Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular ‘interference’ of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization ‘hole b’, also known as ‘b-pockets’, through PEGylated complementary ‘knob B’ mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, K_d, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its K_d but increases extensibility of fibers at concentrations above its K_d. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.     

Evaluation of a Commercial Kit for the Radioimmunoassay of Fibrinopeptide A

Measurement of fibrinopeptide A (FpA) provides a sensitive and specific marker of thrombin generation and is important in the investigation of the mechanisms involved in thrombosis and hemostasis. However, current methods available for determination of FpA by radioimmunoassay (RIA) require rigorous method development. Recently, a commercial kit (Mallinckrodt Corp: M-Kit) for the RIA of FpA has become available which contains all the necessary reagents for the assay. We evaluated this kit and compared it to an assay prepared from a commercial kit (IMCO Corp: I-Kit) which contains only the raw materials. Both assays had similar characteristics and duplicate plasma samples assayed using both methods were not significantly different. Separation of FpA from fibrinogen using bentonite slurry (M-Kit) proved superior to the ethanol precipitation method (I-Kit). The complete kit (M-Kit) will provide the routine hemostasis laboratory with an RIA for FpA which is immediately available.     

Mitogenicity of a spread film of monophosphoryl lipid A

When spread at the air-water interface, monophosphoryl lipid A (MPLA) forms stable insoluble monolayers that collapse at approximately 55 dyn/cm. At collapse, the exclusion area of each molecule is approximately 119 Angstrom(2), consistent with the cross-sectional area of the lipid's 6 acyl chains. The nominal thickness of such films is approximately 22 Angstrom, determined, presumably, by the length of the acyl chains. For biological modeling of MPLA films, a system was developed in which monolayers of the lipid are supported by monodisperse hydrophobic beads of microscopic dimensions. Beads coated with MPLA monolayers within which the nominal area of each molecule is approximately equivalent to the "take-off" area of the lipid at the air-water interface, 280 Angstrom(2), are mitogenic for spleen cells. Given the natural occurrence of lipid A in the bacterial cell wall as well as the inherent stability of lipid A films, it seems reasonable to assume that at least some of the biological activities attributed to the lipid derive from its presentation/operation at an interface, i.e., on a surface. We propose beads coated with adsorbed films of lipid A will prove useful tools for modeling the activities of the lipid both in vitro and in vivo, and for elucidating the surface dependency and structural requirements of those activities.     

Fusion of mApple and Venus fluorescent proteins to the Sindbis virus E2 protein leads to different cell-binding properties

Fluorescent proteins (FPs) are widely used in real-time single virus particle studies to visualize, track and quantify the spatial and temporal parameters of viral pathways. However, potential functional differences between the wild type and the FP-tagged virus may specifically affect particular stages in the virus life-cycle. In this work, we genetically modified the E2 spike protein of Sindbis virus (SINV) with two FPs. We inserted mApple, a red FP, or Venus, a yellow FP, at the N-terminus of the E2 protein of SINV to make SINV-Apple and SINV-Venus. Our results indicate that SINV-Apple and SINV-Venus have similar levels of infectivity and are morphologically similar to SINV-wild-type by negative stain transmission electron microscopy. Both mutants are highly fluorescent and have excellent single-particle tracking properties. However, despite these similarities, when measuring cell entry at the single-particle level, we found that SINV-Apple and SINV-Venus are different in their interaction with the cell surface and FPs are not always interchangeable. We went on to determine that the FP changes the net surface charge on the virus particles, the folding of the spike proteins, and the conformation of the spikes on the virus particle surface, ultimately leading to different cell-binding properties between SINV-Apple and SINV-Venus. Our results are consistent with recent findings that FPs may alter the biological and cellular localization properties of bacterial proteins to which they are fused.     

The effect of sCD4 on the binding and accessibility of HIV-1 gp41 MPER epitopes to human monoclonal antibodies

Two human monoclonal anti-HIV-1 antibodies, 2F5 and 4E10, were utilized to investigate the accessibility and conservation of gp41 MPER epitopes on five different clades of HIV-1 in the absence and presence of sCD4. The binding of human monoclonal antibodies (mAbs) to HIV-1 was dependent upon the virus clade. Soluble CD4 significantly increased the accessibility of gp41 MPER-binding epitopes on several isolates that previously showed little or no binding with 2F5 and 4E10 mAbs as determined by a modified ELISA-based virus capture assay and surface plasmon resonance. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus. However, neither binding per se nor the total envelope content per virion was a predictor of neutralization. The hidden or conformational gp41 MPER epitopes unmasked by sCD4 may provide additional targets for vaccine design.     

