Development of a Simple In Vitro Assay to Assess Digestion of the Extracellular Matrix of the Human Pancreas by Collagenase Enzyme Blends

Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet–exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm3 were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet–exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.

2098

OBJECTIVES/SPECIFIC AIMS: Endogenous RT (eRT) is necessary for the function of retrotransposons, elements that replicate via an RNA intermediate. One source of eRT activity is long interspersed elements (LINE). LINEs, of which there are several subgroups (L1, L2, L3), are retrotransposons that regulate cellular growth and gene expression. Given their diverse and important roles, we hypothesized that L1 elements regulate functional responses in megakaryocytes and platelets; a concept not yet examined in the field. METHODS/STUDY POPULATION: To study eRT in human platelets we used RT activity assays, PCR, and Western blot approaches. Furthermore, we used an RT-inhibitor to dissect the function of eRT, analyzed RT-dependent protein synthetic capacity, and immunoprecipitated RNA-DNA hybrids. RNA-DNA hybrids were also detected by means of ICC and automated analysis using CellProfiler software. RNA-DNA hybrids were validated by PCR and eRT regulated synthesis of target proteins was analyzed using autoradiography and Western blot techniques. Platelets from patients with HIV+ were examined in parallel. RESULTS/ANTICIPATED RESULTS: We identified that highly purified, isolated platelets from healthy subjects possess eRT activity. eRT activity was blocked with the non-nucleoside RT inhibitor nevirapine at concentrations within the therapeutic drug range. L1 elements are bicistronic, containing 2 open reading frames (ORFs), ORF1 and ORF2. Thus, we next identified that human platelets express full-length L1 mRNA containing ORF1 and ORF2. In human platelets, eRT activity was localized to L1 protein containing ribonucleo particles. Platelet eRT reverse transcribed exogenous RNAs, a process inhibited by nevirapine, acting in trans using the 3′-UTR of exogenous mRNAs as a template. To dissect the function of eRT in platelets, we next examined cytoskeletal and protein synthetic events in the presence or absence of nevirapine. Inhibition of eRT in isolated platelets led to characteristically beaded platelets in appearance, strongly resembling bone marrow proplatelets. Parallel increases in platelet reactivity were also observed. As these changes occurred over hours, not minutes, we hypothesized that inhibition of eRT would affect platelet protein synthetic events. Consistent with this, RT inhibition resulted in upregulation of global platelet protein synthesis. We validated upregulation of the synthesis of specific proteins (mitofilin, p-selectin, and L26—a component of the 60S ribosomal subunit essential for mRNA translation). RNA-DNA hybrids, noncanonical nucleic acid structures that regulate gene expression, are enriched in regions where L1 is abundant. RNA-DNA hybrids were present in platelets and expression confirmed via differential digestion of RNAs (eg, with RNase A and RNAse I). Next-generation sequencing of pulled down (eg, immunoprecipitated) platelet RNA-DNA hybrids identified numerous differentially expressed transcripts and we focused on MAP1LC3B (LC3B), a primary regulator of autophagy. Hybrid sequencing results for LC3B were validated using qPCR and we confirmed that LC3B RNA binds to L1-encoded RNA binding protein. Platelets treated with nevirapine had increased total LC3B protein expression. As RT inhibition is an important mechanism to control HIV infection, we examined platelet morphology, activation, and LC3B expression in platelets from HIV+ subjects treated with nevirapine. HIV+ patients treated with RT inhibitors had higher numbers of platelets that were beaded in appearance at baseline, increased platelet reactivity, and differential LC3B expression compared with healthy controls. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, these results demonstrate that platelets possess eRT activity that regulates platelet morphology, platelet hyperreactivity, and protein synthetic events. We postulate that eRT activity in platelets may be a new post-transcriptional regulatory checkpoint. Moreover, our findings have implications in HIV+ patients treated with RT inhibitors, where off-target effects may contribute to platelet activation and an increased risk of thrombosis.

DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

Food and feeding ecology of Northeast Atlantic swordfish (Xiphias gladius) off the Bay of Biscay

As part of a larger project on the feeding ecology of large pelagic predators off the Bay of Biscay, this study analyses the diet of the swordfish, Xiphias gladius. Stomachs were collected from 86 swordfish. The diet was analysed in terms of prey occurrence, relative abundance, reconstituted mass, and size distribution. It consisted mainly of fish, 40.5% by mass (%M) and cephalopods, 59.3%M; crustaceans, 0.2%M, were considered secondary prey. When considering only the fresh fraction to allow for differential digestion rates, these figures were 77.3%M, 22.7%M, and trace amounts, respectively. Lanternfish, including Notoscopelus kroeyeri and Symbolophorus veranyi, were abundant, but paralepidids, Atlantic pomfret (Brama brama), and the squid Todarodes sagittatus, Ommastrephes bartramii, and Gonatus steenstrupi, dominated the diet by mass. The overall prey size range was 11–1420 mm, but sizes of 60–360 mm accounted for 80% of the distribution by number and of 140–760 mm for 80% of the distribution by mass. Intraspecifically, larger swordfish ate larger prey as a result of a change in species composition of the diet. The swordfish appears to show feeding plasticity both between different areas and between animals in the same area.

Differential Digestion of Different LMWHs by Heparinase-I and Heparinase-II: Drug Developmental Implications.

The purpose of this study was to determine the differential digestion of two different batches of branded enoxaparin (Aventis, USA), three generic versions of enoxaparin (GlandPharma, India; Lazar, Argentina; and Biochemie, Brazil), dalteparin (Pfizer, USA) and tinzaparin (Leo, USA) by heparinase-I and heparinase-II. Heparinase-I (Ibex Tech., Montreal, Canada) and heparinase-II (Ram Sasisekharan, MIT, Cambridge, MA) were isolated from Flavobacterium heparinum. The substrate specificity of these enzymes has been inferred from the reducing and non-reducing terminal structures of the di and oligosaccharides formed by digesting heparin. Heparinase-I cleaves the glucosaminidic linkage in GlcN (N-sulfate) a 1–4 IdceA (2-sulfate) and endures C-6 sulfation of hexosamine unit. More susceptibility of polymers such as heparin than oligomers to this enzymatic depolymerization indicates the size dependency of this enzymatic activity. Heparinase-II cleaves all the glucosaminidic linkages in heparin independent of O-and/or N-sulfation as well as the type of uronic acid residue. The non-sulfated substrates are somewhat resistant to this enzyme. The glucosaminidic linkage adjacent to a 3-O-sulfated GlcN residue and the innermost glucosaminidic linkage right next to the glycosaminoglycan-protein linkage region of proteoglycan are resistant to this enzymatic activity (Sugahara et al., 1994, Glycobiology 4, 535–544). In this study, several low molecular weight heparins (LMWHs) produced from different depolymerization methods of unfractionated heparin were digested with heparinase-I and heparinase-II to determine the differential digestion of these two enzymes. Eight different LMWHs with average molecular weight (MW) ranging from 3425 to 6281 Da (in UV detection at 205nm) were prepared at a concentration of 10mg/ml in 0.3M Na2SO4. Each LMWH was incubated with these enzymes (1.0 U/mL) separately for 30 minutes at 37° C in the presence of calcium Following the incubation, the samples were heated at 100° C to inactivate enzymatic activity. The molecular weight profiling of these samples was determined by using a gel permeation chromatography-high performance liquid chromatography (GPC-HPLC) system with UV detection at 205nm. A narrow range calibration method comprised of oligosaccharides and polysaccharides was used to determine the relative molecular profile of the native and digested products. The LMWHs were digested to LMW oligosaccharides with average MW of 1510± 275 Da with heparinase-I and 3071± 1044 Da with heparinase-II. The extent of digestion observed with all the LMWHs was more with heparinase-I than heparinase-II. This may be due to the different specific binding sites available for these enzymes and the requirement of sulfation at different positions in GlcN/uronic acid residues. All the LMWHs were equally digested into oligosaccharides (di, tetra and hexa) with heparinase-I. However heparinase-II resulted in the formation of only hexa, octa and decasaccharides. These results show that the LMWHs were more susceptible to heparinase-I than heparinase-II. The possible reason for the less susceptibility of these compounds to heparinase-II is likely due to the oligosaccharide composition and degree and pattern of sulfation in GlcN/uronic acid residues of these compounds. The heparinase-I and heparinase-II digestion can therefore be used in the profiling of these agents.