Intact Chain Research Articles

Extensional Stress-Induced Ductility of Poly(l-lactide) Films: Role of the Entangled Network in Amorphous Regions.

The relationship between the density of the entangled amorphous network and the ductility of oriented poly(l-lactide) (PLLA) films is explored based on the preferential hydrolysis of the amorphous regions in phosphate buffer solution (PBS). PLLA films with a balance of ductility and stiffness have been prepared by the "casting-annealing stretching" based on mechanical rejuvenation, and the structural evolution and mechanical properties at different hydrolysis durations have been identified. Various stages are found during the transition of ductility to brittleness for hydrolyzed PLLA films. First, the elongation at break for hydrolyzed PLLA films remains unchanged in the first stage of hydrolysis and then gradually decreases. Eventually, the films turn to be brittle in the third stage. The strain-hardening modulus (GR) of the hydrolyzed films is utilized to reflect the density of the entangled amorphous network, and a gradual decrease of GR with hydrolysis time indicates the decisive role of the amorphous entanglement network in the mechanical rejuvenation-induced ductility of PLLA. The quantitative relationship between the entangled amorphous network and the stress-induced ductility of PLLA films is revealed. The dependence of deformation behavior on entangled amorphous network density is closely correlated to activated primary structure during deformation. The intact chain network plays a crucial role in sufficiently activating the primary structure to yield and disentanglement during the subsequent necking. These findings could advance the understanding of the PLLA's ductility induced by mechanical rejuvenation and offer guidance for awakening the intrinsic toughness of PLLA.

Paralia flatoniana sp. nov., a new species from the late Eocene of Texas with discussion on ecology and initial valves

A new Paralia Heiberg species, Paralia flatoniana sp. nov., is documented from a late Eocene diatomite and mudstone deposit in east central Texas. Paralia represents up to 63% of the assemblage in the diatomite samples and 34% in the mudstone. Numerous Paralia initial valves are present adding an unusual component to this assemblage as they are infrequently reported from samples containing populations of fossil Paralia. A third valve type observed attached to Paralia chains is also illustrated and discussed. Intact chains are common and preservation of the frustules is very good with limited indication of dissolution or transport. An average valve diameter of 19.0 µm (7.1–37.7 µm range), the presence of numerous initial valves and preservation of fine morphological structures suggest an environment with often high in situ productivity rather than a transported assemblage. Morphologically similar species Paralia crenulata and Paralia thybergii share many mantle characteristics with P. flatoniana while the valve face details of Paralia fausta are similar. Paleogeography and – ecology of the Eocene Gulf of Mexico and volcanism to the west provided a temperate coastal nutrient-rich shelf environment favourable for Paralia.

MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour.

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.

Ossicular Chain Status in Adults with Chronic Otitis Media Mucosal at a Tertiary Referral Hospital in Nepal

Introduction
 Chronic Otitis Media (COM) is a common condition characterized by perforation of the tympanic membrane and inflammation of middle ear cleft mucosal lining for at least 2-6 weeks. This study was carried out to find out the status of the middle ear ossicles in patients with COM mucosal and to correlate with clinical parameters.
 Materials and Methods
 This retrospective clinical study comprises 106 myringoplasty patients at the department of ENT, Shree Birendra Hospital. Their preoperative PTA, four frequencies (500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz) Air Conduction Thresholds (ACT), and Air Bone Gap (ABG) were recorded. Ossicular chain integrity and mobility evaluated intraoperatively were noted. Ossicular chain status was analyzed with ACT and ABG.
 Results
 The ossicles were intact and mobile in 91.5%, either fixed/ restricted or disrupted in 8.5%. Mean ACT and ABG comparisons in intact and ossicular chain defect groups were statistically significant. Frequency-specific ACT at all four frequencies and ABG except at 4000 Hz in the two groups were also statistically significant to predict the ossicular involvement. The group with ossicular defect had moderate to severe Conductive Hearing Loss (CHL).
 Conclusion
 Mean as well as frequency-specific ACT and ABG can be considered as a potential preoperative predictor for ossicular chain status in COM mucosal.

Our Laboratory Experience: Comparison of Capillary Electrophoresis/Immunosubtraction and Agarose Gel/Immunofixation

Abstract. Monoclonal gammopathies represent a wide spectrum of related diseases. The common denominator is the presence of a monoclonal protein in the serum or urine, which can be in the form of intact immunoglobulin, immunoglobulin fragments, and/or free light chains (κ and λ). We can detect these abnormalities by immunofixation electrophoresis (IFE) in which specific antibodies are overlaid after electrophoresis and the corresponding immunoglobulin. In our study, we compared gel-based and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins in serum samples. We analyzed 500 serum samples by Sebia Hydragel agarose gel electrophoresis (AGE)/immunofixation electrophoresis (IFE) and CAPILLARYS 2 capillary zone electrophoresis (CZE)/immunosubtraction (IS). AGE/IFE and CZE/IS had an overall agreement of 98% on serum protein electrophoresis. In our hands, AGE/IFE and CZE/IS had the same specificity for detection of monoclonal proteins; however, CZE/IS seems to be more sensitive than AGE/IFE for the detection of some critical cases. However, CZE/IS is an analytically suitable alternative to AGE/IFE, although laboratories should need to combine, for singular samples, both electrophoretic methods in their clinical routine. A combined use of AGE/IFE and CZE/IS is necessary to lead to an accurate diagnosis and clinically error-free results.

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.

Back cover: High‐performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements

DOI: 10.1002/elps.202000028 The cover picture shows a new HPCE method for the quali-quantitative analysis of animal origin chondroitin sulfate, detected as intact chains in samples and food supplements. The method is also able to separate and quantify intact chains of keratan sulfate and hyaluronic acid. The keratan sulfate could eventually be present in the samples as residue of low controlled chondroitin sulfate manufacturing processes of extraction from animal tissues, while hyaluronic acid is frequently added in the chondroitin food supplement preparations.

Audiological Outcome in Myringoplasties with an Intact Ossicular Chain: Is there a Difference between Chronic Otitis with or without Cholesteatoma?

Introduction Chronic otitis media (COM) with a central perforation or a concomitant cholesteatoma are both inflammatory lesions, however, with different etiologies. Both entities may present with an intact chain, and the final reconstruction is quite similar. Does it also apply for the hearing outcome? Objectives In a retrospective analysis, we investigated the preoperative hearing and the final hearing outcome of two groups of patients: those with COM and those with cholesteatoma, and compared various factors. Methods Patients operated between 2010 and 2019 were entered prospectively into a research database, and the integrity of the ossicular chain, the extent of the cholesteatoma, and the findings on computed tomography (CT) scans were retrospectively analyzed and correlated to the final hearing outcome. Results Out of 210 tympanoplasties for COM, 162 (80%) presented with an intact chain, and 85 (40%) ears could be analyzed. Out of 283 cholesteatoma surgeries, 53 (19%) ears presented with an intact chain. The preoperative air-bone gap (ABG) was worse in the COM group, but the postoperative ABG over the frequencies of 0.5 kHz and 4 kHz was the same (10 dB to 12 dB) in both groups, and remained within 20 dB in 90% (40 and 78 patients, respectively). The extension of the disease was rather limited in the cholesteatoma group (stages Ch1a and 1b), and better pneumatization and ventilation were beneficial for a good result. Postoperatively, the frequency of 4 kHz had the largest ABG (14 dB and 18 dB). Conclusion Overall, 80% of the patients with COM and less than 20% of those with cholesteatoma had an intact and mobile chain at surgery. Using equivalent surgical techniques for the tympanoplasty, the final outcome was almost the same for both groups, with a mean ABG of 10 dB to 12 dB.

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Mechanical Energy Dissipation Through the Ossicular Chain and Inner Ear Using Laser Doppler Vibrometer Measurement of Round Window Velocity.

