Extracellular Poly Research Articles

Suspended Tissue Open Microfluidic Patterning (STOMP).

Cell-laden hydrogel constructs suspended between pillars are powerful tools for modeling tissue structure and physiology, though current fabrication techniques often limit them to uniform compositions. In contrast, tissues are complex in nature with spatial arrangements of cell types and extracellular matrices. Thus, we present Suspended Tissue Open Microfluidic Patterning (STOMP), which utilizes a removable, open microfluidic patterning channel to pattern multiple spatial regions across a single suspended tissue. The STOMP platform contains capillary pinning features along the open channel that controls the fluid front, allowing multiple cell and extracellular matrix precursors to be pipetted into one tissue. We have used this technique to pattern suspended tissues with multiple regional components using a variety of native and synthetic extracellular matrices, including fibrin, collagen, and poly(ethylene glycol). Here, we demonstrate that STOMP models a region of fibrosis in a functional heart tissue and a bone-ligament junction in periodontal tissues. Additionally, the STOMP platform can be customized to allow patterning of suspended cores and more spatial configurations, enhancing its utility in complex tissue modeling. STOMP is a versatile technique for generating suspended tissue models with increased control over cell and hydrogel composition to model interfacial tissue regions in a suspended tissue.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV)

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Electroconductive and Immunomodulatory Natural Polymer‐Based Hydrogel Bandages Designed for Peripheral Nerve Regeneration

AbstractSuccessful regeneration of the peripheral nerve relies on the collaborative efforts of neural cells and immune cells. Conductive hydrogels have yielded promising results in supporting axonal growth; however, their inability to regulate the immune response and their poor biological integration with tissues hinder the repair of injured peripheral nerves. Herein, an adhesive conductive immunomodulatory nerve hydrogel bandage is developed for nerve regeneration. The nerve bandage hydrogel is prepared from the bioactive material extracellular matrix (ECM), oxidized polysaccharides, and poly(3,4‐ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT:PSS) by self‐assembly and dynamic Schiff base cross‐linking. The drug indole‐3‐propionic acid (IPA) is loaded into nerve bandages and contributes to the rapid chemotaxis of neutrophils in the dorsal root ganglia and modulation of the immune system. In addition, the conductive hydrogel bandage exhibits close conformal contact with the injured nerve, forming a stable and tightly coupled electrical bridge with the electroresponsive neural tissue. In summary, the nerve bandage effectively promote nerve regeneration and enable both anatomical and functional recovery of neural tissue while preventing muscle atrophy. This work provides a new strategy for peripheral nerve regeneration and may have critical clinical applications in the future.

Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Synthesis of hybrid myocardium constructs and in vitro characterization under mechanical stimulation

The myocardium tissue may lose its contraction and mechanical characteristics due to irreversible necrosis in cardiac muscles in patients with infarction history. The repair of such damages by the body is unfortunately not possible since the adult cardiomyocytes fail to divide themselves. Recently, stem cells, cell sheet technologies, decellularized and synthetically produced cardiac constructs for the regenerative solutions for such situations have been widely studied in cardiac tissue engineering. Within the scope of this study, Poly(glycerol-sebacate) (PGS) elastomer impregnated decellularized matrices (hybrid) were produced for the myocardium regeneration. Firstly, the effectiveness of the decellularization approach was investigated, next, chemical, mechanical and morphological characterizations of the hybrid structures were studied. Young modulus was found as 83.3 ± 22.6 kPa, 654.6±16.7 kPa and 340.5 ± 17.8 kPa for native tissue, decellularized tissue and hybrid construct, respectively. The hybrid constructs were recellularized with human cardiomyocytes and mechanically stimulated using a bioreactor. Compared to static cell culture conditions, dynamic cell culture studies showed a remarkable cell proliferation rate up to the 14 th day of the culture period. The applied mechanical stimulation improved the cell proliferation, increased the GAG amount and gene expressions for cardiac markers. • Decellularization process causes a major loss of the tissue architecture • The advantages of extracellular matrix and Poly(glycerol-sebacate) polymer may be combined to improve mechanical properties of cardiac patch • An alternative hybrid candidates as cardiac biomaterials for regenerative medicine

Role of Hfq in glucose utilization, biofilm formation and quorum sensing system in Bacillus subtilis.