Paracoccidioides brasiliensis conidia recognize fibronectin and fibrinogen which subsequently participate in adherence to human type II alveolar cells: involvement of a specific adhesin

We examined the ability of Paracoccidioides brasiliensis conidia to interact with fibronectin, fibrinogen and with A549 cells, in order to establish the nature of the molecules involved. Conidia bound to immobilized proteins in a concentration-dependent manner. Antibodies against fibronectin and fibrinogen inhibited the fungal adherence to the corresponding proteins; as did laminin and fibronectin, but not fibrinogen when added in soluble form; however, the fibrinogen fragment D interfered with adhesion in a significant manner. Various monosaccharides and RGD/RGDS peptides had no effect on adherence to fibronectin or fibrinogen, while N-acetylneuraminic acid (NANA) abolished adherence to both proteins. Additionally, these proteins were detected on the surface of A549 cells. Inhibition assays showed a significant decrease in fungal adherence when A549 cells were treated with anti-fibrinogen, anti-fibronectin antibodies and a purified adhesin of P. brasiliensis (32-kDa protein); or when conidia were treated with these soluble proteins, mAb anti-32-kDa protein, RGD peptides and NANA. These results suggest that fibrinogen and fibronectin facilitate the adherence of conidia to A549 cells probably through the interaction with adhesin-type molecules or a sialic acid based recognition system. These interactions appear to play a role in the initial fungal attachment to the lung, and consequently, also in the pathogenesis of paracoccidioidomycosis.     

Non-invasive method to detect motor unit contractile properties and conduction velocity in human vastus lateralis muscle

The contractile properties and conduction velocity of motor units are estimated by using surface array electrodes during voluntary isometric contractions of the human vastus lateralis muscle. The subjects develop and maintain sufficient force to steadily discharge a given motor unit, assisted by visual feedback from an oscilloscope. The torque curve developed around the knee joint is triggered by an individual motor unit and averaged. 31 motor units in five subects are studied. The twitch tension detected ranges from 3 to 27 m Nm with a mean of 12·3 m Nm. The threshold force ranges from 1·88 to 10·12 Nm with a mean of 5·48 Nm, which is 3% of the maximal voluntary contraction. The rise time ranges from 56 to 106 ms with a mean of 83 ms. The mean value of conduction velocity is 4·64 m s⁻¹. The twitch tension is positively correlated to the threshold force (r=0·839, p<0.01), but has no relation to the other parameters. It is concluded that the use of non-invasive surface array electrodes provides the contractile properties of motor units and muscle fibre conduction velocity during weak contractions.     

Use of enzyme-linked immunosorbent assay to detect antibodies to staphylococcal protein A in the rabbit

An ELISA technique is described which employs both solid phase and labelled free SpA. The technique allows the detection of anti-SpA antibodies in the rabbit. It is not suited for the same purpose with human sera, because human immunoglobulins are not saturated by non-specific interaction with solid phase SpA.     

Surface characterisation of various bone cements prepared with functionalised methacrylates/bioactive ceramics in relation to HOB behaviour

This study reports the relationship between the biocompatibility and surface properties of experimental bone cements. The effect of hydroxyapatite (HA) or alpha-tri-calcium phosphate (alpha-TCP) incorporated into bone cements prepared with methyl methacrylate as base monomer and either methacrylic acid or diethyl amino ethyl methacrylate (DEAEMA) as comonomers was investigated. The in vitro biocompatibility of these composite cements was assessed in terms of the interaction of primary human osteoblasts grown on the materials over a period of 5 days and compared with a control surface. These results were related to the surface properties investigated through a number of techniques, namely Fourier transform infrared, contact angle measurements, X-ray photoelectron spectroscopy and energy dispersive analysis of X-rays. Complementary techniques of thermal analysis and ion chromatography were also performed. Biocompatibility results showed that the addition of alpha-TCP improves biocompatibility regardless of comonomer type. This is in contrast to HA-based cements where cell proliferation was significantly lower. Surface characterisations showed that structural integrity of the materials was maintained in the presence of the acid and base comonomers, and water contact angles were reduced particularly in DEAEMA containing materials. Furthermore, ion chromatography confirmed higher Ca2+ and PO4(3-) ion release by both types of ceramics, particularly for those containing DEAEMA. In conclusion, the incorporation of acidic and basic comonomers to either HA or alpha-TCP ceramics containing bone cements can have differential effects upon the attachment and proliferation of bone cells in vitro. Moreover, those cements consisting of alpha-TCP and containing DEAEMA comonomer indicated the most favourable biocompatibility.     

A nationwide seroprevalence of total antibody to hepatitis A virus from 2005 to 2009: age and area-adjusted prevalence rates

Background/Aims

Recent outbreak of hepatitis A in Korea is clearly related to the epidemiological shift of hepatitis A virus (HAV). However, nationwide seroprevalence data have been limited. This study estimated the nationwide, age- and area-adjusted anti-HAV prevalence from 2005 to 2009.

Methods

Retrospective analysis of the results of total anti-HAV test in 25,140 cases which were requested by 1,699 medical institutions throughout the nation to Seoul Clinical Laboratory from Jan. 1 2005 to Dec. 31 2009 was performed. The estimated seroprevalence was adjusted by area and age of the standard population based on the 2005 Census data from Korea National Statistical Office.

Results

The area-adjusted anti-HAV prevalence in the children younger than 10 years were 33.4% in 2005 and 69.9% in 2009. The most susceptible age groups to HAV infection during the last 5 years were teenagers and the young adults in their age of twenties. The area-adjusted seroprevalence in 2009 were 11.9% in the age group of 20-29 years, 23.4% in the age group of 10-19 years, 48.4% in the age group of 30-39 years. The population in 40-49 years showed geographically different seroprevalence with the lowest rate in Seoul (80%).

Conclusions

The most susceptible age group to HAV infection is 10-29 years, while the young children less than 10 years showed about 70% seropositivity. The changing seroepidemiology should be monitored continuously for the proper vaccination and patient care.     

NR2A亚单位C末端的部分缺失对 NMDA受体功能和表达的影响

目的研究NMDA受体NR2A亚单位羧基末端（以下简称为C末端）不同部位缺失对该受体表面表达和功能的影响。方法以GFP-NR2A为起始质粒构建了4个依次缺失部分c末端的GFP标记的NR2A亚单位表达载体：NR2A-ΔC1（缺失897L至1017S）、NR2A-ΔC2（缺失1024D至1142P）、NR2A-ΔC3（缺失1149D至1347G）及NR2A-ΔC4（缺失1354S至1464V）;通过活细胞免疫化学染色分析这些载体与NR1-1a亚单位共转染后在HEK293细胞和海马神经元的表面表达,并通过电生理检测转染HEK293细胞NMDA受体的功能。结果当NR2A-ΔC1、NR2A-ΔC2、NR2AΔC3、NR2A-ΔC4分别与NR1-1a共转染HEK293细胞时,均可获得细胞膜表面表达,其表面染色阳性的细胞在绿色荧光蛋白细胞中的百分比与共转染NR1-1a／GFP-NR2A时的百分比相比均无显著性降低;并且NR1-1a／NR2A-ΔC2或NR1-1a／NR2A-ΔC4转染的HEK293细胞均能记录到谷氨酸诱发的NMDA受体通道电流。在海马神经元中,当转染NR2A-ΔC1、NR2A-ΔC2或NR2A-ΔC3时,单位长度树突表面表达的受体簇数量均显著低于转染GFP-NR2A的神经元,而转染NR2A-ΔC4的树突表面表达的受体簇数量却无显著性降低。结论在HEK293细胞中,C末端部分缺失的NR2A亚单位不影响含NR2A亚单位的NMDA受体在膜表面表达;而在海马神经元中,含NR2A的NMDA受体的表面表达数量受NR2AC末端的调控。     

Antibodies to Hepatitis A Virus in Immune Serum Globulin

Two hundred one immune serum globulin (ISG) lots manufactured in the US between 1967 and 1977 were tested for antibodies to the hepatitis A virus (anti-HAV) by a competitive-inhibition radioimmunoassay (RIA); a lesser number were also tested by immune adherence hemagglutination (IAHA). The percentage of ISG lots that contained anti-HAV with a titer of 1:100 or greater by RIA was 50% for those manufactured in 1967, 69% for those manufactured in 1972, and 100% for those manufactured in 1977. The percentage of lots with anti-HAV titers equal to or greater than 1:500 by RIA was 7% in 1967, 18% in 1972, and 70% in 1977. Only ten lots of ISG (5%) had anti-HAV titers of 1:1,000 or greater by RIA; seven of these were manufactured in 1977. Both the mean titer of anti-HAV in ISG lots and the percentage of lots containing significant titers of this antibody appear to have increased in the US over the past ten years. This may reflect the increased use of source plasma from paid plasmapheresis donors in the US during this period. The lower titers of anti-HAV in the older lots of ISG studied were shown not to be due to fragmentation of antibody molecules during storage.     

A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials.     

Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles

The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant ⁷⁶HD/AN. The sPLA2 defect in ⁷⁶HD/AN, which severely impairs AAV's infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued ⁷⁶HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.     