Round window velocity measurements should correlate closely with vibration measurements taken at proximal points along an intact chain over a set frequency range. These round window vibration measurements should be similar to the vibration measurements taken of the ossicles if mechanical energy is conserved through the vestibular organ. To date there has not been a study which compares vibratory velocity measurements through an intact ossicular chain to the level of the round window. This study attempted to quantify the degree of mechanical energy transmission and suspected dissipation through the ossicular chain and vestibular organ through incus, stapes, and round window velocity measurements in response to sound stimulus. Five thawed human temporal bones with intact ossicular chain and tympanic membrane underwent complete mastoidectomy and a facial recess approach. A laser Doppler vibrometer (LDV) was mounted on the operating microscope to measure vibration of incus, stapes, and round window in response to a sound stimulus within the external auditory canal. Sound stimulus frequencies ranged from 0.5 to 4 kHz at 90 dB SPL. Vibration velocity was measured across the frequency range for each incus, stapes, and round window. Vibration velocity curves obtained over the frequency range were similar for each of the bones with a notable resonant frequency around 2 kHz. The incus and stapes curve amplitudes were nearly identical with similar maximum velocity and frequency at which this maximal velocity was noted. Round window vibration velocity demonstrated a unique peak velocity. Transfer function measurements of the stapes and round window demonstrated markedly similar curves. The variation in velocity between temporal bones in response to the standardized stimulus was more dramatic in the round window measurements when compared with the incus and stapes. This study supports the concept that round window transfer function is equivalent to stapes footplate transfer function when subjected to the same acoustic stimuli. This study also demonstrates that the round window is a much more difficult target to measure when using LDV technology and improvements in experimental design are required to better understand round window physiology in relation to transfer of acoustic vibratory stimulus transferred throughout the middle ear. A complete and thorough understanding of the biophysical properties of the middle and inner ear are critical for optimal ossiculoplasty outcomes and the development of future ossicular prosthetics.

Effects of Voxelotor (GBT440) Therapy on Laboratory Testing of Sickle Cell Patients

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

Aggregates Dramatically Alter Fibrin Ultrastructure

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH, etc.), it has been suggested that the polydispersity of fibrinogen may play an important role. We propose here a detailed investigation of the influence of this parameter on fibrin multiscale structure. Two commercial fibrinogen preparations were used, a monodisperse and a polydisperse one. First, the respective compositions of both fibrinogen preparations were thoroughly determined by measuring the fibrin-stabilizing factor; fibronectin; α, β, and γ intact chain contents; the γ/γ′ chains ratio; the N-glycosylation; and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found that are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, multiangle laser light scattering-coupled size exclusion chromatography and dynamic light scattering measurements showed that the polydisperse preparation contains significant amounts of aggregates, whereas the other preparation is essentially monodisperse. The multiscale structure of the fibrins produced from those two fibrinogen preparations was determined by using x-ray scattering, spectrophotometry, and confocal microscopy. Results show that fibers made from the aggregate-free fibrinogen present a crystalline longitudinal and lateral structure and form a mikado-like network. The network produced from the aggregates containing fibrinogen looks to be partly built around bright spots that are attributed to the aggregate. The multiscale structure of mixtures between the two preparations shows a smooth evolution, demonstrating that the quantity of aggregates is a major determining factor for fibrin multiscale structure. Indeed, the effect of a few percent in the mass of aggregates is larger than any other effect because of compositional differences under the same reaction conditions. Finally, we propose a mechanistic interpretation of our results, which points at a direct role of the aggregates during polymerization, which disrupts the ideal ordering of monomers inside fibrin protofibrils and fibers.

Abstract 330: A HaloTag-TEV Genetic Cassette for Mechanically Probing Native Titin

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces interact with titin in the sarcomere, enabling muscle contraction. However, testing these mechanical hypotheses on native titin in its natural environment has remained inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in titin. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors.

Polyhedral oligomeric butyl stannoxane cages (Sn-POSS) as oxidation-activated linear repairing units or crosslinking nano-building blocks, depending on structure of the polymer matrix

The chemically active heavier POSS homolog, the n-butylstannoxane dodacamer cage, shows unusual stabilizing properties during an oxidizing treatment of organic-inorganic hybrid polymer materials, into which it is incorporated as nano-building block. In this work, we dispersed Sn_POSS in several matrixes, which mainly had all-carbon backbones (based on polystyrene, polyacrylates and polyethers). Sn_POSS concentration was varied, and it was incorporated either as non-functional cage (blending), or via covalent bonding as a large co-monomer. The stannoxane cages' reactivity in the different matrixes was compared, and the authors propose matrix-dependent mechanisms of the stabilizing effect of Sn_POSS, which make possible to predict the behavior of this cage in different polymers, if their degradation mechanism and eventual reactivity of pendant groups can be estimated. Under oxidizing conditions, the studied matrixes yielded either pendant radicals on (at first) intact chains, or terminal radicals on ends of fragmented chains, depending on the exact polymer structure. The stannoxane cages, if present, subsequently underwent either crosslinking, or chain-repair reactions (fragmented chains re-connection) with the radical sites of the matrix. Another reaction type, which yielded additional crosslinking in the hybrids, was observed in case that the polymer matrix contained suitable pendant functional groups, which were able to react with Sn_POSS after activation either by heat or by oxidation.

LO08: Effect of an intact “chain of survival” sequence on survival to discharge from out-of-hospital cardiac arrest

Introduction: The “chain of survival” is a 5-link theoretical construct that has been central to cardiac arrest resuscitation for over 40 years. Although the role of each link has been extensively studied, little is known about the impact of performing the chain of survival in sequence. The purpose of this study was to estimate the proportion of out-of-hospital cardiac arrest (OHCA) responses by Emergency Medical Services (EMS) that had an intact chain of survival sequence response, and the effect of this on survival to hospital discharge. Methods: We conducted a prospective cohort study of adult (>age 20 years) OHCA patients using data collected between 2005-2007 by the Resuscitation Outcomes Consortium (ROC). ROC is a research network involving 10 research sites and 264 EMS agencies across North America. Using routinely collected data, we coded cases as receiving an intact or non-intact chain of survival sequence based on EMS cardio pulmonary resuscitation (CPR), rhythm analysis or defibrillation, epinephrine administration or endotracheal intubation, and transport to a hospital with an electrophysiology lab or percutaneous coronary intervention capability, contingent on the patient’s condition when EMS arrived. Multiple variable logistic regression was performed, adjusting for known (Utstein) survival predictors, to estimate the independent effect of intact chain of survival sequence on survival to hospital discharge. REB approval was obtained. Results: We enrolled12,821 OHCA cases, of which, 29.4% (n=3,773) had an intact chain of survival and 7.6% (n=972) survived to hospital discharge. Cases with an intact chain of survival were younger, and more likely to arrest in public, receive bystander CPR, occur in the USA and specific ROC sites, and had faster EMS response times. The adjusted odds ratio of survival to hospital discharge with an intact chain of survival sequence was 2.4 (95% CI: 2.1-2.8). A sensitivity analysis of 4,056 cases with known timing of endotracheal intubation found a similar adjusted odds ratio of 2.1 (95% CI: 1.6-2.8). Conclusion: Our results indicate that OCHA resuscitation with an intact chain of survival occurs in approximately 1/3 of cases, and results in over a two-fold increase in the odds of surviving to hospital discharge. Initiatives to improve EMS teamwork and increase the proportion of OHCA resuscitation with an intact chain of survival appear to be warranted.

Presumed magnetic biosignatures observed in magnetite derived from abiotic reductive alteration of nanogoethite

The oriented chains of nanoscale Fe-oxide particles produced by magnetotactic bacteria are a striking example of biomineralization. Several distinguishing features of magnetite particles that comprise bacterial magnetosomes have been proposed to collectively constitute a biosignature of magnetotactic bacteria (Thomas-Keprta et al., 2001). These features include high crystallinity, chemical purity, a single-domain magnetic structure, well-defined crystal morphology, and arrangement of particles in chain structures. Here, we show that magnetite derived from the inorganic breakdown of nanocrystalline goethite exhibits magnetic properties and morphologies remarkably similar to those of biogenic magnetite from magnetosomes. During heating in reducing conditions, oriented nanogoethite aggregates undergo dehydroxylation and transform into stoichiometric magnetite. We demonstrate that highly crystalline single-domain magnetite with euhedral grain morphologies produced abiogenically from goethite meets several of the biogenicity criteria commonly used for the identification of magnetofossils. Furthermore, the suboxic conditions necessary for magnetofossil preservation in sediments are conducive to the reductive alteration of nanogoethite, as well as the preservation of detrital magnetite originally formed from goethite. The findings of this study have potential implications for the identification of biogenic magnetite, particularly in older sediments where diagenesis commonly disrupts the chain structure of magnetosomes. Our results indicate that isolated magnetofossils cannot be positively distinguished from inorganic magnetite on the basis of their magnetic properties and morphology, and that intact chain structures remain the only reliable distinguishing feature of fossil magnetosomes.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Lung Heparin and Porcine Intestine Heparin

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin–derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin–derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

Dehydrogenative Homocoupling of Alkyl Chains on Cu(110).

