Template-dependent multiple displacement amplification for profiling human circulating RNA

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material. </jats:p

Cutinase-Catalyzed Polyester-Polyurethane Degradation: Elucidation of the Hydrolysis Mechanism

open5siPolyurethanes (PU) are one of the most-used classes of synthetic polymers in Europe, having a considerable impact on the plastic waste management in the European Union. Therefore, they represent a major challenge for the recycling industry, which requires environmentally friendly strategies to be able to re-utilize their monomers without applying hazardous and polluting substances in the process. In this work, enzymatic hydrolysis of a polyurethane-polyester (PU-PE) copolymer using Humicola insolens cutinase (HiC) has been investigated in order to achieve decomposition at milder conditions and avoiding harsh chemicals. PU-PE films have been incubated with the enzyme at 50 degrees C for 168 h, and hydrolysis has been followed throughout the incubation. HiC effectively hydrolysed the polymer, reducing the number average molecular weight (M-n) and the weight average molecular weight (M-w) by 84% and 42%, respectively, as shown by gel permeation chromatography (GPC), while scanning electron microscopy showed cracks at the surface of the PU-PE films as a result of enzymatic surface erosion. Furthermore, Fourier Transform Infrared (FTIR) analysis showed a reduction in the peaks at 1725 cm(-1), 1164 cm(-1) and 1139 cm(-1), indicating that the enzyme preferentially hydrolysed ester bonds, as also supported by the nuclear magnetic resonance spectroscopy (NMR) results. Liquid chromatography time-of-flight/mass spectrometry (LC-MS-Tof) analysis revealed the presence in the incubation supernatant of all of the monomeric constituents of the polymer, thus suggesting that the enzyme was able to hydrolyse both the ester and the urethane bonds of the polymer.openDi Bisceglie, Federico; Quartinello, Felice; Vielnascher, Robert; Guebitz, Georg M.; Pellis, AlessandroDi Bisceglie, Federico; Quartinello, Felice; Vielnascher, Robert; Guebitz, Georg M.; Pellis, Alessandr

Treatment of hepatocellular carcinoma

Fifty-one patients with unresectable hepatocellular carcinoma were treated by embolization of the hepatic artery with Gelfoam powder, contrast material and three chemotherapeutic agents (doxorubicin, mitomycin, cisplatin). Twelve patients (24%) had a partial response with a decrease in the tumor diameter by at least 50%, 13 patients (26%) had only minor responses, 12 (24%) had stabilization of disease and the remainder had progressive disease. Tumor liquefaction was noted on computed tomographic scanning in 70% of patients, and 23 of 34 patients with elevations in serum alphafetoprotein values had a greater than 50% reduction following treatment. The median patient survival time from treatment was 207 days. Most patients experienced transient pain, fever, nausea and elevations in serum aminotransferase activities as a result of therapy. Ascites developed in 14 patients. There were two treatment-related deaths: one from tumor hemorrhage and one from liver failure. Chemoembolization therefore appears to have significant activity in patients with hepatocellular carcinoma and is relatively well tolerated. This study reports the results of resection in 72 cirrhotic patients with hepatocellular carcinoma from Europe. One and 3 year survival rates were 68% and 51% respectively. Survival was significantly higher in Child's class A than in class B or C patients. Patients with a thickly encapsulated tumor lived longer than those with an infiltrating tumor and also had a significantly lower recurrence rate. There was no relationship between the size of the tumor or the presence of symptoms and survival. These data suggest that good results can be achieved by resection of hepatocellular carcinoma in European cirrhotic patients. A thickly encapsulated tumor and good liver function are the main determinants of low cancer recurrence and high survival.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38355/1/1840130431_ftp.pd

High-resolution quantification of hepatitis C virus genome-wide mutation load and its correlation with the outcome of peginterferon-alpha2a and ribavirin combination therapy

