Degradation Fragments Research Articles

3D printed double-network alginate hydrogels containing polyphosphate for bioenergetics and bone regeneration

Low mechanical strength, poor processability, and low bioactivity of hydrogels limit their application in bone tissue engineering severely. Herein, a new 3D-printable, osteoinductive, and bioenergetic-active double-network (DN) hydrogel containing sodium alginate (SA), poly (ethylene glycol) diacrylate (PEGDA), and sodium polyphosphate (PolyP) was developed via a two-step method. The synergy of the covalent cross-linking network and the ionic cross-linking network improves the mechanical properties of the hydrogel. And the pre-gel with Ca2+ has better 3D printing performance to print complex tissue engineering scaffolds than common hydrogels. In addition, the incorporation of PolyP into DN hydrogel matrix significantly improves the bioactivity of hydrogels. The bioenergetic effect of PolyP improves adenosine triphosphate content of cells significantly to promote cell activities such as migration. The in vitro osseointegration investigation suggests that the orthophosphate monomer units, which are degradation fragments of PolyP, provide enough phosphoric acid units for the formation of calcium phosphate and accelerate the osteogenic differentiation of cells greatly. Therefore, the proposed printable, bioenergetic-active, osteoinductive DN hydrogel is potential to solve the problems of complex tissue engineering scaffolds and be applied in energy-crucial bone tissue regeneration.

The prokaryotic ubiquitin-like protein presents poor cleavage sites for proteasomal degradation.

In an event reminiscent of eukaryotic ubiquitination, the bacterial prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS) marks target proteins for proteasomal degradation by covalently attaching Pup, the bacterial tagging molecule. Yet, ubiquitin is released from its conjugated target following proteasome binding, whereas Pup enters the proteasome and remains conjugated to the target. Here, we report that although Pup can be degraded by the bacterial proteasome, it lacks favorable 20S core particle (CP) cleavage sites and is thus a very poor 20S CP substrate. Reconstituting the PPS invitro, we demonstrate that during pupylated protein degradation, Pup can escape unharmed and remain conjugated to a target-derived degradation fragment. Removal of this degradation fragment by Dop, a depupylase, facilitates Pup recycling and re-conjugation to a new target. This study thus offers a mechanistic model for Pup recycling and demonstrates how a lack of protein susceptibility to proteasome-mediated cleavage can play a mechanistic role in a biological system.

Abstract 386: Evaluation of CPa9-HNE - a peripheral biomarker based on neutrophil extracellular traps (NETs) - for predicting outcome in patients with metastatic melanoma treated with anti-PD-1 therapy

Abstract Background: Peripheral biomarkers associated with response and resistance to anti-PD-1 therapy in metastatic melanoma patients represent an unmet medical need. High neutrophil activity is associated with immune checkpoint inhibitor failure, amongst others due to release of neutrophil extracellular traps (NETs) that have been shown to protect tumor cells against cytotoxic attacks. Human neutrophil elastase (HNE) and calprotectin are the major NETs constituents. We hypothesized that HNE mediated degradation of calprotectin (CPa9-HNE), when measured in serum, has peripheral biomarker potential in metastatic melanoma patients treated with anti-PD-1 therapy. Methods: A monoclonal antibody was raised against a specific HNE mediated degradation fragment of calprotectin (CPa9-HNE) and used to develop an ELISA for quantifying CPa9-HNE in serum. CPa9-HNE was then measured in pre-treatment serum from metastatic melanoma patients treated with anti-PD-1 therapy (pembrolizumab) (n=35). The biomarker levels were associated with progression-free survival (PFS) and overall survival (OS) by univariate and multivariate Cox regression analyses adjusting for PDL1 expression (≥1%), lactate dehydrogenase (LDH), BRAF mutational status, and c-reactive protein (CRP). Results: High pre-treatment CPa9-HNE levels predicted worse PFS (HR=3.32, 95%CI=1.25-8.82, p=0.016) and OS (HR=11.31, 95%CI=2.27-56.33, p=0.003) when compared to low CPa9-HNE levels by univariate Cox regression. By multivariate Cox regression, high CPa9-HNE was found to be independently predictive of poor PFS (HR=8.22, 95%CI=1.30-52.14, p=0.025) and OS (HR=76.87, 95%CI=4.73-1248.57, p=0.002) when adjusted for PDL1 expression, LDH, BRAF mutations, and CRP. Conclusions: A non-invasive biomarker of HNE mediated degradation of calprotectin (CPa9-HNE) was associated with poor PFS and OS in metastatic melanoma patients treated with anti-PD-1 therapy. These findings support a link between neutrophil activity/NETs and response to anti-PD-1 and indicate promising non-invasive biomarker potential to stratify melanoma patients for anti-PD-1 therapy in future clinical studies. Citation Format: Christina Jensen, Anders Kverneland, Marco Donia, Joachim H. Mortensen, Morten A. Karsdal, Inge Marie Svane, Nicholas Willumsen. Evaluation of CPa9-HNE - a peripheral biomarker based on neutrophil extracellular traps (NETs) - for predicting outcome in patients with metastatic melanoma treated with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 386.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/−, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

P165 Non-invasive biomarkers of type III collagen cross-linking reflects fibrolysis in patient with luminal CROHN’S DISEASE

Abstract Background Type III collagen of the interstitial matrix is highly expressed in fibrotic tissue of Crohn’s disease (CD) and suggested to undergo remodelling during the characteristic deep tissue inflammation. Periods of inflammatory flares and remission could thus be reflected by varying degrees of type III collagen degradation and formation in relation to fibrogenesis and resolution of fibrosis. The aim was to investigate the association between type III collagen remodelling and CD disease phenotypes based on the Montreal classification Methods 40 CD patients were endoscopically assessed according to the Montreal B classification (luminal or stricturing phenotype) and Harvey Bradshaw Index for clinical disease activity (<4 = inactive; >4 = active). Patients were stratified based on both the Montreal B classification and HBI scores. Biomarkers were assessed by ELISA: 1) CTX-III (cross-linked protease degraded fragments of type III collagen), PC3X (cross-linked type III collagen formation), 3) PRO-C3 (type III collagen formation), and 4) C3M (MMP-9 degradation fragments) Results Biomarker levels of CTX-III was significantly elevated in CD patients with luminal- and clinically inactive- disease compared to patients with stricturing- and clinically inactive- disease and luminal- and clinically active- disease (p<0.01 and p<0.05, fig 1A). The levels of net fibrolysis of cross-linked type III collagen (CTX-III/PC3X) was significantly higher in patients with luminal- and clinically inactive- disease compared to patients with stricturing- and clinically inactive- disease (p<0.001), luminal- and clinically active- disease (p<0.001), and patients with stricturing- and clinically active- disease (p<0.05, fig 1C). Significantly elevated levels of net type III collagen degradation (C3M/PRO-C3) were observed in patients with a luminal- and clinically active- disease compared to luminal- and clinically inactive- as well as stricturing- and clinically inactive- disease (p<0.05). Furthermore, net type III collagen degradation was elevated compared to patients with stricturing- and clinically active- disease (p<0.05, fig 1F). Biomarker levels of PC3X, C3M, and PRO-C3 were unable to differentiate between patient groupings (fig 1B, D, and E) Conclusion Proteolytic degradation of cross-linked type III collagen (CTX-III) reflecting resolution of fibrosis in CD patients with luminal disease, may be associated with a protective effect against fibrogenesis and progression to fibrostenosis. These data indicate differences in cross-linked and non-cross-linked type III collagen remodelling in CD patients, and their potential use for differentiating patient phenotypes and disease activity

P278 Type IV collagen formation/degradation ratio predicts response to infliximab induction therapy in patients with Ulcerative Colitis

Abstract Background Up to 50% of patients with ulcerative colitis (UC) do not respond to biological therapies, including TNF-α-antagonists (e.g., infliximab, IFX) and migration inhibitors (e.g., the α4β7-integrin inhibitor vedolizumab, VEDO). Therefore, serological biomarkers that predict therapy response are urgently needed. In this respect, biomarkers of extracellular matrix (ECM) remodeling, including basement membrane (BM) remodeling, and intestinal inflammation may hold promise as these processes play an important role in the pathophysiology of UC. In this study, we aimed to assess the predictive ability of serological biomarkers of ECM turnover and intestinal inflammation with regard to clinical response to IFX and VEDO therapy in patients with UC. Methods Serological biomarkers of collagen formation (PRO-C3, PRO-C4), matrix metalloproteinase (MMP)-mediated collagen degradation (reC1M, C3M, C4M, C4G, C6Ma3) and intestinal inflammation (macrophage activity marker [VICM] and calprotectin degradation fragments [CPa9-HNE]) were measured using neo-epitope solid-phase competitive enzyme-linked immunosorbent assay (ELISA) technology in 80 patients with UC who received either infliximab (IFX, n=26) or vedolizumab (VEDO, n=54) induction therapy. Clinical response to therapy was defined by composite assessment of available Simple Clinical Colitis Activity Index (SCCAI) scores and physician’s global assessments (PGA) derived from clinical records. Logistic regression modelling and receiver operating characteristics (ROC) statistics were used to assess discriminative capacity regarding response to IFX and VEDO. Results Only baseline ratios of type IV collagen formation and degradation (PRO-C4/C4M) were significantly elevated in patients with UC who clinically responded to IFX therapy (P<0.05). In addition, PRO-C4/C4M ratios accurately discriminated responders and non-responders to IFX therapy (area under the curve [AUC]=0.77, P<0.05), also after adjustment for confounding factors (AUC=0.88, P<0.01). Serum VICM levels were elevated in patients not responding to IFX therapy, but did not reach statistical significance (AUC=0.76, P=0.06). None of the biomarkers predicted response to VEDO therapy. Conclusion The baseline ratio of type IV collagen formation/degradation demonstrated high predictive accuracy with regard to response to IFX therapy in patients with UC, which may imply that patients with increased BM formation vs. degradation are more likely to respond. Furthermore, VICM may hold promise as a predictor for non-response to IFX therapy. Serological biomarkers of ECM turnover and intestinal inflammation may aid in predicting response to biological therapy and deserve validation in larger, prospective cohort studies.

P123 Type I collagen degradation fragments and type IV collagen formation/degradation ratio are serological biomarkers for stricturing (Montreal B2) Crohn’s disease

Abstract Background Crohn’s disease (CD) is characterized by increased extracellular matrix (ECM) remodeling, which is a key pathophysiological mechanism underlying intestinal stricture formation and other fibrotic disease complications. Alterations in intestinal ECM turnover may be reflected by products of collagen formation and degradation that are released into the systemic circulation. In this study, we aimed to investigate associations between serological biomarkers of collagen turnover and disease behavior subtypes according to the Montreal classification in patients with CD. Methods Serological biomarkers of collagen formation (PRO-C3, PRO-C4) and matrix metalloproteinase (MMP)-mediated collagen degradation (reC1M, C3M, C4M, C4G, C6Ma3) were measured using neo-epitope solid-phase competitive enzyme-linked immunosorbent assay (ELISA) technology in 101 patients with CD (Montreal B1: n=47; B2: n=28; B3: n=26) who were characterized according to the Montreal classification. Logistic regression modelling and receiver operating characteristics (ROC) statistics were used to assess discriminative power. Results Specific fragments of MMP-2,9,13-mediated degradation of type I collagen (reC1M) were significantly reduced in patients with stricturing (Montreal B2) disease compared to other disease phenotypes (B1 and B3) (P<0.001). Ratios of type IV collagen formation and degradation (PRO-C4/C4M) were significantly elevated in Montreal B2 patients (P<0.001). No differences in remaining serological biomarkers were observed between the different Montreal subtypes. Both reC1M and PRO-C4/C4M ratios demonstrated high discriminative power to distinguish inflammatory CD (B1) from stricturing (B2) CD, also after adjustment for confounding factors (area under the curve [AUC] 0.85 and 0.84, respectively). Conclusion Elevated degradation of type I collagen and excessive (relative) formation of type IV collagen strongly associate with stricturing CD (Montreal B2) and accurately discriminate it from inflammatory CD (Montreal B1). Therefore, reC1M and PRO-C4/C4M ratios could serve as serological biomarkers for stricturing CD. Our results should be validated in additional prospective, larger patient cohorts to corroborate these findings.

Combined Effects of Microplastics and Biochar on the Removal of Polycyclic Aromatic Hydrocarbons and Phthalate Esters and Its Potential Microbial Ecological Mechanism.

Microplastics (MPs) have been attracting wide attention. Biochar (BC) application could improve the soil quality in the contaminated soil. Currently, most studies focused on the effect of MPs or BC on the soil properties and microbial community, while they neglected the combined effects. This study investigated the combined effects of BC or ball-milled BC (BM) and polyethylene plastic fragments (PEPFs) and degradable plastic fragments (DPFs) on the removal of polycyclic aromatic hydrocarbons (PAHs) and phthalate esters (PAEs) from the PAH-contaminated soil and the potential microbial ecological mechanisms. The results showed that BC or BM combined with PEPF could accelerate the removal of PAHs and PAEs. PEPF combined with BM had the most significant effect on the removal of PAHs. Our results indicating two potential possible reasons contribute to increasing the removal of organic pollutants: (1) the high sorption rate on the PEPF and BC and (2) the increased PAH-degrader or PAE-degrader abundance for the removal of organic pollutants.

Designer Anticancer Nanoprodrugs with Self-Toxification Activity Realized by Acid-triggered Biodegradation and In Situ Fragment Complexation.

Prodrugs that allow in situ chemical conversion of less toxic precursors into active drugs in response to certain stimuli are promising anticancer candidates. Herein, we present a novel design of nanoprodrugs with a "degradation-mediated self-toxification" strategy, which realizes intracellular synthesis of anticancer agents using the nanoparticles' own degradation fragments as the precursors. To fulfill this concept, a metal complexing dicyclohexylphosphine (DCP) organosilane is carefully screened out from various ligands to conjugate onto Pd(OH)2 nanodots confined hollow silica nanospheres (PD-HSN). This constructed nanoprodrug shows acid-triggered degradation in lysosomes and neutralizes protons to induce lysosomes rupturing, generating predesigned less toxic fragments (Pd2+ and DCP-silicates) that complex into DCP/Pd complex in situ for inducing DNA damage, leading to enhanced anticancer activity against various cancer cell lines as well as in a xenograft tumour model.

Designer Anticancer Nanoprodrugs with Self‐Toxification Activity Realized by Acid‐triggered Biodegradation and In Situ Fragment Complexation

AbstractProdrugs that allow in situ chemical conversion of less toxic precursors into active drugs in response to certain stimuli are promising anticancer candidates. Herein, we present a novel design of nanoprodrugs with a “degradation‐mediated self‐toxification” strategy, which realizes intracellular synthesis of anticancer agents using the nanoparticles’ own degradation fragments as the precursors. To fulfill this concept, a metal complexing dicyclohexylphosphine (DCP) organosilane is carefully screened out from various ligands to conjugate onto Pd(OH)2 nanodots confined hollow silica nanospheres (PD‐HSN). This constructed nanoprodrug shows acid‐triggered degradation in lysosomes and neutralizes protons to induce lysosomes rupturing, generating predesigned less toxic fragments (Pd2+ and DCP‐silicates) that complex into DCP/Pd complex in situ for inducing DNA damage, leading to enhanced anticancer activity against various cancer cell lines as well as in a xenograft tumour model.

Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme.

The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage.

Environmental RNAi pathways in the two-spotted spider mite

BackgroundRNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design.ResultsBiochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented.ConclusionsUsing a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

Characterization of Materials for Shape-memory and Biodegradable Ureteral Stent

Ureteral stent is one of the commonly used instruments in urology, and its clinical efficacy is confirmed. However, there are also a series of complications or deficiencies, including infection after catheterization, formation of stent shell, and secondary endoscopy for tube extraction. The biodegradable ureteral stent can play the same role as the double-J tube, and it will be completely degraded after a certain time. The degradation fragments will be discharged with the urine, and the physiological functions of the ureter will not be affected. At the same time, there is no need to perform a second cystoscope for tube extraction. Therefore, patients do not have to undergo an invasive operation again, so as to alleviate the pain of patients and save time and cost.

Association of Diabetic Kidney Disease Markers and Urinary Beta-crossLaps in Type 2 Diabetes

Introduction: Diabetic Kidney Disease (DKD) is a chronic complication in Type 2 diabetes. The Chronic Kidney Disease (CKD273) peptide classifier has been found to predict development of DKD even before microalbuminuria develops. Seventy four percent of peptides in the CKD273 classifier are Collagen degradation fragments. The Beta-CrossLaps (β-CTx) Enzyme Llinked Immunosorbent Assay (ELISA) assay detects the specific collagen degradation product, C terminal telopeptide of Type 1 collagen. In light of the Capillary Electrophoresis/Mass Spectrometry (CE-MS) findings, linking collagen degradation fragments excretion to early detection of DKD, the significance of urinary β-CTx levels as a DKD biomarker needs to be evaluated. Aim: To study the urinary excretion of β-CTx in type 2 diabetes patients and to evaluate its relation to Microalbuminuria status and estimated Glomerular Filtration Rate (eGFR) of the patients. Materials and Methods: This descriptive cross-sectional study was undertaken at a tertiary care hospital, with enrollment of 82 type 2 diabetes patients from the diabetes Out Patient Department (OPD) Participants were divided into groups based on their Urinary Albumin Creatinine Ratio (UACR) and eGFR levels. The study participants were tested for Urinary β-CTx level, UACR and eGFR. Mean or median was calculated for the parameters with normal and non-normal distribution, respectively. All statistical testing was performed on online calculators available at the site; https://www.socscistatistics.com/. Results: The median urinary β-CTx level observed was 100.6 ng/mmol of creatinine. Among the 82 participants, 15 participants had urinary β-CTx level 15pg/mL, the sensitivity of the kit. Among the remaining 67 participants, the minimum Urinary Beta-CrossLaps: Creatinine ratio observed was 2.6 ng/mmol and the maximum value observed was 2071 ng/mmol (i.e., 2.1 μg/mmol). The median urinary β-CTx level was highest (100.6 ng/mmol creatinine) in the patient group with eGFR in the normal range. The urinary β-CTx level was found to decline with decline in eGFR, with median urinary β-CTx 65.5 ng/mmol creatinine in the patient group with mildly decreased eGFR and 7.2 ng/mmol creatinine in the patient group with moderately decreased eGFR. Conclusion: The Urinary β-CTx concentration in type 2 diabetes patients is dispersed over a wide range. The Urinary β-CTx concentration correlates with the eGFR of the patient and is not influenced by age, gender or duration of diabetes. This parameter is a potential early DKD biomarker.

Urinary troponin concentration as a marker of cardiac damage in pregnancies complicated with preeclampsia

Pregnant women with preeclampsia experience significant hemodynamic changes which lead to an increased myocardial workload. In response to increased demands in pregnancy, the heart muscle responds with ventricular remodeling process which involves cardiac muscle hypertrophy. Opposed to occurrence of eccentric ventricular hypertrophy in normal pregnancy, myocardial remodeling in a form of concentric hypertrophy will occur in pregnant patients with preeclampsia. Increased myocardial workload is manifested by an increased troponin release. As process of troponin degradation continue, filtration of degradation fragment through glomerular membrane occur, raising the possibility of it’s detection in urine. Degradation fragments of troponin molecules are estimated to be 20 kDa with preserved immunoreactivity to high-sensitivity assays. Some of the authors suggest that serum levels of cardiac troponin I might be elevated in patients with hypertension, as well as in preeclamptic pregnant women. It is to be expected that evaluation of severity of the myocardial damage in pregnant woman with preeclampsia may be performed by measuring levels of troponin in the urine using high-sensitivity assays. Designing of urine dipstick will help to detect an early phase of myocardial involvement in preeclamptic pregnancies.

The degradation fragments of gamma-glutamyl transpeptidase from Bacillus subtilis BU108 have antimicrobial activity against Streptomyces scabiei

Bacillus species are often regarded as ideal biocontrol agents against plant pathogens, which can produce various antimicrobial peptides (AMPs). In this study, strain BU108 with good antimicrobial activity against Streptomyces scabiei was isolated from the rhizosphere soil of a potato field, which was then identified as Bacillus subtilis through 16S rDNA sequence analysis. An antimicrobial protein was purified by ammonium sulfate precipitation, ion exchange chromatography and ultrafiltration. The active sample showed a single band on SDS-PAGE gel, which was identified by mass spectrometry as partial fragments of gamma-glutamyl transpeptidase (GGT). To verify this new finding, three potential AMPs (KT20, IF20 and AE26) were predicted based on the sequence and structure of GGT, and the antimicrobial activity test confirmed two AMPs (KT20 and IF20). As GGT was involved in the metabolism of glutathione in former references, in this study, we reported the antimicrobial activity of its degradation fragments for the first time, which not only enriched the antimicrobial substance of Bacillus subtilis, but also provided a new choice for potato common scab biocontrol.

Microplastic content of Kutum fish, Rutilus frisii kutum in the southern Caspian Sea

The occurrence of microplastics (MPs) in the digestive tract of commercial Kutum fish, Rutilus frisii kutum was investigated. Fish samples, ranging from 33 to 48.5 cm fork length which sold for human consumption, were collected from local fish markets in Bandar-e Torkaman (the south-eastern of Caspian Sea) on November 2017, and March 2018. The MPs were characterized using optical microscopy, NR staining, and SEM-EDS for number, shape, color, surface morphology, and elemental composition. On average, 11.4 MP items per fish (0.015 items per 1 g fish wet weight) were found in Kutum's stomach at an individual detection rate of 80%. Around 66% of all identified MP items were < 500 μm, and 53% possessed light colors. Morphological researches indicated that fish ingested the degradation fragments from larger plastic pieces, fibers, and manufactured microbeads. Microfibers are the most dominant items accounting for over 75% of all MPs. The SEM images indicated the various degrees of erosions upon environmental exposure. Some MPs had surface cracks, broken margins, scaly appearances, and obvious pores. Considering the commercial importance which the Kutum plays for Iran's fishery, the potential effect of MPs on the trophic food web, particularly for human consumption and health, should be urgently investigated.

Abstract 3091: Liquid biopsy reflecting a T-cell permissive tumor microenvironment identifies metastatic melanoma patients responding to immune checkpoint inhibitor therapy

Abstract Background: Non-invasive biomarkers for predicting response to immune checkpoint inhibitors (ICI) are urgently needed. Crucial for response to ICI's is a T-cell permissive tumor microenvironment (hot tumor), characterized by presence of activated T-cells and low fibrotic activity. Activated T-cells release the protease granzyme B (GzB), which can cleave type IV collagen during tumor infiltration. We evaluated the potential of measuring GzB generated type IV collagen degradation fragments in a liquid biopsy for identifying metastatic melanoma (MM) patients responding to ipilimumab. Methods: A monoclonal antibody was raised against a GzB generated neo-epitope on type IV collagen (C4G), and used to develop a technically robust competitive electro-chemiluminescence immunoassay (ECLIA). C4G was measured in serum from MM patients (n=54) before initiation of ipilimumab treatment. C4G was combined with the fibrosis biomarker PRO-C3, measuring type III collagen formation. Biomarker levels were associated with objective response rate (ORR) and overall survival (OS) outcomes. Results: The C4G assay was specific for a neo-epitope on type IV collagen degradation fragments generated by GzB. The ORR was 2.6 fold higher (18% vs 7%) in patients with high C4G levels (&amp;gt;25th percentile) vs low levels (≤25th percentile). Likewise, high C4G levels at baseline were associated with longer OS, with a median OS of 646 days vs 290 days for low C4G levels (HR=0.48, 95%CI: 0.24-0.98, p=0.045). When combining high C4G with low PRO-C3, the HR dropped to 0.30 (95%CI: 0.15-0.60, p=0.0006), and remained significant when adjusted for the covariates age, lactate dehydrogenase levels and prior treatment (HR: 0.35, 95%CI: 0.18-0.72, p=0.004). Conclusions: A liquid biopsy measuring granzyme B degraded type IV collagen (C4G) as a surrogate of active immune infiltration into the tumor microenvironment is associated with response to the ICI therapy ipilimumab. When combining C4G with the fibrosis biomarker PRO-C3, patients with this special phenotype - low fibrosis and high immune infiltration - have an even better chance of responding compared to high C4G levels alone. If validated, this suggests that specific collagen remodeling biomarkers (C4G+PRO-C3) have potential for predicting response to ICI's in clinical cancer trials. Citation Format: Christina Jensen, Dovile Sinkeviciute, Daniel H. Madsen, Patrik Önnerfjord, Morten Hansen, Henrik Schmidt, Inge Marie Svane, Morten A. Karsdal, Nicholas Willumsen. Liquid biopsy reflecting a T-cell permissive tumor microenvironment identifies metastatic melanoma patients responding to immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3091.

