Effect of surface treatments on microtensile bond strength of repaired aged silorane resin composite.

OBJECTIVE: This laboratory study compared the repaired microtensile bond strengths of aged silorane resin composite using different surface treatments and either silorane or methacrylate resin composite. METHODS: One hundred eight silorane resin composite blocks (Filtek LS) were fabricated and aged by thermocycling between 8°C and 48°C (5000 cycles). A control (solid resin composite) and four surface treatment groups (no treatment, acid treatment, aluminum oxide sandblasting, and diamond bur abrasion) were tested (N=12 blocks, 108 beams/group). Each treatment group was randomly divided in half and repaired with either silorane resin composite (LS adhesive) or methacrylate resin composite (Filtek Z250/Single Bond Plus). After 24 hours in 37°C distilled water, microtensile bond strength testing was performed using a non-trimming technique. Surface topography after surface treatment was analyzed using scanning electron microscopy (SEM). Failure mode was examined using optical microscopy (50×). RESULTS: Weibull-distribution survival analysis revealed that aluminum oxide sandblasting followed by silorane or methacrylate resin composite and acid treatment with methacrylate resin composite provided insignificant differences from the control (p>0.05). All other groups were significantly lower than the control. Failure was primarily adhesive in all groups. CONCLUSION: Aluminum oxide sandblasting produced microtensile bond strength not different from the cohesive strength of silorane resin composite. After aluminum oxide sandblasting, aged silorane resin composite can be repaired with either silorane resin composite with LS system adhesive or methacrylate resin composite with methacrylate dental adhesive.

Quantitative proteomic analysis of the tizoxanide effect in vero cells.

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes.

The E2 glycoprotein of the alphavirus Sindbis is a typical type I membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. The carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6K and E1. These two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the lumen of the endoplasmic reticulum (b). We have investigated the orientation of this glycoprotein domain with respect to cell membranes by substituting a tyrosine for the normally occurring serine, four amino acids upstream of the carboxyl terminus. Using radioiodination of this tyrosine as an indication of the exposure of the glycoprotein tail, we have provided evidence that this domain is initially translocated into a membrane and is returned to the cytoplasm after export from the ER. This is the first demonstration of such a transient translocation of a single domain of an integral membrane protein and this rearrangement explains some important aspects of alphavirus assembly.

Membrane characteristics and osmotic behavior of isolated rod outer segments.

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na(+) influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na(+) influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na(+) permeability constant of about 2.8 x 10(-6) cm/s and an osmotic permeability coefficient of greater than 2 x 10(-3) cm/s.

Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport.

Native microtubules prepared from extruded and dissociated axoplasm have been observed to transport organelles and vesicles unidirectionally in fresh preparations and more slowly and bidirectionally in older preparations. Both endogenous and exogenous (fluorescent polystyrene) particles in rapid Brownian motion alight on and adhere to microtubules and are transported along them. Particles can switch from one intersecting microtubule to another and move in either direction. Microtubular segments 1 to 30 microns long, produced by gentle homogenization, glide over glass surfaces for hundreds of micrometers in straight lines unless acted upon by obstacles. While gliding they transport particles either in the same (forward) direction and/or in the backward direction. Particle movement and gliding of microtubule segments require ATP and are insensitive to taxol (30 microM). It appears, therefore, that the mechanisms producing the motive force are very closely associated with the native microtubule itself or with its associated proteins. Although these movements appear irreconcilable with several current theories of fast axoplasmic transport, in this article we propose two models that might explain the observed phenomena and, by extension, the process of fast axoplasmic transport itself. The findings presented and the possible mechanisms proposed for fast axoplasmic transport have potential applications across the spectrum of microtubule-based motility processes.

Histone gene switching in murine erythroleukemia cells is differentiation specific and occurs without loss of cell cycle regulation.

We investigated the expression characteristics of the fully replication-dependent (FRD) and the partially replication-dependent (PRD) histone gene variants by measuring changes in steady-state mRNA levels during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells. Between 24 and 60 h after induction, there was a dramatic switch in histone gene expression, such that the ratio of PRD to FRD transcripts increased severalfold over that found in uninduced MEL cells. We demonstrated that this gene switching was not simply a partial or complete uncoupling of PRD gene expression from DNA synthesis. PRD and FRD transcript levels were regulated coordinately upon treatment of uninduced or induced MEL cells with inhibitors of DNA synthesis, protein synthesis, or both. Using several criteria, we were unable to detect any difference in PRD and FRD gene expression under any conditions except in cells undergoing differentiation. MEL cells were arrested at a precommitment stage of differentiation by induction with HMBA in the presence of dexamethasone (DEX). If DEX was subsequently removed, DNA synthesis resumed, the cells underwent commitment, and histone gene switching was observed. In contrast, if both DEX and HMBA were removed, DNA synthesis still resumed, but commitment did not occur and no gene switching was observed. These results imply that histone gene switching is intimately related to the differentiation process.

Changes in the levels of three different classes of histone mRNA during murine erythroleukemia cell differentiation.

We used a gene-specific S1 nuclease assay to study the changes in steady-state mRNA levels of several core histone variants during the differentiation of murine erythroleukemia cells. These studies allowed us to distinguish three distinct expression classes of histone genes. The expression of the major replication-dependent class of histone genes was tightly linked to DNA synthesis. The concentrations of these transcripts decreased rapidly as cell division slowed during the process of differentiation. In contrast, the replication-independent H3.3 transcript levels were constitutively maintained throughout differentiation and were unaffected by inhibitors of DNA or protein synthesis. We also identified among the cloned histone genes used as probes a third expression class, the partially replication-dependent variants. Expression of these transcripts became transiently uncoupled from the reduced rate of DNA synthesis accompanying the early stages of differentiation. We show that their synthesis is sensitive to the DNA synthesis inhibitor hydroxyurea but that selective uncoupling from DNA synthesis of these histone mRNAs occurs at a specific stage of differentiation. We present several hypotheses to explain how this might be accomplished. The expression characteristics of the mRNAs studied coincided with those of the proteins for which they code, indicating that changes in the relative levels of the different variants is mediated at least in part by changes in mRNA levels.