Hepatitis C virus (HCV) is a highly mutable RNA virus and circulates as a heterogeneous population in individual patients. The magnitude of such population heterogeneity has long been proposed to be linked with diverse clinical phenotypes, including antiviral therapy. Yet data accumulated thus far are fairly inconclusive. By the integration of long RT-PCR with 454 sequencing, we have built a pipeline optimized for the quantification of HCV genome-wide mutation load at 1% resolution of mutation frequency, followed by a retrospective study to examine the role of HCV mutation load in peginterferon-alpha2a and ribavirin combination antiviral therapy. Genome-wide HCV mutation load varied widely with a range from 92 to 1639 mutations and presented a Poisson distribution among 56 patients (Kolmogorov-Smirnov statistic  = 0.078, p = 0.25). Patients achieving sustained virological response (n = 26) had significantly lower mutation loads than that in null responders (n = 30) (mean and standard derivation: 524±279 vs. 805±271, p = 0.00035). All 36,818 mutations detected in 56 patients displayed a power-law distribution in terms of mutation frequency in viral population. The low-frequency mutation load, but not the high-frequency load, was proportional firmly to the total mutation load. In-depth analyses revealed that intra-patient HCV population structure was shaped by multiple factors, including immune pressure, strain difference and genetic drift. These findings explain previous conflicting reports using low-resolution methods and highlight a dominant role of natural selection in response to therapeutic intervention. By attaining its signatures from complex interaction between host and virus, the high-resolution quantification of HCV mutation load predicts outcomes from interferon-based antiviral therapy and could also be a potential biomarker in other clinical settings

Prospects for personalizing antiviral therapy for hepatitis C virus with pharmacogenetics

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. HCV infection is currently treated with IFNα plus ribavirin for 24 to 48 weeks. This demanding therapy fails in up to 50% of patients, so the use of pharmacogenetic biomarkers to predict the outcome of treatment would reduce futile treatment of non-responders and help identify patients in whom therapy would be justified. Both IFNα and ribavirin primarily act by modulating the immune system of the patient, and HCV uses multiple mechanisms to counteract the antiviral effects stimulated by therapy. Therefore, response to therapy is influenced by variations in human genes governing the immune system and by differences in HCV genes that blunt antiviral immune responses. This article summarizes recent advances in understanding how host and viral genetic variation affect outcome of therapy. The most notable human associations are polymorphisms within the IL28B gene, but variations in human leukocyte antigen and cytokine genes have also been associated with treatment outcome. The most prominent viral genetic association with outcome of therapy is that HCV genotype 1 is much less sensitive to treatment than genotypes 2 and 3, but genetic differences below the genotype level also influence outcome of therapy, presumably by modulating the ability of viral genes to blunt antiviral immune responses. Pharmacogenetic prediction of the outcome of IFN-based therapy for HCV will require integrating the efficacies of the immunosuppressive mechanisms of a viral isolate, and then interpreting the viral resistance potential in context of the genetic profile of the patient at loci associated with outcome of therapy. Direct-acting inhibitors of HCV that will be used in combination with IFNα are nearing approval, so genetic prediction for anti-HCV therapy will soon need to incorporate viral genetic markers of viral resistance to the new drugs

Which route of antibiotic administration should be used for third molar surgery? A split-mouth study to compare intramuscular and oral intake

Objectives. To compare the effectiveness of two different routes of antibiotic administration in preventing septic complications in patients undergoing third molar extraction. Materials and Methods. Twenty-four healthy patients requiring bilateral surgical removal of impacted mandibular third molars were successfully enrolled for this study. Depth of impaction, angulation, and relationship of the lower third molars with the mandibular branch had to be overlapping on both sides. A split-mouth design was chosen, so each patient underwent both the first and second surgeries, having for each extraction a different antibiotic route of administration. The second extraction was carried out 1 month later. To compare the effects of the two routes of antibiotic administration, inflammatory parameters, such as edema, trismus, pain, fever, dysphagia and lymphadenopathy were evaluated 2 and 7 days after surgery. Side effects of each therapy were evaluated 48h after surgery. Results. oral and intramuscular antibiotic therapies overlap in preventing post-operative complications in dental surgery (p>0.05), even if the oral intake, seems to promote the onset of significant gastrointestinal disorders (p=0.003). Conclusions. This study could help dentists in their ordinary practice to choose the right route of antibiotic administration in the third molar surgery. At the same effectiveness, the higher cost and the minor compliance of the patient seem not to justify a routine antibiotic intramuscular therapy, reserving it for patients with gastrointestinal disorders

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3) fucosylation. Increases in core (alpha-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification