Degradation Fragments Research Articles

Analysis of blood clot degradation fragment sizes in relation to plasma flow velocity

Thrombolytic therapy aims to dissolve blood clots and restore vessel patency. Our hypothesis is that the therapy depends not only on chemical reactions of the fibrinolytic system, but also on mechanical forces exerted by streaming blood on the clot surface. The aim of the study was to analyze the role of mechanical forces in promoting thrombolysis and their relation to the maximum size of removed clot fragments. Non-occlusive whole-blood clots were exposed to flow of plasma containing the thrombolytic agent rt-PA. Plasma, perfusing through the clot, was collected and the sizes of clot degradation fragments in the plasma were analyzed by optical microscopy. Theoretical models for the maximum clot fragment size as a function of blood flow velocity were developed based on the relation between surface or volume clot binding forces and the opposing forces of the streaming plasma in the laminar or turbulent flow regime. The best agreement between experimental results and models was obtained for the volume binding forces and the laminar flow model, in which the maximum clot fragment size was linearly dependent on the plasma flow velocity. Such result could not be obtained if thrombolysis would be purely a biochemical process. Therefore, the result confirms our hypothesis that thrombolysis is also strongly influenced by the mechanical forces of streaming plasma.

The Detection of Myocardial Fibrosis

Both type 1 and type 2 diabetes mellitus (DM) are associated with a significant increase in cardiovascular risk. Glycemic control has, unfortunately, not been convincingly shown to reduce cardiovascular risk.1–3 The level at which blood pressure should be controlled also is controversial.4 Thus, there may be a need to focus on new therapeutic targets if we are to reduce cardiovascular risk in patients with either type 1 or type 2 DM. Cardiac and large arteries fibrosis is a frequent occurrence in patients with DM and one of the major factors predisposing to the development of heart failure (HF). Cardiac fibrosis is the consequence of extracellular cardiac matrix remodeling resulting from pathological processes, including ischemia; stretch; inflammation; and specifically in DM, oxidative stress, advanced glycation end products, and several neurohormonal mediators. Cardiac fibrosis may cause myocyte slippage, tissue heterogeneity and dyssynchrony, ventricular dilatation, and contractile dysfunction. Myocardial fibrosis alters myocardial compliance, and associated fibrosis-related large artery stiffness may predispose to diastolic dysfunction and HF with a preserved left ventricular ejection fraction. Cardiac fibrosis also may result in alteration of gap junctions and predispose to arrhythmias and sudden cardiac death.5 Bioimaging and biomarkers of cardiac fibrosis, therefore, may become clinically useful tools, particularly given the potential for cardioprotective and cardioreparative pharmacological strategies.6,7 Article see p 51 The measurement of various serum peptides arising from the metabolism of collagen types I and III, of degradation fragments (carboxy-terminal telopeptide of type I collagen), and of specific metalloproteinases may provide a noninvasive assessment of fibrosis.8–10 A strong correlation exists between changes in serum levels of biomarkers for the turnover of extracellular cardiac matrix proteins and ongoing cardiac remodeling. Diagnostic approaches for diabetic cardiomyopathy …

Will posttranslational modifications of brain proteins provide novel serological markers for dementias?

Drug development for dementias is significantly hampered by the lack of easily accessible biomarkers. Fluid biomarkers of dementias provide indications of disease stage, but have little prognostic value, cannot detect early pathological changes, and can only be measured in CSF (cerebrospinal fluid) which significantly limits their applicability. In contrast, imaging based biomarkers can provide indications of probability of disease progression, yet are limited in applicability due to cost, radiation and radio-tracers. These aspects highlight the need for other approaches to the development of biomarkers of dementia, which should focus on not only providing information about pathological changes, but also on being measured easily and reproducibly. For other diseases, focus on development of assays monitoring highly specific protease-generated cleavage fragments of proteins has provided assays, which in serum or plasma have the ability to predict early pathological changes. Proteolytic processing of brain proteins, such as tau, APP, and α-synuclein, is a key pathological event in dementias. Here, we speculate that aiming biomarker development for dementias at detecting small brain protein degradation fragments of generated by brain-derived proteases specifically in blood samples could lead to the development of novel markers of disease progression, stage and importantly of treatment efficacy.

