Intact Chain Research Articles

A robust system reliability analysis using partitioning and parallel processing of Markov chain

AbstractThis paper presents a robust reliability analysis method for systems of multimodular redundant (MMR) controllers using the method of partitioning and parallel processing of a Markov chain (PPMC). A Markov chain is formulated to represent the N distinct states of the MMR controllers. Such a Markov chain has N2 directed edges, and each edge corresponds to a transition probability between a pair of start and end states. Because N can be easily increased substantially, the system reliability analysis may require large computational resources, such as the central processing unit usage and memory occupation. By the PPMC, a Markov chain's transition probability matrix can be partitioned and reordered, such that the system reliability can be evaluated through only the diagonal submatrices of the transition probability matrix. In addition, calculations regarding the submatrices are independent of each other and thus can be conducted in parallel to assure the efficiency. The simulation results show that, compared with the sequential method applied to an intact Markov chain, the proposed PPMC can improve the performance and produce allowable accuracy for the reliability analysis on large-scale systems of MMR controllers.

Fragmented proteins as food emulsion stabilizers: A theoretical study

Using a model based on the primary structure of αs1 -casein, the colloid stabilizing ability of fragmented protein and that of the intact chains are compared. We perform self consistent field (SCF) calculations to obtain the induced interaction potentials between the oil droplets, resulting from the overlap of adsorbed protein layers in each case. For the intact αs1 -casein, we confirm the known result, that the mediated interparticle interaction potential develops a deep attractive energy minimum at high salt concentrations and pH values close to the isoelectric point of the protein. The same does not occur for the appropriately fragmented systems, with improved emulsion stability predicted as a result, even at pH values close to pI. It is shown that this superior performance, for the case considered, is due to the diblock-type behavior of one of the fragments. However, it is well known that αs1 -casein more closely resembles the less favorable triblock structure. However, it is also demonstrated that the presence of a "diblock" like fragment by itself may not always be enough to produce a better emulsion stabilizer. It is seen that the hydrolysis of some peptide bonds may indeed lead to a suitable polypeptide, but that this is displaced from the interface by the structurally less desirable ones, also generated by the fragmentation process. The displacement occurs due to the competitive adsorption between different fragments. The removal of the undesirable fragments from the solution is found to greatly enhance the predicted colloid stabilizing ability of the remaining polypeptide.

The influence of incudostapedial joint separation on the middle ear transfer function.

ObjectivesOne of the risks in middle ear surgery is high frequency hearing loss. It is believed that manipulations on the middle ear ossicles with the instruments may cause overstimulation of the inner ear and damage of the hear cells. Controversy arises whether temporary separation of the ossicles has any impact on middle ear transfer function and hearing threshold after surgery. The aim of the study is to evaluate the influence of incudostapedial joint (ISJ) separation on middle ear function in an experimental model.MethodsWith the use of single point laser Doppler vibrometer (LDV) stapes velocity in the intact chain and after ISJ separation was measured in 5 fresh human cadaver temporal bones.ResultsIn all cases there was a decrease in stapes velocity after ISJ separation. Mead stapes velocity was reduced for 1 dB in 800 Hz to 9 dB in frequencies above 1,000 Hz. The decrease of velocity was greater in higher frequencies.ConclusionSeparation of the ISJ does not reduce significantly the middle ear function.

Evaluation Of Mass Spectrometry Based Approaches For The Diagnosis Of Hemoglobinopathies

