Enzyme‐assisted aqueous extraction of Kalahari melon seed oil: optimization using response surface methodology

Enzymatic extraction of oil from Kalahari melon seeds was investigated and evaluated by response surface methodology (RSM). Two commercial protease enzyme products were used separately: Neutrase® 0.8 L and Flavourzyme® 1000 L from Novozymes (Bagsvaerd, Denmark). RSM was applied to model and optimize the reaction conditions namely concentration of enzyme (20–50 g kg−1 of seed mass), initial pH of mixture (pH 5–9), incubation temperature (40–60 °C), and incubation time (12–36 h). Well fitting models were successfully established for both enzymes: Neutrase 0.8 L (R 2 = 0.9410) and Flavourzyme 1000 L (R 2 = 0.9574) through multiple linear regressions with backward elimination. Incubation time was the most significant reaction factor on oil yield for both enzymes. The optimal conditions for Neutrase 0.8 L were: an enzyme concentration of 25 g kg−1, an initial pH of 7, a temperature at 58 °C and an incubation time of 31 h with constant shaking at 100 rpm. Centrifuging the mixture at 8,000g for 20 min separated the oil with a recovery of 68.58 ± 3.39%. The optimal conditions for Flavourzyme 1000 L were enzyme concentration of 21 g kg−1, initial pH of 6, temperature at 50 °C and incubation time of 36 h. These optimum conditions yielded a 71.55 ± 1.28% oil recovery

Expectations and needs of patients with a chronic disease toward self-management and eHealth for self-management purposes

Background: Self-management is considered as an essential component of chronic care by primary care professionals. eHealth is expected to play an important role in supporting patients in their self-management. For effective implementation of eHealth it is important to investigate patients’ expectations and needs regarding self-management and eHealth. The objectives of this study are to investigate expectations and needs of people with a chronic condition regarding self-management and eHealth for self-management purposes, their willingness to use eHealth, and possible differences between patient groups regarding these topics. Methods: Five focus groups with people with diabetes (n = 14), COPD (n = 9), and a cardiovascular condition (n = 7) were conducted in this qualitative research. Separate focus groups were organized based on patients’ chronic condition. The following themes were discussed: 1) the impact of the chronic disease on patients’ daily life; 2) their opinions and needs regarding self-management; and 3) their expectations and needs regarding, and willingness to use, eHealth for self-management purposes. A conventional content analysis approach was used for coding. Results: Patient groups seem to differ in expectations and needs regarding self-management and eHealth for self-management purposes. People with diabetes reported most needs and benefits regarding self-management and were most willing to use eHealth, followed by the COPD group. People with a cardiovascular condition mentioned having fewer needs for self-management support, because their disease had little impact on their life. In all patient groups it was reported that the patient, not the care professional, should choose whether or not to use eHealth. Moreover, participants reported that eHealth should not replace, but complement personal care. Many participants reported expecting feelings of anxiety by doing measurement themselves and uncertainty about follow-up of deviant data of measurements. In addition, many participants worried about the implementation of eHealth being a consequence of budget cuts in care. Conclusion: This study suggests that aspects of eHealth, and the way in which it should be implemented, should be tailored to the patient. Patients’ expected benefits of using eHealth to support self-management and their perceived controllability over their disease seem to play an important role in patients’ willingness to use eHealth for self-management purposes

TGF-ß induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression.

Abstract TGF-ß/Activin induces epithelial-to-mesenchymal transition (EMT) and stemness in pancreatic ductal adenocarcinoma (PDAC). However, the microRNAs (miRNAs) regulated during this response have remained yet undetermined. Here, we show that TGF-ß transcriptionally induces MIR100HG lncRNA, containing miR-100, miR-125b and let-7a in its intron, via SMAD2/3. Interestingly, we find that although the pro-tumourigenic miR-100 and miR-125b accordingly increase, the amount of anti-tumourigenic let-7a is unchanged, as TGF-ß also induces LIN28B inhibiting its maturation. Notably, we demonstrate that inactivation of miR-125b or miR-100 affects the TGF-ß-mediated response indicating that these miRNAs are important TGF-ß effectors. We integrated AGO2-RIP-seq with RNA-seq to identify the global regulation exerted by these miRNAs in PDAC cells. Transcripts targeted by miR-125b and miR-100 significantly overlap and mainly inhibit p53 and cell-cell junctions’ pathways. Together, we uncover that TGF-ß induces an lncRNA, whose encoded miRNAs, miR-100, let-7a and miR-125b, play opposing roles in controlling PDAC tumourigenesis

Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.This work was supported by the Medical Research Council (UK). S.F. was supported by a Herchel Smith Research Studentship and K.F. by an MRC Career Development Award. E.R.M is supported by the ERC Advanced Researcher award 323004–ONCOTREAT. P.H.M. is supported by Senior Investigator Awards from the Wellcome Trust and NIHR. The Cambridge Human Research Tissue Bank and A.W. are supported by the NIHR Cambridge Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Nature Publishing at http://dx.doi.org/10.1038/nature19353

Ablation of Dicer from murine Schwann cells increases their proliferation while blocking myelination