Thymic stromal lymphopoietin exerts antimicrobial activities

Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.

Novel ex-vivo mechanobiological intervertebral disc culture system

Intervertebral disc degeneration, a leading cause of low back pain, poses a significant socioeconomic burden with a broad array of costly treatment options. Mechanical loading is important in disease progression and treatment. Connecting mechanics and biology is critical for determining how loading parameters affect cellular response and matrix homeostasis. A novel ex-vivo experimental platform was developed to facilitate in-situ loading of rabbit functional spinal units (FSUs) with relevant biological outcome measures. The system was designed for motion outside of an incubator and validated for rigid fixation and physiologic environmental conditions. Specimen motion relative to novel fixtures was assessed using a digitizer; fixture stiffness exceeded specimen stiffness by an order of magnitude. Intradiscal pressure (IDP), measured using a fiber-optic pressure transducer, confirmed rigidity and compressive force selection. Surrounding media was controlled at 37°C, 5% O2/CO2 using a closed flow loop with an hypoxic incubator and was validated with probes in the specimen chamber. FSUs were subjected to cyclic compression (20 cycles) and four-hour creep at 1.0MPa. Disc tissue was analyzed for cell viability using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which showed high viability (>90%) regardless of loading. Conditioned media was assayed for type-II collagen degradation fragments (CTX-II) and an aggrecan epitope (CS-846) associated with new aggrecan synthesis. CTX-II concentrations were not associated with loading, but CS-846 concentrations appeared to be increased with loading. Preservation of the full FSU allows physiologic load transmission and future multi-axis motion and identification of load-responsive proteins, thereby forming a new niche in intervertebral disc organ culture.

Determinants of undercarboxylated and carboxylated osteocalcin concentrations in type 1 diabetes

To determine whether undercarboxylated osteocalcin (UC-OC) or gamma-carboxyglutamic-carboxylated-type osteocalcin (GLA-OC) concentrations deviate from normal in type 1 diabetes (T1D), serum levels were compared between 115 subjects with T1D and 55 age-matched healthy controls. UC-OC and GLA-OC concentrations were similar between groups; however, in T1D, UC-OC correlated positively with markers of insulin exposure, either endogenously produced or exogenously administered. A study was conducted to determine whether dysregulation of circulating concentrations of UC-OC or GLA-OC occurs in patients with type 1 diabetes, a condition of insulin deficiency without insulin resistance. We measured serum concentrations of UC-OC and GLA-OC in 115 subjects with T1D, ages 14-40 years, and in 55 age-matched healthy control subjects. Relationships between UC-OC and GLA-OC concentrations and patient characteristics (gender and age), indices of glycemic control (hemoglobin A1c (HbA1c), fasting plasma glucose, C-peptide concentration, 3-day average glucose measured by a continuous glucose sensor, total daily insulin dose) and circulating indices of skeletal homeostasis (total calcium, 25-OH vitamin D, parathyroid hormone, insulin-like growth factor 1 (IGF-1), type 1 collagen degradation fragments (CTX), adiponectin, leptin) were examined. Between group differences in the concentrations of UC-OC and GLA-OC were the main outcome measures. Although adiponectin levels were higher in the T1D group, between-group comparisons did not reveal statistically significant differences in concentration of UC-OC, GLA-OC, CTX or leptin between the T1D and control populations. Instead, by multivariate regression modeling, UC-OC was correlated with younger age (p < 0.001), higher CTX (p < 0.001), lower HbA1c (p = 0.013), and higher IGF-1 (p = 0.086). Moreover, within the T1D subgroup, UC-OC was positively correlated with C-peptide/glucose ratio (reflecting endogenous insulin secretion), with IGF-1 (reflecting intra-portal insulin sufficiency), and with total daily insulin dose. In T1D, UC-OC appears to correlate positively with markers of insulin exposure, either endogenously produced or exogenously administered.

Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

BackgroundCollagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful information than traditional serological assays that crudely measure total protein. In the present study, we developed an ELISA for the quantification of a neoepitope generated by MMP degradation of type IV collagen and evaluated the association of this neoepitope with liver fibrosis in two animal models.MethodsType IV collagen was degraded in vitro by a variety of proteases. Mass spectrometric analysis revealed more than 200 different degradation fragments. A specific peptide sequence, 1438'GTPSVDHGFL'1447 (CO4-MMP), in the α1 chain of type IV collagen generated by MMP-9 was selected for ELISA development. ELISA was used to determine serum levels of the CO4-MMP neoepitope in two rat models of liver fibrosis: inhalation of carbon tetrachloride (CCl4) and bile duct ligation (BDL). The levels were correlated to histological findings using Sirius red staining.ResultsA technically robust assay was produced that is specific to the type IV degradation fragment, GTPSVDHGFL. CO4-MMP serum levels increased significantly in all BDL groups compared to baseline, with a maximum increase of 248% seen two weeks after BDL. There were no changes in CO4-MMP levels in sham-operated rats. In the CCl4 model, levels of CO4-MMP were significantly elevated at weeks 12, 16 and 20 compared to baseline levels, with a maximum increase of 88% after 20 weeks. CO4-MMP levels correlated to Sirius red staining results.ConclusionThis ELISA is the first assay developed for assessment of proteolytic degraded type IV collagen, which, by enabling quantification of basement membrane degradation, could be relevant in investigating various fibrogenic pathologies. The CO4-MMP degradation fragment was highly associated with liver fibrosis in the two animal models studied.

Transfer RNA-derived Small RNAs: Degradation Fragments or Novel Regulatory Molecules?*

Transfer RNAs (tRNAs) with classic clover structure, have been researched as a key component of cellular protein synthesis machine for decades. However, the pace of progress in the understanding of tRNA function is still staggering, especially in the role of tRNA as the precursor of potential molecules to regulate gene expression globally. Several recent studies suggest that some tRNA-derived small RNAs have been detected in many cell lines by deep sequencing. These cleavage products of tRNAs are indicated to interact with some important protein factors like Dicer and Ago family in microRNA processing system. In additional, luciferase reporter assay results implied that the small RNAs derived from tRNA might play some roles in response to the stress as regulatory molecules with microRNA-like features. If their regulatory functions were to be confirmed by more evidence, these newly revealed RNAs would provide expanding insight into the repertoire of small noncoding RNAs.

In Vitro Proteolysis of Myofibrillar Proteins from Beef Skeletal Muscle by Caspase-3 and Caspase-6

The objective of the study was to investigate in vitro degradation of myofibrils by caspase-3 or -6. Myofibrillar proteins prepared from beef skeletal muscle were incubated with caspase-3 or -6 at 30 °C for 2 or 12 h, and subsequently, protein degradation was detected. Results showed that caspase-3 and -6 reproduced the degradation patterns of titin and nebulin observed during normal postmortem (PM) aging; however, they only reproduced the 28 kDa fragment derived from troponin-T. Caspase-3 induced only minor degradation of desmin. However, caspase-6 caused increasing degradation of desmin with extended incubation time and produced three degradation fragments (45, 29, and 27 kDa) of which only the 45 kDa fragment has been reported in aged beef. Therefore, caspase-3 or -6 could only reproduce a part of myofibrillar protein degradation or degradation fragments observed in naturally aged meat and may be involved in PM proteolysis of muscle proteins together with other endogenous proteases.

Separating melanins into their primary colors: quantifying contributions from eumelanin and pheomelanin

Separating melanins into their primary colors: quantifying contributions from eumelanin and pheomelanin

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen—A novel biomarker of atherosclerotic plaque remodeling

Objective Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine. Design and methods A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice. Results The lower limit of detection was 0.5 ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20 weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p < 0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice. Conclusion We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.

Measurement of matrix metalloproteinase 9-mediated Collagen type III degradation fragment as a marker of skin fibrosis

BackgroundThe current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies.MethodsSkin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels.ResultsCO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65).ConclusionIncreased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice.

Serum Bone Biomarkers and Oral/Systemic Bone Loss in Humans

We recently reported that subantimicrobial-dose doxycycline (SDD) significantly reduced serum bone-resorption biomarkers in subgroups of post-menopausal women. We hypothesize that changes in serum bone biomarkers are associated not only with systemic bone mineral density (BMD) changes, but also with alveolar bone changes over time. One hundred twenty-eight eligible post-menopausal women with periodontitis and systemic osteopenia were randomly assigned to receive SDD or placebo tablets twice daily for two years, adjunctive to periodontal maintenance. Sera were analyzed for bone biomarkers. As expected, two-year changes in a serum bone biomarker were significantly associated with systemic BMD loss at the lumbar spine (osteocalcin, bone-turnover biomarker, p = 0.0002) and femoral neck (osteocalcin p = 0.0025). Two-year changes in serum osteocalcin and serum pyridinoline-crosslink fragment of type I collagen (ICTP; bone-resorption biomarker) were also significantly associated with alveolar bone density loss (p < 0.0001) and alveolar bone height loss (p = 0.0008), respectively. Thus, we have shown that serum bone biomarkers are associated with not only systemic BMD loss, but with alveolar bone loss as well. Clinical Trial Registration Information: Protocol registered at ClinicalTrials.gov, NCT00066027. Abbreviations: bone mineral density (BMD), bone-specific alkaline phosphatase (BSAP), computer-assisted densitometric image analysis (CADIA), confidence interval (CI), deoxypyridinoline-containing degradation fragment of the C-terminal telopeptide region of type I collagen (CTX), coefficient of variation (CV), dual-energy x-ray absorptiometry (DXA), pyridinoline-crosslink-containing degradation fragment of the C-terminal telopeptide region of type I collagen (ICTP), and subantimicrobial-dose doxycycline (SDD).

Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials

Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2-6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20ng/ml to 20μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.

Comparison of Mass Spectrometry and Clinical Assay Measurements of Circulating Fragments of B-Type Natriuretic Peptide in Patients With Chronic Heart Failure

Multiple B-type natriuretic peptide (BNP) fragments circulate in patients with heart failure (HF) but the types and relative quantities, particularly in relation to bioactive BNP 1-32, remain poorly defined. The purpose of the study was to relate clinically available BNP values with quantitative information on the concentration of pre-secretion and post-processed fragments of BNP detected by mass spectrometry. Seventy Class I-IV patients were prospectively enrolled with blood drawn into tubes containing a preservative to protect against BNP degradation. Samples were analyzed by quantitative mass spectrometry (MS) immunoassay for intact BNP 1-32 and its fragments. Clinical BNP 1-2 was measured by standard clinical laboratory methods. ProBNP 1-108, corin, and clinically measured BNP levels were elevated, but MS BNP 1-32 levels were low and differed from clinical BNP (P=0.01). Intact MS BNP 1-32 correlated modestly with clinical BNP (r=0.46, P<0.001). MS BNP fragments 3-32, 4-32, and 5-32 demonstrated the best associations with clinical BNP; fragment 5-32 with a correlation coefficient of r=0.81 (P<0.001). ProBNP 1-108 is measured by clinical BNP assays and contributes to the cumulative results of the BNP assay. However, the observation that clinically measured BNP correlates best with MS degradation fragments and relatively poorly with MS BNP 1-32 suggests that a significant component of circulating clinical BNP is composed of such fragments that are known to demonstrate little biological activity. There appear to be multiple pathways involved in the dysregulation of proBNP in HF, and both the processing of proBNP and the downstream degradation to BNP 1-32 appear to be critical.

Palmitoylation Controls Dopamine Transporter Kinetics, Degradation, and Protein Kinase C-dependent Regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.

Troponin I degradation in serum of patients with acute ischemic stroke

Background. Although troponin is a cornerstone biomarker in the assessment and management of patients with acute coronary syndrome, much remains to be learned about the biology of this widely used biomarker, including its post-release modification. Degradation of troponin following release in patients with acute coronary syndrome has been described; however whether such post-release modification occurs in other non-acute coronary syndrome states remains unknown. The aim of this study was to define troponin degradation in patients with acute ischemic stroke. Materials and methods. Troponin I (cTnI) was measured daily during the first 5 days of admission in 244 patients with acute ischemic stroke. Western blot analysis was performed using anti-cTnI antibodies and compared with serum concentrations of cTnI in seven patients and one patient with myocardial infarction (positive control). Results. Elevated levels of troponin were detected in 25 (10 %) patients; in all, both intact cTnI and cTnI degradation products were detected, with up to seven degradation fragments found. Samples with the highest total cTnI levels gave the strongest and most numerous western-blotting bands. All fragments were comparable with the degradation pattern of the positive control in terms of position. Conclusions. Immunoblotting of blood samples from patients with acute ischemic stroke reveals similar degradation patterns of cTnI as has been described in patients with acute myocardial infarction. The biological ramification and potential clinical impact of this finding bears further scrutiny.

Autophagy in herpesvirus immune control and immune escape

Autophagy delivers cytoplasmic constituents for lysosomal degradation, and thereby facilitates pathogen degradation and pathogen fragment loading onto MHC molecules for antigen presentation to T cells. Herpesviruses have been used to demonstrate these novel functions of autophagy, which previously has been primarily appreciated for its pro-survival role during starvation. In this review, we summarize recent findings how macroautophagy restricts herpesvirus infections directly, how macroautophagy and chaperone mediated autophagy contribute to herpesviral antigen presentation on MHC molecules, and which mechanisms herpesviruses have developed to interfere with these pathways. These studies suggest that herpesviruses significantly modulate autophagy to escape from its functions in innate and adaptive immunity.

Measurement of CO3-610, a Potential Liver Biomarker Derived from Matrix Metalloproteinase-9 Degradation of Collagen Type III, in a Rat Model of Reversible Carbon-Tetrachloride-Induced Fibrosis

Background and aim:The current study utilized a carbon tetrachloride (CCl4)-induced liver fibrosis model to measure levels of the MMP9-mediated collagen type III degradation fragment CO3-610 (site of cleavage: KNGETGPQGP), during disease progression and regression, and to investigate a potential prognostic role of the biomarker.Materials and methods:72 female Sprague-Dawley rats aged 6 months old were injected with CCl4 twice a week over different periods of time to induce varying degrees of liver fibrosis. After 4, 6 and 8 weeks of treatment, administration of CCl4 was stopped. The 6- and 8-week treatment groups were left to regress for a further 6 or 12 weeks at which point they were sacrificed and livers removed and sectioned. Liver fibrosis was quantified using Visiopharm software to analyse Sirius red-stained sections. Serum levels of CO3-610 were measured in all animals using an ELISA assay as described by Barascuk et al.1Results:Quantitative histology revealed total collagen deposition in the liver increased as fibrosis progressed. In animals treated with CCl4 for 4 weeks, collagen comprised on average 4.94% of the total tissue in liver sections, while after 6 weeks the mean was 8.25%, and after 8 weeks, 9.11%. During the regression phase, the total collagen deposition gradually decreased to a mean of 6.9% and 5.09% for animals regressing 6 and 12 weeks respectively after 6 weeks treatment, and 6.27% for animals regressed 12 weeks after 8 weeks treatment. CO3-610 values increased progressively in rats treated for 4 weeks (by a mean of 55.0 ng/ml), 6 weeks (mean 61.1 ng/ml) and 8 weeks (mean 70.2 ng/ml). During the regression phase, CO3-610 values rapidly decreased by a mean of 28.9 ng/ml at 6 weeks and 21.6 ng/ml at 12 weeks in animals previously treated for 6 weeks, and by a mean of 19.52 ng/ml in animals treated for 8 weeks and regressed for 12 weeks. CO3-610 levels were statistically significantly correlated with total collagen during disease progression (r = 0.5701, P < 0.0001). No statistically significant correlation was observed during regression (r = 0.2081, P = 0.1138).Conclusion:Levels of the MMP-9 generated fragment of collagen type III, CO3-610, correlated with the degree of liver fibrosis in rats during the progression phase, but were not correlated with total collagen levels during regression. CO3-610 seems to be produced only under the CCL4 stimulus, and signifies CO3-610 as a potential marker of progression rather than regression. The corresponding steep elevations in levels of CO3-610 total collagen and collagen type III during liver fibrosis progression underline a potential prognostic capacity of the biomarker.

Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.