Hfq is an RNA-binding protein, its main function is to participate in post-transcriptional regulation of bacteria and regulate small regulatory RNA (sRNA) and messenger RNA (mRNA) stability, but the Hfq function of Bacillus subtilis (B. subtilis) has not been fully explained. In this study, we used the strains of B. subtilis168 (BS168), BS168Δhfq and BS168Δhfq-C to explore the effects of Hfq on the glucose utilization, biofilm formation and quorum sensing (QS) system of B. subtilis. The results showed that the knockout of hfq resulted in growth defects when bacteria were cultured in the Luria-Bertani (LB) medium, but we did not observe the same effects in Nitrogen medium (NM) and Inorganic Salt-free medium (ISM). We further found that the growth of strains under different glucose concentrations was also different, which was related to the expression of CcpA. Interestingly, the hfq mutant showed increased resistance to a high-glucose environment. Furthermore, the biofilm and extracellular poly saccharides (EPS) formation of BS168Δhfq decreased significantly. At the same time, changes were observed in the morphology of the biofilm, such as larger intercellular space of the biofilm and thinner edge. The qRT-PCR results confirmed that the hfq knockout caused significant up-regulation or down-regulation of gene expression in QS system, and down-regulated genes were involved in the positive regulation of biofilm formation. Taken together, we demonstrated that Hfq plays a vital role in glucose utilization, biofilm formation and QS of B. subtilis, which provides a new perspective for subsequent related research.

Poly (ADP-Ribose) and α-synuclein extracellular vesicles in patients with Parkinson disease: A possible biomarker of disease severity.

Background/ObjectiveDespite multiple attempts, no surrogate biomarker of Parkinson disease (PD) has been definitively identified. Alternatively, identifying a non-invasive biomarker is crucial to understanding the natural history, severity, and progression of PD and to guide future therapeutic trials. Recent work highlighted alpha synuclein-containing extracellular vesicles and Poly (ADP-ribose) polymerase (PARP-1) activity as drivers of PD pathogenesis and putative PD biomarkers. This exploratory study evaluated the role of alpha-synuclein-positive extracellular vesicles and PARP-1 activity in the plasma of PD patients as non-invasive markers of the disease’s severity and progression.MethodsWe collected plasma of 57 PD patients (discovery cohort 20, replication cohort 37) and compared it with 20 unaffected individuals, 20 individuals with clinically diagnosed Alzheimer’s disease, and 20 individuals with dementia with Lewy bodies. We analyzed alpha-synuclein-positive extracellular vesicles from platelet-free plasma by nanoscale flow cytometry and blood concentrations of poly ADP-ribose using sandwich ELISA kits.ResultsMedian concentration of α-synuclein extracellular vesicles was significantly higher in PD patients compared to the other groups (Kruskal-Wallis, p < .0001). In the discovery cohort, patients with higher α-synuclein extracellular vesicles had a higher Unified Parkinson Disease Rating Scale score (UPDRS III median = 22 vs. 5, p = 0.045). Seven out of 20 patients (35%) showed detectable PAR levels, with positive patients showing significantly higher levels of α-synuclein extracellular vesicles. In the replication cohort, we did not observe a significant difference in the PAR-positive cases in relationship with UPDRS III.ConclusionsNon-invasive determination of α-synuclein-positive extracellular vesicles may provide a potential non-invasive marker of PD disease severity, and longitudinal studies are needed to evaluate the role of α-synuclein-positive extracellular vesicles as a marker of disease progression.

Transcriptional Expression Profiles of Olive Flounder (Paralichthys olivaceus) Toll-like Receptor 3 (TLR3) and TLR22 in Response to Extracellular Poly I:C Stimuli

Toll-like receptors (TLRs) recognize extracellular and intracellular double-stranded RNA (dsRNA) and play crucial roles in innate and adaptive immune systems. Here, we aimed to investigate the changes in the transcriptional profile of TLR3 and TLR22 genes of olive flounder (Paralichthys olivaceus) in response to polyinosinic:polycytidylic acid (poly I:C; a dsRNA analog) stimuli in vivo, using healthy olive flounders and in vitro, using Hirame natural embryonic (HINAE) cells. The role of JfTLR3 and JfTLR22 in inducing type I interferon response in HINAE cells was analyzed through construction and transfection of short-hairpin RNA (shRNA)-producing vector targeting the JfTLR22 and JfTLR3. Poly I:C stimuli upregulated the JfTLR3 and JfTLR22 genes both in vivo and in vitro, and JfTLR22 showed an earlier (at 6 and 12 h post-stimulation) response than JfTLR3, suggesting that JfTLR22 is a more sensitive receptor and recognizes the extracellular dsRNA more quickly than JfTLR3. After 12 h of poly I:C stimulation, the expression of ISG15 and Mx genes in HINAE cells harboring TLR22-targeting shRNA vector was markedly reduced, wherein the reduction in expression of these genes was weakly reduced in HINAE cells carrying TLR3-targeting shRNA vector. These results suggested that JfTLR22 plays an important role, wherein JfTLR3 partially induces type I interferon response to extracellular dsRNA at 12 h after poly I:C stimulation.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-β in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Cartilage Extracellular Matrix Scaffold With Kartogenin-Encapsulated PLGA Microspheres for Cartilage Regeneration.

Repair of articular cartilage defects is a challenging aspect of clinical treatment. Kartogenin (KGN), a small molecular compound, can induce the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Here, we constructed a scaffold based on chondrocyte extracellular matrix (CECM) and poly(lactic-co-glycolic acid) (PLGA) microspheres (MP), which can slowly release KGN, thus enhancing its efficiency. Cell adhesion, live/dead staining, and CCK-8 results indicated that the PLGA(KGN)/CECM scaffold exhibited good biocompatibility. Histological staining and quantitative analysis demonstrated the ability of the PLGA(KGN)/CECM composite scaffold to promote the differentiation of BMSCs. Macroscopic observations, histological tests, and specific marker analysis showed that the regenerated tissues possessed characteristics similar to those of normal hyaline cartilage in a rabbit model. Use of the PLGA(KGN)/CECM scaffold may mimic the regenerative microenvironment, thereby promoting chondrogenic differentiation of BMSCs in vitro and in vivo. Therefore, this innovative composite scaffold may represent a promising approach for acellular cartilage tissue engineering.

Effect of C/N substrates for enhanced extracellular polymeric substances (EPS) production and Poly Cyclic Aromatic Hydrocarbons (PAHs) degradation.

Extracellular Polymeric Substances (EPS) influenced Poly Cyclic Aromatic Hydrocarbons (PAHs) degrading Klebsiella pneumoniae was isolated from the marine environment. To increase the EPS production by Klebsiella pneumoniae, several physicochemical parameters were tweaked such as different carbon sources (arabinose, glucose, glycerol, lactose, lactic acid, mannitol, sodium acetate, starch, and sucrose at 20g/L), nitrogen sources (ammonium chloride, ammonium sulphate, glycine, potassium nitrate, protease peptone and urea at 2g/L), different pH, carbon/nitrogen ratio, temperature, and salt concentration were examined. Maximum EPS growth and biodegradation of Anthracene (74.31%), Acenaphthene (67.28%), Fluorene (62.48%), Naphthalene (57.84%), and mixed PAHs (55.85%) were obtained using optimized conditions such as glucose (10g/L) as carbon source, potassium nitrate (2g/L) as the nitrogen source at pH 8, growth temperature of 37°C, 3% NaCl concentration and 72h incubation period. The Klebsiella pneumoniae biofilm architecture was studied by confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM). The present study demonstrates the EPS influenced PAHs degradation of Klebsiella pneumoniae.