Decreased RPM reduces von Willebrand factor degradation with the EVAHEART LVAS: implications for device-specific LVAD management.

Continuous-flow left ventricular assist devices (LVADs) produces supraphysiologic shear stress that causes von Willebrand factor (VWF) degradation and a bleeding diathesis. Reduction of revolutions per minute (RPM) with axial-flow LVADs does not decrease shear stress enough to reduce VWF degradation and bleeding. However, it is unknown if RPM reduction with centrifugal flow LVADs may minimize VWF degradation. We tested the hypothesis that RPM reduction preserves VWF multimers in the centrifugal-flow EVAHEART left ventricular assist system (LVAS), which is designed to minimize shear stress and blood trauma. Whole blood samples were collected from humans (n = 28). Blood was circulated in ex vivo mock circulatory loops for 6 hours with an EVAHEART LVAS at 2300 (n = 12), 2100 (n = 8), or 1800 RPM (n = 8). Immunoblotting was used to resolve and quantify VWF multimers and degradation fragments. RPM reduction from 2300 to 2100 to 1800 RPM significantly decreased EVAHEART blood flow from 5.8 ± 0.4 to 4.3 ± 0.6 to 4.1 ± 0.5 L/min (analysis of variance [ANOVA], P = .03). RPM reduction protected VWF from pathologic degradation. At lower RPMs, significantly greater levels of VWF multimers were observed (ANOVA, P = .001). Similarly, at lower RPMs, significantly fewer VWF fragments, a product of VWF degradation, were observed (ANOVA, P = .007). RPM reduction significantly reduced VWF degradation with the centrifugal-flow EVAHEART LVAS, an LVAD specifically designed with low shear stress. Different LVADs have unique hematologic footprints and should be managed with device-specific protocols. Adjustment of RPM to minimize blood trauma while still maintaining physiologic hemodynamics has the potential to decrease complications related to LVAD-associated von Willebrand's disease, such as gastrointestinal bleeding and hemorrhagic stroke.

Fibrogenesis and inflammation contribute to the pathogenesis of cirrhotic cardiomyopathy.

Fibrogenesis and inflammation contribute to the progression of cirrhosis. However, it is unknown if these processes also contribute to the development of cirrhotic cardiomyopathy (CCM). Novel magnetic resonance imaging with quantification of the extracellular volume (ECV) provides an estimate of the fibrotic remodelling in the liver and heart. To investigate the relationship between liver and cardiac ECV in cirrhosis and their association with collagen turnover and inflammation. A prospective study of 52 patients with cirrhosis and 14 healthy controls. All patients underwent contrast-enhanced MRI with T1-mapping and quantification of myocardial and liver ECV, biochemical assessments of collagen turnover (PRO-C3, PRO-C5, PRO-C6, collagen type IV degradation fragment, collagen type V degradation fragment, LG1M) and inflammation (TNFα, IL-1β, IL-6, IL-8, IL-18, SDF1α, sCD163, sMR, soluble macrophage mannose receptor). Myocardial and liver ECV were increased in patients compared with healthy controls (myocardial ECV 31.2±5.5% vs 27.4±2.9%, P=0.037; liver ECV 44.1±9.6% vs 33.7±6.7%, P<0.001). Myocardial ECV correlated strongly with liver ECV (r=0.48, P=0.001) and biomarkers of collagen formation and inflammation (P<0.005). Similarly, liver ECV correlated with biomarkers of collagen formation and inflammation (P<0.003). In a multivariate analysis, liver ECV was predicted by biomarkers of collagen formation (PRO-C3 and PRO-C6), whereas myocardial ECV was predicted by biomarkers of collagen formation (PRO-C6) and inflammation (IL-6 and sMR). Structural myocardial changes seem closely related to liver fibrosis in patients with cirrhosis. The strong associations with biomarkers of collagen formation and inflammation provide new insight into the role of inflammation and fibrogenesis in the development of structural cardiac abnormalities, potentially leading to CCM.