Mass spectrometry (MS) is becoming an accepted technique for clinical applications. It has already been shown to be a valuable technique for the characterisation of structural hemoglobin variants, and its applicability for screening purposes has been demonstrated by several research groups. It has not yet been implemented as an approved clinical laboratory technique to screen for hemoglobinopathies, especially the thalassemias. In order to replace existing methods the proposed approach has to provide improved technical, financial and medical benefits. For the purposes of antenatal/neonatal screening laboratories, all methods must at least detect HbS, HbC, HbD-Punjab, HbE, HbO-Arab, Hb Lepore, elevated HbF, and elevated HbA2 (to distinguish α and β thalassemia). Three mass spectrometry based approaches have been evaluated against a current standard method (cation exchange liquid chromatography, HPLC, and gel electrophoresis) in a clinical trial incorporating 2017 patient samples, in 12 batches. The MS methods used were:1.Measurement of the mass of intact globin chains and the relative proportion of β and δ globin chains. When any α or β variant was detected, this was followed by tandem MS of tryptic peptides to confirm identity.2.Ultra performance liquid chromatography+MS/MS experiments using pre-determined tryptic peptides to screen for clinically important structural variants.3.Mass measurement of intact globin chains followed by top down electron transfer dissociation (ETD) fragmentation of selected globin chains for confirmation of variant (no complex pre-analytical sample digestion/separation, or second stage analysis). Variant detection The clinical samples were all tested using methods 1 and 2. Method 3 has been used on a more limited sample set which, however, included all samples which were identified to have clinically significant variants. All methods agreed with the hospital lab results for >99.5% of samples. However, a number of additional observations could be made using the mass spectrometry data. The current clinical laboratory method was found to give false results on three samples (0.15%), two of which were identified by the existing approach as HbD-Punjab but were in fact characterised unambiguously by MS as HbG-Philadelphia. One sample was identified as containing elevated HbF by the existing method but was characterised as a β-chain variant using MS. In addition, seven patient samples were found by mass spectrometry to contain non-significant Hb variants which were not detected using the existing hospital method. HbA2 measurement (based on ratio of δ chains to ‘δ+β' chains) The correlation coefficient (R2) for HbA2 measurement between HPLC and MS in the 12 batches varied from 0.57-0.90 (method 1), and 0.72-0.90 (method 2, using the T5 tryptic digest peptide pair), but with significant systematic bias requiring either calibration to a standard or adjustment of the normal range. However, a cut-off between normal and abnormal was clearly evident, and there was complete agreement between HPLC and MS methods in categorisation of samples as beta-thalassemia trait. Method 3 has shown potential to measure HbA2levels but has not yet been tested on a large batch of clinical samples. The approaches differ in their instrumentation requirements, sample introduction, sample preparation and data interpretation. Key issues to be considered when selecting the most appropriate method to develop for future hospital use include cost, speed, sensitivity, selectivity, potential for automation and diagnostic information provided. Disclosures: Smith: Bruker UK Limited: Employment.

Insights into the electrooxidation of glycolaldehyde on platinum in acidic media

Here we investigate the electrooxidation of glycolaldehyde on platinum in acidic medium. In order to do that, we follow the oxidation of glycolaldehyde by using in situ FTIR spectroscopy. Results show that CO is formed as soon as glycolaldehyde is adsorbed, covering nearly half of platinum surface. This intermediate generates CO2 that is apparently produced through multiple pathways. At least one of these pathways involves the formation of CO as an intermediate. On the other hand, adsorbates of intact chain generate important amounts of glycolic acid. Overall, the spectroscopic study allows the identification of some adsorbed species (formed at low potentials) and helps to understand the oxidation of glycolaldehyde in terms of the voltammetric response.

The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1−/−, but not Sulf2−/−, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1−/− mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Comparative molecular dynamic simulations of wild type and Thr114Val mutated Scaptodrosophila lebanonensis alcohol dehydrogenase

Enzyme kinetics studies have shown that Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH) and other drosophilid alcohol dehydrogenases function by a compulsory-ordered mechanism where the coenzyme binds to the free enzyme, and that a proton is released upon formation of the binary enzyme-NAD+ complex. A proton relay mechanism for the proton abstraction has been suggested that includes an eight-membered chain of water molecules connecting the active site with the bulk solvent. Thr114 bridges between two water molecules in the water chain. In a previous structural and enzyme kinetic study of a Thr114 Val mutant of SlADH, we showed that an intact water chain is essential for full enzyme activity. In the present study, comparative molecular dynamic (MD) simulations of the wild type and the SlADHT114V were performed. The simulations showed differences in hydrogen bonding properties and dynamics between the wild type and the SlADHT114V. Differences in molecular dynamical behaviour were seen in the loop of importance for binding the nicotinamide part of NAD+, in the region important for binding the adenine part of NAD+, and in the region of the amino acid at position 114. The substrates also had more freedom for conformational changes in active site of the wild type SlADH than of the SlADHT114V. The differences in hydrogen bonding properties and MDs between the wild type and mutant could not have been observed from the X-ray crystal structures only.