3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. Bio-based materials that support cellular adhesion, differentiation, and proliferation - including gelatin, collagen, hyaluronic acid, and alginate - have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states. The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Stability and performance of poly γ-(glutamic acid) in the presence of sulfate ion for enhanced heavy oil recovery

First, the extracellular poly γ-(glutamic acid) (PGA) was synthesized by bacillus licheniformis LMG 7559. Then, the applicability of this biopolymer in the microbial enhanced oil recovery (MEOR) was evaluated. The impact of sulfate ions, as strong potential determining ions (PDI's) toward carbonate surfaces, on the stability and performance of PGA flooding, was investigated. The stability of the PGA solution was reduced by increasing the biopolymer content and adding salts. The contact angle measurement showed that sulfate ions played an important role in altering of wetting state to a water-wet condition (less oil-wet). After the treatment of glass and carbonate rock plates in the PGA solution (0.25% w/v), the contact angles were changed about 18 and 44 units, respectively. Also, the PGA solution with a two-fold concentration of typical sulfate ions content (SBP#2 S) changed the contact angle about 47-fold and 92-fold at the glass and rock surfaces, respectively. It was concluded that the SBP#2 S decreased the surface charge of carbonate rock to the value of −6.5 mV compared to the sulfate-free sea brine-PGA solution (SBP#0 S) with the value of −2.3 mV. Furthermore, the adsorption of PGA onto the carbonate particles and the fluids viscosity as well as the interfacial tension (IFT) between PGA solution and crude heavy oil were examined. We observed that as the PGA content increased to 0.5% w/v, PGA adsorption on the carbonate rock surface increased to 21.1 mg/g. The Freundlich isotherm model was more acceptable for the experimental results than the Langmuir model. Also, the results of viscosity measurement showed that the viscosity of PGA solutions decreased in the presence of the salts. Stable fluids were injected in a quarter five-spot heterogeneous glass micro-model. The PGA solution with the content of 0.5% (w/v) recovered 31.45% of oil in place and the SBP#2 S produced 27.82% of oil in place compared to the SBP#0 S solution (19.1%).

Cell Surface Display of Poly(3-hydroxybutyrate) Depolymerase and its Application.

We have expressed extracellular poly(3-hydroxybutyrate) (PHB) depolymerase of Ralstonia pickettii T1 on the Escherichia coli surface using Pseudomonas OprF protein as a fusion partner by C-terminal deletion-fusion strategy. Surface display of depolymerase was confirmed by flow cytometry, immunofluorescence microscopy and whole cell hydrolase activity. For the application, depolymerase was used as an immobilized catalyst of enantioselective hydrolysis reaction for the first time. After 48 h, (R)-methyl mandelate was completely hydrolyzed, and (S)-mandelic acid was produced with over 99% enantiomeric excess. Our findings suggest that surface displayed depolymerase on E. coli can be used as an enantioselective biocatalyst.

Production and physicochemical characterization of bacterial poly gamma- (glutamic acid) to investigate its performance on enhanced oil recovery

Bacillus licheniformis LMG 7559, which is capable of producing extracellular poly gamma- (glutamic acid) (PGA), was provided for the biopolymer synthesis. Using a modified PGA medium for PGA production, the isolated biopolymer, undergone dialysis process mainly for desalination and removal of other impurities. The bacteria produced high molecular weight biopolymers with a weight average molecular weight (M̅n) of 1.6 × 105 g/mole identified by gel permeation chromatography (GPC). Furthermore, GPC analysis was utilized to determine the poly-dispersity of PGA as well as molecular weight variation by cultivation time. The heavy weight fraction of 1.85 × 105 g/mole with poly-dispersity index of 7.42 was distinguished. For the extracted and dialyzed biopolymer, thermal properties were studied using DSC/TGA by which a mass loss of 36 percent was observed. Eventually, the biopolymer solution was injected into the oil saturated heterogeneous porous medium to evaluate the recovery factor enhancement by PGA flooding. It was found that 31.45% of oil in place was recovered by biopolymer flooding, whereas only 16.6% of oil in place was obtained by water flooding.

