated fat in particular, with suggestions that recommended intakes be considered not at a total fat intake within the diet, but at a food-specific level. A large body of evidence exists examining the impact of dairy consumption on markers of metabolic health, both at a total-dairy-intake level and also at a food-item level, with mixed findings to date. However the evidence suggests that the impact of saturated fat intake on health differs both across food groups and even between foods within the same food group such as dairy. The range of nutrients and bioactive components in milk and dairy foods are found in different levels and are housed within very different food structures. The interaction of the overall food structure and the nutrients describes the concept of the 'food matrix effect' which has been well-documented for dairy foods. Studies show that nutrients from different dairy food sources can have different effects on health and for this reason, they should be considered individually rather than grouped as a single food category in epidemiological research. This narrative review examines the current evidence, mainly from randomised controlled trials and meta-analyses, with respect to dairy, milk, yoghurt and cheese on aspects of metabolic health, and summarises some of the potential mechanisms for these findings.
Assuntos
Laticínios/análise , Dieta Saudável/métodos , Gorduras na Dieta/análise , Ingestão de Alimentos/fisiologia , Nutrientes/análise , Animais , Humanos , Metanálise como Assunto , Leite/química , Ensaios Clínicos Controlados Aleatórios como Assunto , Recomendações NutricionaisRESUMO
Sickle cell disease (SCD) is an autosomal recessive disorder caused by a mutation in ß-globin (HBB) gene. We have generated an induced pluripotent stem cell (iPSC) line, IGIBi001-A from an Indian sickle cell patient with a homozygous HBB gene mutation using Sendai virus reprogramming system. Characterization of IGIBi001-A showed that these iPSCs are transgene-free and expressed pluripotent stem cell markers. They had a normal karyotype and were able to differentiate into all three germ layers. This new SCD-iPSC line will contribute to better understanding of the disease biology of sickle cell anemia and for screening of small molecule drugs.
Assuntos
Anemia Falciforme/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Globinas beta/genética , Homozigoto , Humanos , Cariotipagem , Repetições de Microssatélites/genética , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ß-hemoglobin disorders, such as ß-thalassemia and sickle cell anemia are among the most prevalent inherited genetic disorders worldwide. These disorders are caused by mutations in the gene encoding hemoglobin-ß (HBB), a vital protein found in red blood cells (RBCs) that carries oxygen from lungs to all parts of the human body. As a consequence, there has been an enduring interest in this field in formulating therapeutic strategies for the treatment of these diseases. Currently, there is no cure available for hemoglobin disorders, although, some patients have been treated with bone marrow transplantation, whose scope is limited because of the difficulty in finding a histocompatible donor and also due to transplant-associated clinical complications that can arise during the treatment. On account of these constraints, reactivation of fetal hemoglobin (HbF) synthesis holds immense promise and is a viable strategy to alleviate the symptoms of ß-hemoglobin disorders. Development of new genomic tools has led to the identification of important natural genetic modifiers of hemoglobin switching which include BCL11A, KLF1, HBSIL-MYB, LRF, LSD1, LDB1, histone deacetylases 1 and 2 (HDAC1 and HDAC2). miRNAs are also promising therapeutic targets for development of more effective strategies for the induction of HbF production. Many new small molecule pharmacological inducers of HbF production are already under pre-clinical and clinical development. Furthermore, recent advancements in gene and cell therapy includes targeted genome editing and iPS cell technologies, both of which utilizes a patient's own cells, are emerging as extremely promising approaches for significantly reducing the burden of ß-hemoglobin disorders.
Assuntos
Anemia Falciforme/terapia , Hemoglobina Fetal/genética , Terapia Genética/métodos , Fármacos Hematológicos/uso terapêutico , Hemoglobinas Anormais/genética , Mutação , Talassemia beta/terapia , Anemia Falciforme/sangue , Anemia Falciforme/genética , Animais , Hemoglobina Fetal/biossíntese , Edição de Genes , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fenótipo , Talassemia beta/sangue , Talassemia beta/genéticaRESUMO
Heterojunction devices of Ge(1-x)Sn(x) / n-Ge were grown by solid source molecular beam epitaxy (MBE), and the mid-infrared (IR) photocurrent response was measured. With increasing Sn composition from 4% to 12%, the photocurrent spectra became red-shifted, suggesting that the bandgap of Ge(1-x)Sn(x) alloys was lowered compared to pure Ge. At a temperature of 100 K, the wavelengths of peak photocurrent were shifted from 1.42 µm for pure Ge (0% Sn) to 2.0 µm for 12% Sn. The bias dependence of the device response showed that the optimum reverse bias was > 0.5 volts for saturated photocurrent. The responsivity of the Ge(1-x)Sn(x) devices was estimated to be 0.17 A/W for 4% Sn. These results suggest that Ge(1-x)Sn(x) photodetectors may have practical applications in the near/mid IR wavelength regime.