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Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries
No full textAbstract
Background
During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery.
Results
Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous \u3b2-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw.
Conclusions
In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei . With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases
Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process
No full textAbstract
Background
Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively.
Results
Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95\ua0\ub0C for 30\ua0min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer.
Conclusions
Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples
The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry
No full textAbstract
Background
RRx-001, a dinitroazetidine derivative, is a novel anticancer agent currently in phase II clinical trials. It mediates immunomodulatory effects either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a radio- and chemo-sensitizer to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. Here, we identified another mechanism, viral mimicry, which refers to the \u201cunsilencing\u201d of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anticancer activity of RRx-001.
Results
RRx-001 inhibited the growth of colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases DNMT1 and DNMT3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5\ua0\u3bcM RRx-001 for 24\ua0h significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4\ua0weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retrovirus ERV-Fc2 and LTRs from the ERV-L family (MLT2B4 and MLT1C49) was induced by RRx-001. The induction of ERV-Fc2-env was through demethylation of ERV-Fc2 LTR as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA.
Conclusions
Transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this \u201cpseudo-pathogenic\u201d stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination. The striking safety profile of RRx-001 in comparison to other more toxic epigenetic and immunomodulatory agents such as azacitidine makes it a leading candidate for such clinical applications
The epigenomic basis of common diseases
No full textAbstract
A report of the 6
th
Epigenomics of Common Diseases Conference held at the Wellcome Genome Campus in Hinxton, Cambridge, UK, on 1\u20134 November 2016
The impact of reconstruction and scanner characterisation on the diagnostic capability of a normal database for [ 123 I]FP-CIT SPECT imaging
No full textAbstract
Background
The use of a normal database for [
123
I]FP-CIT SPECT imaging has been found to be helpful for cases which are difficult to interpret by visual assessment alone, and to improve reproducibility in scan interpretation. The aim of this study was to assess whether the use of different tomographic reconstructions affects the performance of a normal [
123
I]FP-CIT SPECT database and also whether systems benefit from a system characterisation before a database is used.
Seventy-seven [
123
I]FP-CIT SPECT studies from two sites and with 3-year clinical follow-up were assessed quantitatively for scan normality using the ENC-DAT normal database obtained in well-documented healthy subjects. Patient and normal data were reconstructed with iterative reconstruction with correction for attenuation, scatter and septal penetration (ACSC), the same reconstruction without corrections (IRNC), and filtered back-projection (FBP) with data quantified using small volume-of-interest (VOI) (BRASS) and large VOI (Southampton) analysis methods. Test performance was assessed with and without system characterisation, using receiver operating characteristics (ROC) analysis for age-independent data and using sensitivity/specificity analysis with age-matched normal values. The clinical diagnosis at follow-up was used as the standard of truth.
Results
There were no significant differences in the age-independent quantitative assessment of scan normality across reconstructions, system characterisation and quantitative methods (ROC AUC 0.866\u20130.924). With BRASS quantification, there were no significant differences between the values of sensitivity (67.4\u201383.7%) or specificity (79.4\u201391.2%) across all reconstruction and calibration strategies. However, the Southampton method showed significant differences in sensitivity between ACSC (90.7%) vs IRNC (76.7%) and FBP (67.4%) reconstructions with calibration. Sensitivity using ACSC reconstruction with this method was also significantly better with calibration than without calibration (65.1%). Specificity using the Southampton method was unchanged across reconstruction and calibration choices ..
Is health care a necessary or luxury product for Asian countries? An answer using panel approach
No full textAbstract
A number of studies have estimated the income elasticity of health care expenditure to identify whether health care is a necessary or luxury product. However, the issue has received less attention in developing countries, especially in Asian economies. The current study for the first time has used the panel data covering 36 Asian countries for the period 1995\u20132013 for revealing the nature of health care as a product. Along with conventional econometric techniques we have addressed the issue of cross section dependence and used Westerlund (2007) panel cointegration test which is robust against cross section dependence and heterogeneity for detecting the presence of panel cointegration. By applying Fully Modified OLS (FMOLS) and Dynamic OLS (DOLS) it was found that the long run elasticity of Health Care Expenditure (HCE) with Gross Domestic Product (GDP) is less than unit implying that the health care can be regarded as necessary in nature for these countries.