Through the interplay of high-resolution scanning tunneling microscopy imaging and density functional theory calculations, the stepwise dehydrogenative homocoupling of alkyl chains on Cu(110) is demonstrated, proceeding from the intact chain, via the dehydrogenative intermediates, to the formation of the divers final coupling products.

Properties of magnetite nanoparticles produced by magnetotactic bacteria

The magnetic nanoparticles (magnetite) were prepared through the fermentation of the Magnetospirillum strain WM-1 newly isolated by our group. The samples were characterized by TEM, SAED, XRD, rock magnetic analysis, and Mossbauer spectroscopy. TEM and SAED measurements showed that the magnetosomes formed by strain WM-1 were single crystallites of high perfection with a cubic spinel structure of magnetite. X-ray measurements also fitted very well with standard Fe3O4 reflections with an inverse spinel structure of the magnetite core. The size of crystal as calculated by the Debye-Scherrer’s equation was approximately 55 nm. Rock magnetic analysis showed WM-1 synthesized single-domain magnetite magnetosomes, which were arranged in the form of linear chain. The high delta ratio ((δFC / δZFC = 4) supported the criteria of Moskowitz test that there were intact magnetosomes chains in cells. The Verwey transition occurred at 105 K that closed to stoochiometric magnetite in composition. These observations provided useful insights into the biomineralization of magnetosomes and properties of M. WM-1 and potential application of biogenic magnetite in biomaterials and biomagnetism.

Abstract 1156: Shed syndecan-1 downregulates histone acetyltransferase activity and shuttles HGF to the nucleus of tumor and host cells

Abstract The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) is expressed by myeloma tumor cells, is proteolytically shed from the cell surface and accumulates in the serum. High levels of serum syndecan-1 are an independent indicator of poor prognosis. Our lab has demonstrated that shed syndecan-1 drives myeloma tumor progression in vivo, but the mechanism(s) mediating this are not fully understood. The role of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix has been extensively studied, but their presence and function within the nucleus is often overlooked. However, recent findings indicate that nuclear heparan sulfate has important regulatory functions. Based on preliminary findings, we hypothesized that shed syndecan-1 binds to the tumor cell surface and translocates to the nucleus where it regulates gene expression thereby promoting aggressive tumor behavior. We utilized murine myeloma cells which were grown in the presence of medium containing human shed syndecan-1. Strikingly, the shed syndecan-1 was rapidly taken up by these cells and translocated to the nucleus. Additionally, exogenous human shed syndecan-1 added to murine stromal cells also bound to the cell surface and translocated to the nucleus. This is the first demonstration that shed proteoglycans can translocate to the nucleus of cells. When stromal cells were grown in the presence of tumor-derived shed syndecan-1, acetylated histone H3 was decreased. Additionally, tumor cells engineered to express high levels of shed SDC1 have a 58% reduction in histone acetyltransferase activity. This may be due to syndecan-1 directly binding to p300/CBP-associated factor, a HAT enzyme. Moreover, HGF bound to shed syndecan-1 was shuttled to the nucleus of myeloma cells as a complex. Shed syndecan-1 and HGF translocation to the nucleus is dependent on intact heparan sulfate chains present on the syndecan-1 core protein. These data reveal a novel mechanism of tumor-host crosstalk whereby syndecan-1, shed by tumor cells, alters histone acetyltransferase activity and shuttles heparin-binding factors to the nucleus to reprogram cells and alter gene expression. Therapeutic regulation of syndecan-1 shedding, its binding to the cell surface or translocation to the nucleus represent novel strategies to control the progression of myeloma and other cancers. Citation Format: Mark Stewart, Vishnu Ramani, Ralph Sanderson. Shed syndecan-1 downregulates histone acetyltransferase activity and shuttles HGF to the nucleus of tumor and host cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2014-1156