Influence of fiber architecture and growth factor formulation on osteoblastic differentiation of mesenchymal stem cells in coacervate-coated electrospun fibrous scaffolds

In order to achieve effective bone regeneration, the architectural/structural and biological environment must be considered. In this study, we investigated the influence of structure of extracellular matrix-mimicking electrospun poly (l-lactic acid) (PLLA) fibrous scaffolds and incorporated growth factors (bone morphogenetic protein2 (BMP2) and platelet-derived growth factor (PDGF)) (GFs) on in vitro differentiation of human mesenchymal stem cells (hMSCs) cultured onto the scaffolds. Furthermore, Cargo GFs were first encapsulated into coacervate (Coa). Coa-coated nano-sized PLLA (Coa-nPLLA) exhibited relatively higher coating efficacy, than Coa-coated micro-sized PLLA (Coa-mPLLA) due to large mesh hole area of mPLLA. Consequently, a faster GF release pattern was observed in nPLLA groups. As a result of osteogenic differentiation of hMSC which cultured onto GFs loaded Coa-PLLA, the architectural cue of fiber diameters effectively modulated early osteogenic differentiation of hMSCs. Facilitated long-term differentiation of hMSCs could be achieved by synergistic effect of dual BMP2 and PDGF in nanofbrous environments.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization

SummaryBiomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

RETRACTED ARTICLE: A switch in the poly(dC)/RmlB complex regulates bacterial persister formation

Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex physiological state (from poly(dC)/RmlB to P-poly(dC)/RmlB or RmlB). In response to the switch, bacteria decrease proton motive force and intracellular ATP levels, forming dormant cells. This alteration in complex status is linked to a (p)ppGpp-controlled signaling pathway that includes inorganic polyphosphate, Lon protease, exonuclease VII (XseA/XseB), and the type III secretion system. The persistence might be also an adaptive response to the lethal action of the dTDP-l-rhamnose pathway shutdown, which occurs due to switching of poly(dC)/RmlB.

Purification and characterization of new bio-plastic degrading enzyme from Burkholderia cepacia DP1

Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0–10 and at 35–55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.

Abstract 5585: Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening

Abstract Extracellular vesicles (EV) are a promising biomarker source and present in several biological fluids such as plasma (8.0 x 1011 particles/mL, 121 ± 2.3 nm in diameter) and urine (1.1 x 1011 particles/mL, 152 ± 11.2 nm in diameter). In order to streamline marker screening and validation in translational studies, we developed and optimized high throughput filter-based EV capture, poly(A)+ RNA isolation and gene expression assay protocols for RT-qPCR and RNA-seq. First, we confirmed that plasma and urinary EV mRNA profiles (12 to 15 genes by RT-qPCR) are stable up to 1 week at 4°C, and up to 5 repeats of freeze-thaw cycles, and urinary EV was stable up to 10 months at -80°C, indicating EV mRNA could be a useful biomarker source. Although EV mRNA is less abundant (550 pg mRNA from 1 mL plasma) compared to tissue mRNA or EV non-coding RNA, the optimized protocol allows quantification of about 9,000 genes in average by RNA-seq from 2 mL frozen plasma (N=3) with high reproducibility among the replicates (Spearman correlation: 0.9086 to 0.9326) and between the commercial EV kits (Spearman correlation: 0.9207 to 0.9683). About 77% of the 1000 most abundant genes in plasma EV are expressed ubiquitously throughout various organs but some are expressed specifically in blood (12%), spleen (4.4%), esophagus mucosa (1.2%), etc. according to the Genotype-Tissue Expression (GTEx) database. In urinary EV, other than ubiquitously expressed genes (88%), the genes specific to liver (1.5%), bladder (1%), brain (1%), etc. are present. These data suggest EV mRNA are released from various organs and circulating in biological fluids. The protocol was further applied to biomarker discovery for bladder cancer, and differential gene expression analysis of EV mRNA was conducted using bladder cancer (N=4), cancer remission (N=2), and healthy/disease control urine samples (N=4). Ingenuity pathway analysis indicated that cancer-related molecular and cellular functions are activated in bladder cancer urinary EV but not in remission, while immune-related functions are activated both in cancer and remission. These data indicate that EV mRNA is a promising biomarker source for cancer biomarker screening, and could be used in translational studies. Citation Format: Taku Murakami, Cindy Yamamoto, Mieko Ogura, Melanie Oakes, Hiroshi Harada. Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5585.