JEL Classification: I10, C51
Saturation effect and transmembrane conversion of Monascus pigment in nonionic surfactant aqueous solution
No full textAbstract
Extractive fermentation in a nonionic surfactant aqueous solution provides a promising and efficient method to produce Monascus pigments. The behaviour of pigment secretion during the extractive cultivation was investigated in the present work. The results revealed that the secretion of intracellular pigment was limited by its saturation concentration in the nonionic surfactant aqueous solution. The intracellular pigment was completely extracted to the outside of the cell at a low cell density and high concentration of Triton X-100 (TX) in fermentation broth; otherwise, a restriction for pigment extraction would occur. The decrement of the intracellular orange and yellow pigments was inconsistent with the increment of extracellular pigments with an increase in the TX concentration. It could be inferred that the intracellular orange pigment was converted to extracellular yellow pigment during the transmembrane secretion process, which might be attributed to the enzyme catalysis in the non-aqueous phase solution. This study helps explain the mechanism of variation of pigment characteristic and extraction capacity in extractive fermentation
Central venous access related adverse events after trabectedin infusions in soft tissue sarcoma patients; experience and management in a nationwide multi-center study
No full textAbstract
Background
Trabectedin has shown efficacy against soft tissue sarcomas (STS) and has manageable toxicity. Trabectedin is administered through central venous access devices (VAD), such as subcutaneous ports with tunneled catheters, Hickman catheters and PICC lines. Venous access related adverse events are common, but have not yet been reported in detail.
Methods
A retrospective analysis of patient files of STS patients receiving trabectedin monotherapy between 1999 and 2014 was performed in all five STS referral centers in the Netherlands. This survey focused on adverse events related to the VAD and the actions taken in response to these events.
Results
In the 127 patients included in this analysis, 102 venous access ports (VAP), 15 Hickman catheters and 10 PICC lines were used as primary means of central venous access. The most frequently reported adverse events at the VAD site were erythema (30.7%), pain (28.3%), inflammation (11.8%) and thrombosis (11.0%). Actions taken towards these adverse events include oral antibiotics (17.3%), VAD replacement (15.0%) or a wait-and-see policy (13.4%). In total, 45 patients (35.4%) with a subcutaneous port developed a varying degree of inflammation along the trajectory of the tunneled catheter. In all but three patients, this was a sterile inflammation, which was considered a unique phenomenon for trabectedin. Microscopic leakage of trabectedin along the venous access device and catheter was considered the most plausible cause for this adverse event. Placing the catheter deeper under the skin resolved the issue almost completely.
Conclusion
Trabectedin infusion commonly leads to central venous access related adverse events. Sterile inflammation along the catheter trajectory is one of the most common adverse events and can be prevented by placing the catheter deeper under the skin
Relation between presence of extended-spectrum \u3b2-lactamase-producing Enterobacteriaceae in systematic rectal swabs and respiratory tract specimens in ICU patients
No full textAbstract
Background
The choice of empirical antimicrobial therapy for pneumonia in intensive care unit (ICU) is a challenge, since pneumonia is often related to multidrug-resistant pathogens, particularly extended-spectrum \u3b2-lactamase-producing Enterobacteriaceae (ESBL-E). To prevent the overuse of broad-spectrum antimicrobial therapy, the main objective of this study was to test the performance of digestive colonization surveillance as a predictor of ESBL-E presence or absence in respiratory samples performed in ICU and to evaluate the impact of time sampling (\u22645\ua0days or >5\ua0days) on such prediction. Design: Multicentric retrospective observational study, including every patient with a respiratory tract specimen positive culture and a previous rectal ESBL-E screening performed within 7\ua0days before the respiratory sample, between January 2012 and December 2014. Results were analyzed in two groups: respiratory samples obtained during the first 5\ua0days of ICU stay (early group) and respiratory samples obtained after 5\ua0days (late group). Interventions: none.
Results
Among 2498 respiratory tract samples analyzed corresponding to 1503 patients, 1557 (62.3%) were performed early (\u22645\ua0days) and 941 (37.7%) later (>5\ua0days). Positivity rates for ESBL-E were 15.0 and 36.8% for rectal swabs in the early and late groups, respectively. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values and likelihood ratios were calculated for ESBL-E digestive colonization as a predictor of ESBL-E presence in respiratory samples. PPVs of ESBL-E digestive colonization were 14.5% (95% CI [12.8; 16.3]) and 34.4% (95% CI [31.4; 37.4]), for the early and late groups, respectively, whereas NPVs were 99.2% (95% CI [98.7; 99.6]) and 93.4% (95% CI [91.9; 95.0]), respectively.
Conclusions
Systematic surveillance of ESBL-E digestive colonization may be useful to limit the use of carbapenems when pneumonia is suspected in ICU. When rectal swabs are negative, the risk of having ESBL-E in respiratory samples is very low even after 5\ua0days of ICU stay
The error analysis of Crank-Nicolson-type difference scheme for fractional subdiffusion equation with spatially variable coefficient
No full textAbstract
A Crank-Nicolson-type difference scheme is presented for the spatial variable coefficient subdiffusion equation with Riemann-Liouville fractional derivative. The truncation errors in temporal and spatial directions are analyzed rigorously. At each time level, it results in a linear system in which the coefficient matrix is tridiagonal and strictly diagonally dominant, so it can be solved by the Thomas algorithm. The unconditional stability and convergence of the scheme are proved in the discrete
L
2
norm by the energy method. The convergence order is
min
{
2
\u2212
\u3b1
2
,
1
+
\u3b1
}
in the temporal direction and two in the spatial one. Finally, numerical examples are presented to verify the efficiency of our method.
MSC: 65M06, 65M12, 65M15