Overall Distribution of Trauma‐Related Deaths in Berlin 2010: The Weakest Links of the Chain of Survival are Emergency Medicine and Critical Care: Reply

Dear Editor,We are very happy to have received such a greatresponse since the publication of our article about trau-matic deaths in Berlin 2010 [1]. Furthermore, we expectmore controversy and scientific discussion after publicationof the second part, preventable trauma deaths (article inreview, World Journal of Surgery).In our opinion, the major finding of our first study is theunderestimation of the importance of preclinical trauma man-agement. The results of our second study will clarify that thequalityofpreclinicaltraumamanagementisoverestimated,andtherefore the significance and potential to reduce traumaticdeaths is not recognized. With the primary focus on advance-ments in critical care, one of the two ‘‘hot spots’’ of traumamanagement, the main problem, preclinical trauma manage-ment,isdisregarded[2].Inarecentstudy,werevealedtheneedfor individualized preclinical treatment of trauma patients withinvasive preclinical measures, e .g., intubation and chest tubing[3]. Therefore, we conclude that the education of paramedicsand emergency physicians, and the active contribution oftrauma surgeons to preclinical trauma management are crucialfactors for improving the quality of national trauma manage-ment, and that adding these components has the potential tosignificantly lower mortality after severe trauma. All organi-zational, structural, and management efforts (national traumanetwork, trauma registry, national interdisciplinary guideline)rely on effective preclinical trauma management. Thus, wehave to secure the preclinical treatment of trauma patients andadvocate for their needs.Referring to the letter from Brambillasca et al. [4]wetotally agree with their striving for structures and nationalprotocols necessary for a modern trauma system, but thebasis of all is an intact chain of survival. The weakest link isalways crucial, and in our study, preclinical trauma care wasthe weakest link. Therefore, we recommend advancingpreclinical trauma management further by educating para-medics and emergency physicians in the administration ofsafe and effective preclinical treatment of patients aftersevere trauma. Self-evidently there are regional or nationaldifferencesdemandingananalysisoflocaltraumasystemstoreveal their weakest link(s). To our minds, the new bimodaltemporal distribution of traumatic deaths is not only aregional phenomenon but also the logical consequence ofcontinuous advancement in the treatment and logistics oftraumapatientsrevealingthetwohotspots,orweakest links:preclinicaltraumamanagementandintensivecaremedicine.References

Scaling laws in simple and complex proteins: size scaling effects associated with domain number and folding class

The native states of the most compact globular proteins have been described as being in the so-called “collapsed-polymer regime,” characterized by the scaling law Rg ~ nν, where Rg is radius of gyration, n is the number of residues, and ν ≈ 1/3. However, the diversity of folds and the plasticity of native states suggest that this law may not be universal. In this work, we study the scaling regimes of: (i) one to four-domain protein chains, and (ii) their constituent domains, in terms of the four major folding classes. In the case of complete chains, we show that size scaling is influenced by the number of domains. For the set of domains belonging to the all-α, all-β, α/β, and α + β folding classes, we find that size-scaling exponents vary between 0.3 ≤ ν ≤ 0.4. Interestingly, even domains in the same folding class show scaling regimes that are sensitive to domain provenance, i.e., the number of domains present in the original intact chain. We demonstrate that the level of compactness, as measured by monomer density, decreases when domains originate from increasingly complex proteins.

Abstract 4577: Targeting N-glycosylation of tyrosine kinase receptors for preventing tumorigenesis and malignant transformation of neurofibromatosis type I

Abstract Neurofibromatosis type I (NF1) is a dominantly inherited disease affecting 1 in every 2,500 to 3,000 individuals, representing the most common familial cancer predisposition syndrome. The hallmark of clinical manifestation of NF1 is the development of multiple neurofibromas, which are highly heterotypic benign tumors of peripheral nerve sheath mainly composed of immature Schwann cells, fibroblasts, perineurial and inflammatory matrix. NF1 patients are also at high risk for the development of certain maligancies such as pheochromocytomas, childhood myeloid leukemias, neuroblastomas, rhabdomyosarcomas, and malignant peripheral nerve sheath tumors (MPNSTs). Because of the potential involvement of underlying nerves and blood vessels, surgical removal of tumors is not always an option. Once progressing to MPNSTs, although the resection is possible, most patients will eventually relapse locally or systemically. There is no effective treatment for NF1, nor effective approaches for predicting or preventing the occurrence of devastating complications. Thus, to develop agents for preventing or reversing the tumorigenesis and malignant transformation of NF1 are critically needed. In this report, we found HGF, c-MET, EGF and EGFR are aberrantly expressed in clinical specimens of neurofibromas and MPNSTs with NF and that autocrine and/or paracrine HGF/c-MET and EGF/EGFR loops promote MPSNT cell migration and invasion in vitro (Fig1). We also found that the loss of Nf1 in neurofibromas and MPNSTs results in hyperactive of Ras (Fig2). Compared with normal Schwann cells, MPNST cells express more N-glycoproteins and display different N-glycoprotein signatures (Fig3). In order to confirm our hypothesis, we treated cells with Tunicamycin, or 2-DG (N-glycosylation inhibitors) respectively in variable doses for different time. We found that both Tunicamycin and 2-DG inhibited the proliferation and promoted apoptosis of MPNST cell lines but had no effect on normal human Schwann cells (Fig4); inhibited the glycosylation and phosphorylation of EGFR and c-MET and impaired receptor-mediated MEK-ERK1/2 and PI3K-AKT signaling in a dose-dependent manner (Fig5). Moreover, 2-DG inhibited the maturation of c-MET as demonstrated by the intact premature chain, failing to cleave into one mature α-chain and another mature α-chain. In addition, 2-DG inhibited the translocation of the receptors from the cytoplasm onto the cell surface and retained receptors in the ER and Golgi apparatus (Fig6). To date, 2-DG inhibition of N-glycosylation and phosphorylation of tyrosine kinase receptors has not been reported. These novel findings prompted us to explore whether 2-DG disrupts Ras-directed N-glycosylation of tyrosine receptors in Schwann cell progenitors and tumor cells and to evaluate its roles in preventing the tumorigenesis and malignant transformation of NF1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4577. doi:1538-7445.AM2012-4577

Cell therapy for opportunistic infection and recurrent disease following allogeneic stem cell transplantation

Allogeneic stem cell transplantation (the process of transferring a blood and immune system from a normal donor to a patient with a serious illness) is most often performed for acute leukaemia or lymphoma and occasionally for other serious but non-malignant genetic disorders. There are approximately 400 transplants of this type performed annually in Australia. The most serious problems facing recipients of allogeneic stem cell transplants are the dual risks of opportunistic infection and disease recurrence that arise as a result of delayed maturation of the immune system after transplant. For the first year or more after transplant, patients are particularly susceptible to serious illness or death due to viral and fungal infections. We have generated virus and fungus specific immune system cells in the laboratory starting with normal donor cells and stimulating with vaccines, extracts or peptides derived from known infectious pathogens such as cytomegalovirus, Epstein-Barr virus and varicella zoster. After expansion of cells in the laboratory, cells can be reinfused to rapidly reconstitute immunity and reduce the risk of serious infections following transplant. Stimulation and expansion of cells is currently done in plastic dishes and normal incubators but bioreactor technology is likely to supersede current methods in the near future. Infused cells effectively prevent and treat viral infections in this patient group. Tumour antigens are generally weaker than antigens derived from infections. Nevertheless, newer developments including the transfer of T cell receptors (either intact alpha and beta chains or single chain chimaeric antigen receptors) or stimulation of T cells with overlapping peptides of known tumour antigens also show promise in generating cells that can be used to reduce the risk of recurrence of malignant disease post-transplant. Methods to expand the number of targets of cell generation will allow for the re-establishment of broad based immunity soon after transplant and reduce deaths from opportunistic infection and malignant disease.

New Total Ossicular Replacement Prostheses With a Resilient Joint

New flexible total ossicular prostheses with an integrated microjoint can compensate for large static displacements in the reconstructed ossicular chain. When properly designed, they can mimic the function of the joints of the intact chain and ensure good vibration transfer in both straight and bent conditions. Prosthesis dislocations and extrusions are frequently observed after middle ear surgery. They are mainly related to the altered distance between the coupling points because of large static eardrum displacements. The new prostheses consist of 2 titanium shafts, which are incorporated into a silicone body. The sound transfer function and stapes footplate displacement at static loads were evaluated in human temporal bones after ossicular reconstruction using prostheses with 2 different silicones with different hardness values. The stiffness and bending characteristics of the prostheses were investigated with a quasi-static load. The sound transfer properties of the middle ears with the prostheses inserted under uncompressed conditions were comparable with those of ears with intact ossicular chains. The implant with the soft silicone had improved acoustic transfer characteristics over the implant with the hard silicone in a compressed state. In the quasi-static experiments, the minimum medial footplate displacement was found with the same implant. The bending characteristics depended on the silicone stiffness and correlated closely with the point and angle of the load incidence. The titanium prostheses with a resilient joint that were investigated in this study had good sound transfer characteristics under optimal conditions as well as in a compressed state. As a result of joint bending, the implants compensate for the small changes in length of the ossicular chain that occur under varying middle ear pressure. The implants require a stable support at the stapes footplate to function properly.

The Serum Free Light Chain Assay Allows Earlier Detection of Relapse/Progression of Multiple Myeloma After Autologous Hematopoietic Cell Transplantation

Abstract 4466 The International Myeloma Working Group (IMWG) guidelines use serum free light chain (SFLC) response criteria only in multiple myeloma (MM) patients without measurable M-protein in serum or urine and to satisfy stringent complete remission (sCR) criteria. Current response criteria use changes in intact immunoglobulin measured by serum protein electrophoresis and immunofixation (SIF). The role of the SFLC assay during follow up of MM patients with measurable intact immunoglobulin after autologous hematopoietic cell transplantation (auto-HCT) is not well defined. In order to assess the role of the SFLC assay (Freelite; The Binding Site Group Ltd, Birmingham, UK) during the follow up of MM patients (with measurable M protein in serum or urine), and its role in detecting relapse compared to SIF test, a retrospective analysis of 20 MM patients after melphalan 200 mg/m2 conditioned first auto-HCT was conducted. Patients were followed for a median 15 months (range 3–36 months) between 2008 and 2011. Each patient had both the SFLC assay and SIF performed every 3 months at each routine clinic visit. No patient had renal failure (defined as serum creatinine 2 mg/dl or more). No patient deaths occurred during the follow up period. Two groups were identified. 10 patients without relapse had the following characteristics: median age at HCT 55 years (range 46–68 years); median time from diagnosis to auto-HCT 6.5 months (3–18 months); Durie-Salmon stage IIIA (n=7), stage IA (n=3); IgG kappa (n=5), IgG lambda (n=5); response to HCT very good partial remission VGPR 1 (n=5), CR1 (n=3), and sCR1 (n=2). All patients without relapse maintained a normal SFLC ratio (0.26–1.65) after HCT with negative SIF (n=5), or stable SIF (n=3) and in 2 patients (who were on lenalidomide maintenance), the SIF became negative 12 months and 18 months after HCT. 10 MM patients relapsed after auto-HCT: median age at HCT 55 years (range 46–68 yrs); Durie-Salmon stage IIIA (n=9), IA (n=1); IgG kappa (n=4), IgG lambda (n=2), kappa (n=2), lambda (n=1), IgA kappa (n=1); median time from diagnosis to HCT 8 months (range 4–28 months); response to HCT VGPR1 (n=7), CR1 (n=3). In all the patients who relapsed, the SFLC ratio became abnormal (<0.26 or >1.65) at a median of 3 months (range 3–12 months) before any other test detected relapse, (including SIF, bone marrow biopsy) and the SFLC ratio remained abnormal and continued to worsen over the follow up period. Salvage chemotherapy was begun at a median of 3 months (range 3–12 months) after the SFLC ratio became abnormal after confirmation of relapse by current approved methods (elevation of SIF or bone marrow biopsy). In follow up after HCT, 5 patients who relapsed developed an abnormal SFLC ratio (<0.26 or >1.65) without a concomitant increase in SIF and these patients had bone marrow biopsy confirmation of relapse (25% increase of plasma cells from baseline). In one patient, the SFLC ratio became abnormal at 3 months post HCT with negative SIF, and negative bone marrow biopsy at 3 months after HCT and multiple extramedullary plasmacytomas were found at 4 months after HCT accounting for the relapse. Conclusion: Abnormal SFLC ratio allowed earlier detection of relapse/progression of MM after auto-HCT even in those with measurable M protein in serum and urine and not only in light chain MM but also in those with intact heavy chain immunoglobulins compared to SIF and this can be critical for earlier intervention with salvage strategies. There are currently no guidelines for use of the SFLC assay in follow up of MM patients with measurable serum and urine M protein. The serum free light chain assay should be used concomitantly with serum protein electrophoresis and immunofixation in the follow up of MM patients after auto-HCT. Disclosures: No relevant conflicts of interest to declare.

Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

In vivo protein stabilization based on fragment complementation and a split GFP system

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.

An infrequent relapse of multiple myeloma predominantly manifesting as light chain escape: clinical experience from two Chinese centers

Patients with intact immunoglobulin (Ig) multiple myeloma (MM) usually show parallel fluctuations of their intact Ig and light chain (LC) concentrations. Herein we report 11 patients with relapsed, intact Ig MM with a marked increase in urinary LCs in the absence of a parallel rise in serum intact Ig, known as light chain escape (LCE). A major feature accompanying the presentation of LCE was conversion of plasma cell morphology. An important feature preceding the onset of LCE was the use of biological therapies. These patients were not associated with a highly aggressive course. Median overall survival times after the diagnosis of MM and after LCE were 48 months and 24 months, respectively. These cases represent a documented series of a rare but clinically important mode. More attention should be directed toward patients with intact Ig MM when the clinical course deteriorates despite a reduction of intact monoclonal immunoglobins.

Thermally activated breakdown in a simple polymer model

We consider the thermally activated fragmentation of a homopolymer chain. In our simple model the dynamics of the intact chain is a Rouse one until a bond breaks and bond breakdown is considered as a first passage problem over a barrier to an absorbing boundary. Using the framework of the Wilemski-Fixman approximation we calculate activation times of individual bonds for free and grafted chains. We show that these times crucially depend on the length of the chain and the location of the bond yielding a minimum at the free chain ends. Theoretical findings are qualitatively confirmed by Brownian dynamics simulations.

ALκI (UNK) – primary structure of an AL-amyloid protein presenting an organ-limited subcutaneous nodular amyloid syndrome of long duration. Case report and review

Slowly progressing subcutaneous nodules all over the body were detected in 1994 in an otherwise healthy, now 66-year-old woman (UNK). A first biopsy was taken 10 years ago and revealed amyloid. Immunohistochemistry was suggestive for ALκ. From a nodular excisate, performed in the same year for cosmetic reasons, amyloid fibrils were extracted. Protein separation according to their size revealed multiple protein fragments below the MW of an intact κ-light chain. They were identified as κ-fragments by Western blotting. The κ-fragments were cleaved into overlapping peptides using tryptic, N-Asp and chymotryptic digests. Peptides were sequenced by Edman-degradation and mass spectrometry. The complete amino acid sequence of the variable region and most of the constant region of ALκ (UNK) was identified in various fragments comprising positions 1 to 207 of a monoclonal κI-light chain. Four novel and several rare amino acid exchanges have been identified as compared to 17 amyloidogenic and >100 non-amyloidogenic κI-sequences published, leading to increased hydrophobicity of ALκ (UNK). Sequence analysis of C-region peptides allowed one to determine the κ-allotype as being invb+.A rabbit antibody was produced against ALκI (UNK). It strongly reacted with amyloid on formalin-fixed paraffin embedded tissue sections of the same patient and detected ALκ-amyloid of many other patients. In contrast, antibodies produced against κBJP of subclasses κI–κIV failed to label ALκ (UNK) amyloid deposits.The patient continues to be free of systemic disease, already for 14 years until today.

Molecular characterization of the protease from Clostridium botulinum serotype C responsible for nicking in botulinum neurotoxin complex

A protease was purified from the culture medium of Clostridium botulinum serotype C strain Stockholm (C-St). The purified protease belonged to the cysteine protease family based on assays for enzyme inhibitors, activators and kinetic parameters. The protease formed a binary complex consisting of 41- and 17-kDa proteins held together non-covalently. The DNA sequence encoding the protease gene was shown to be a single open reading frame of 1593 nucleotides, predicting 530 amino acid residues including a signal peptide. The N-terminal region of the native enzyme underwent further proteolytic modification after processing by a signal peptidase. The protease introduced intermolecular cleavage into an intact single chain botulinum neurotoxin (BoNT) at a specific site. Homology modeling and docking simulation of C-St BoNT and C-St protease demonstrated that the specific nicking-site of the BoNT appears to fit into the deep pocket in the active site of the protease.

Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.