The myelin sheaths that surround the thick axons of the peripheral nervous system are produced by the highly specialized Schwann cells. Differentiation of Schwann cells and myelination occur in discrete steps. Each of these requires coordinated expression of specific proteins in a precise sequence, yet the regulatory mechanisms controlling protein expression during these events are incompletely understood. Here we report that Schwann cell-specific ablation of the enzyme Dicer1, which is required for the production of small non-coding regulatory microRNAs, fully arrests Schwann cell differentiation, resulting in early postnatal lethality. Dicer(-/-) Schwann cells had lost their ability to myelinate, yet were still capable of sorting axons. Both cell death and, paradoxically, proliferation of immature Schwann cells was markedly enhanced, suggesting that their terminal differentiation is triggered by growth-arresting regulatory microRNAs. Using microRNA microarrays, we identified 16 microRNAs that are upregulated upon myelination and whose expression is controlled by Dicer in Schwann cells. This set of microRNAs appears to drive Schwann cell differentiation and myelination of peripheral nerves, thereby fulfilling a crucial function for survival of the organism

Improving gene-set enrichment analysis of RNA-Seq data with small replicates

Deregulated pathways identified from transcriptome data of two sample groups have played a key role in many genomic studies. Gene-set enrichment analysis (GSEA) has been commonly used for pathway or functional analysis of microarray data, and it is also being applied to RNA-seq data. However, most RNA-seq data so far have only small replicates. This enforces to apply the gene-permuting GSEA method (or preranked GSEA) which results in a great number of false positives due to the inter-gene correlation in each gene-set. We demonstrate that incorporating the absolute gene statistic in one-tailed GSEA considerably improves the false-positive control and the overall discriminatory ability of the gene-permuting GSEA methods for RNA-seq data. To test the performance, a simulation method to generate correlated read counts within a gene-set was newly developed, and a dozen of currently available RNA-seq enrichment analysis methods were compared, where the proposed methods outperformed others that do not account for the inter-gene correlation. Analysis of real RNA-seq data also supported the proposed methods in terms of false positive control, ranks of true positives and biological relevance. An efficient R package (AbsFilterG- SEA) coded with C++ (Rcpp) is available from CRAN.open

Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: Cellular model of pathology

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials

Lamin B1 regulates somatic mutations and progression of B-cell malignancies

Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B-cell activation and formation of lymphoid germinal centres. Chromatin immunoprecipitation-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level correlated with progression-free and overall survival in chronic lymphocytic leukaemia, and was strongly involved in the transformation of follicular lymphoma. In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a 'mutational gatekeeper', suppressing the aberrant mutations that drive lymphoid malignancy

Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

Purpose: Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods: Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5-9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ketoconazole. Panobinostat was administered as single agent three times a week on day 15 and onward. Results: In the presence of ketoconazole, there was 1.6- and 1.8-fold increase in Cmaxand AUC of panobinostat, respectively. No substantial change in Tmaxor half-life was observed. No difference in panobinostat-pharmacokinetics between patients carrying CYP3A5*1/*3 and CYP3A5*3/*3 alleles was observed. Most frequently reported adverse events were gastrointestinal related. Patients had asymptomatic hypophosphatemia (64%), and urine analysis suggested renal phosphate wasting. Conclusions: Co-administration of panobinostat with CYP3A inhibitors is feasible as the observed increase in panobinostat PK parameters was not considered clinically relevant. Considering the variability in exposure following enzyme inhibition and the fact that chronic dosing of panobinostat was not studied with CYP3A inhibitors, close monitoring of panobinostat-related adverse events is necessary

The role country of birth plays in receiving disability pensions in relation to patterns of health care utilisation and socioeconomic differences: a multilevel analysis of Malmo, Sweden

BACKGROUND: People of low socioeconomic status have worse health and a higher probability of being granted a disability pension than people of high socioeconomic status. It is also known that public and private general physicians and public and private specialists have varying practices for issuing sick leave certificates (which, if longstanding, may become the basis of disability pensions). However, few studies have investigated the influence of a patient's country of birth in this context. METHODS: We used multilevel logistic regression analysis with individuals (first level) nested within countries of birth (second level). We analysed the entire population between the ages of 40 and 64 years (n = 80 212) in the city of Malmo, Sweden, in 2003, and identified 73% of that population who had visited a physician at least once during that year. We studied the associations between individuals and country of birth socioeconomic characteristics, as well as individual utilisation of different kinds of physicians in relation to having been granted a disability pension. RESULTS: Living alone (OR(women )= 1.72, 95% CI: 1.62–1.82; OR(men )= 2.64, 95% CI: 2.46–2.83) and having limited educational achievement (OR(women )= 2.14, 95% CI: 2.00–2.29; OR(men )= 2.12, 95% CI: 1.98–2.28) were positively associated with having a disability pension. Utilisation of public specialists was associated with a higher probability (OR(women )= 2.11, 95% CI: 1.98–2.25; OR(men )= 2.16, 95% CI: 2.01–2.32) and utilisation of private GPs with a lower probability (OR(men )= 0.76, 95% CI: 0.69–0.83) of having a disability pension. However, these associations differed by countries of birth. Over and above individual socioeconomic status, men from middle income countries had a higher probability of having a disability pension (OR(men )= 1.61, 95% CI: 1.06–2.44). CONCLUSION: The country of one's birth appears to play a significant role in understanding how individual socioeconomic differences bear on the likelihood of receiving a disability pension and on associated patterns of health care utilisation