Type A and B Subjects' Self-Reported Cognitive/Affective/Behavioral Responses to Descriptions of Potentially Frustrating Situations

Heightened psychophysiological reactivity to the novel or unfamiliar is a leading characteristic of shy or behaviorally inhibited individuals. To assess one aspect of the physiological stress response in shyness, the authors compared the morning plasma beta-endorphin levels of 15 extremely shy, healthy elderly individuals with beta-endorphin levels of 15 extremely outgoing persons on three pairs of 2 successive days. The primary finding was that shy participants exhibited significantly higher levels of beta-endorphin on the 1st days of each pair of days, compared with the 2nd days in the laboratory. No main effect for shyness or interaction between shyness and diet on endorphin levels was found. The findings are consistent with a peripheral opioid hyperreactivity to novelty in shy elderly persons. Shyness may constitute a risk factor for panic disorder in younger adults and for nasal allergies and certain cancers in older adults. Experimental design and interpretation of future studies of shy individuals' stress responses may need to consider novelty versus familiarity of the procedures and setting.     

Histocompatibility antigens and alleles in Japanese haemophilia A patients with or without factor VIII antibodies

We carried out human leukocyte antigen (HLA)-A, B, Cw, DR and DQ serological typing and HLA-DQA1, DQB1, DRB1 and DPB1 genetic typing for 46 Japanese haemophilia A patients, including 20 who had developed an antibody to factor VIII. It appears that anti FVIII inhibitor formation is associated with the major histocompatibility complex in Japanese haemophilia A patients. Absence of HLA-A24 is a principal risk factor for inhibitor formation in Japanese haemophilia A patients. As supplemental risk factors, HLA-DR4.1, DQ4 and DQA1*0301=2 are positively associated with patients exhibiting inhibitor compared with normal subjects. This and previous studies show that the association between HLA antigens and the formation of inhibitor depends on race. Data of HLA typing may be useful for the recognition of groups at high risk for the possible formation of inhibitor among Japanese haemophilia A patients.     

A probabilistic approach to measure the strength of bone cell adhesion to chemically modified surfaces

Patterned surfaces with alternating regions of amino silanes [N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS)] and alkyl silanes [dimethyldichlorosilane (DMS)] have been used to alter the kinetics of spatial distribution of cellsin vitro. In particular, we have previously observed the preferential spatial distribution of bone cells on the EDS regions of EDS/DMS patterned surfaces (10). In this study, we examined whether the mechanism of spatial distribution of cells on the EDS regions was adhesion mediated. Homogeneous layers of EDS and DMS were immobilized on quartz substrates and characterized by contact angle, X-ray photoelectron spectroscopy, and spectroscopic ellipsometry. The strength of bone cell attachment to the modified substrates was examined using a radial flow apparatus, within either 20 min or 2 hr of cell incubation in the presence of serum. A Weibull distribution was chosen to characterize the strength of cell-substratum adhesion. Within 20 min of cell exposure, the strength of adhesion was significantly larger on EDS and clean surfaces, compared with DMS surfaces (p<0.0001). Within 2 hr of cell incubation, there was no statistical difference between the strength of cell adhesion to EDS, DMS, and clean surfaces. The results of this study suggest that the surface chemistry mediates adhesion-based spatial cell arrangement through a layer of adsorbed serum proteins.     

Effect of monoamine oxidase A knockout on resistance to long-term exposure to ethanol

It was shown that transgenic Tg8 mice with monoamine oxidase A (MAO A) gene knockout demonstrate higher resistance to acute ethanol exposure compared to wild type C3H mice. This difference was observed at the early age (28-30 days). Long-term ethanol treatment changed the resistance to hypnotic, but not hypothermic action of this agent. Seven-day exposure increased the resistance to ethanol-induced narcotic sleep in Tg8 and C3H mice. After 30-day ethanol treatment the duration of narcotic sleep sharply decreased in C3H mice and increased in Tg8 mice, which attested to their decreased tolerance to ethanol.     

Idiopathic Amyloidosis Due to AA Protein: A Report of 2 Cases

Abstract

Two cases of idiopathic amyloidosis are described. AA protein was found to be the major constituent of tissue amyloid in both, based on sensitivity of Congo red birefringence to pretreatment of slides with potassium permanganate and immunoperoxidase staining with a monospecific antiserum. However, an underlying inflammatory, infectious or neoplastic disorder was not identified. The occurrence of AA amyloidosis without clearcut underlying disease is rare, having been described in only thirteen previous instances. Recent clinical series suggest that such cases may have a different course and response to therapy from “primary” amyloidosis due to systemic light chain deposition. Such cases may also underscore the importance of genetic factors in the pathogenesis of AA amyloid and the importance of characterizing the amyloidoses biochemically, as well as clinically. (The J Histotechnol 12:137, 1989.)