The elastic properties of morsellised cortico-cancellous bone graft are dependent on its prior loading.

Confined compression experiments were carried out on cortico-cancellous bone taken from bovine femoral condyles to assess the effect of prior loading on the elastic confined modulus, E(c) of morsellised cortico-cancellous bone (MCB). Measurements were taken to find the values of E(c) for MCB subjected to cyclic loading resulting in axial stresses in the range of 0.5-3.0 N mm(2). Two values of E(c) were considered: E(ic), the instantaneous modulus, and E(dc), the delayed modulus allowing for stress relaxation effects. It was found that the values of E(c) increased with increasing maximum axial stress. It was also found that for each stress level the values of E(c) increased as the number of load cycles increased. The dependence of E(c) on the maximum axial stress and the number of load cycles is seen to explain the wide range of values for the apparent modulus of MCB found in previous studies. Tests examining the stress relaxation behaviour of MCB are also discussed. The results indicate that a minimum of 10 compaction episodes are required for MCB to achieve around 90% of its predicted maximum stiffness for a given compaction force.

Overproduction of histone H1 variants in vivo increases basal and induced activity of the mouse mammary tumor virus promoter.

BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving a reporter gene were constructed. Expression vectors in which either of two H1 variants, H10 or H1c, were under control of an inducible promoter were introduced into these lines. Surprisingly, overproduction of either variant resulted in a dramatic increase in basal and hormone-induced expression from the MMTV promoter. H1 overproduction also slowed the loss of MMTV promoter activity associated with prolonged hormone treatment. Transiently transfected MMTV reporter genes, which do not adopt a phased nucleosomal arrangement, do not display increased activity upon H1 overproduction. Thus the effects observed for stable constructs most likely represents a direct effect of H1 on a chromatin-mediated process specific to the nucleosomal structure of the integrated constructs. Induction of increased levels of acetylated core histones by treatment with trichostatin A also potentiated MMTV activity and this effect was additive to that caused by H1 overproduction. However, the effects of TSA treatment, in control or H1-overproducing cells, were eliminated by inhibiting protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter activity by increasing core histone acetylation within the MMTV promoter but perhaps by altering the synthesis of an unlinked transcriptional regulator.

Identification of a protein binding site on the surface of the alphavirus nucleocapsid and its implication in virus assembly.

BACKGROUND: Many enveloped viruses exit cells by budding from the plasma membrane. The driving force for budding is the interaction of an inner protein nucleocapsid core with transmembrane glycoprotein spikes. The molecular details of this process are ill defined. Alphaviruses, such as Sindbis virus (SINV) and Semliki Forest virus (SFV), represent some of the simplest enveloped viruses and have been well characterized by structural, genetic and biochemical techniques. Although a high-resolution structure of an alphavirus has not yet been attained, cryo-electron microscopy (cryo-EM) has been used to show the multilayer organization at 25 A resolution. In addition, atomic resolution studies are available of the C-terminal domain of the nucleocapsid protein and this has been modeled into the cryo-EM density. RESULTS: A recombinant form of Sindbis virus core protein (SCP) was crystallized and found to diffract much better than protein extracted from the virus (2.0 A versus 3.0 A resolution). The new structure showed that amino acids 108 to 111 bind to a specific hydrophobic pocket in neighboring molecules. Re-examination of the structures derived from virus-extracted protein also showed this 'N-terminal arm' binding to the same hydrophobic pocked in adjacent molecules. It is proposed that the binding of these capsid residues into the hydrophobic pocket of SCP mimics the binding of E2 (one of two glycoproteins that penetrate the lipid bilayer of the viral envelope) C-terminal residues in the pocket. Mutational studies of capsid residues 108 and 110 confirm their role in capsid assembly. CONCLUSIONS: Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

Organization of the Sindbis virus nucleocapsid as revealed by bifunctional cross-linking agents.

Purified Sindbis virus nucleocapsids were reacted with a variety of bifunctional protein-specific cross-linking agents. The products were analyzed in concentration-gradient polyacrylamide gels and amounts of various products determined. These studies indicated that available lysine residues within adjacent capsid proteins in purified intact nucleocapsids are separated by 6 A. The capsid proteins in intact nucleocapsids are cross-linked in a pattern predicted for discrete monomeric entities, rather than in dimeric or trimeric aggregates. Purified, soluble capsid protein exists in a conformation that differs from the arrangement of protein within nucleocapsids. These conformational differences suggest that topological changes may occur in the capsid protein during virus maturation. Cross-linked nucleocapsids that were treated with RNases resulted in the generation of RNA-free protein shells that retained hexagonal morphology, indicating that, together, the RNA and protein form the outer surface of the nucleocapsid. These data are used to produce a model of the Sindbis virus nucleocapsid in which the proteins are arranged quasi-equivalently in a T = 4 icosahedral shell.

Effects of coupling methods on galvanic corrosion behavior of commercially pure titanium with dental precious alloys.

BACKGROUND: When a dissimilar couple is exposed to corrosive environment, it will normally exhibit a galvanic corrosion. The galvanic corrosion might be influenced by various factors, including type and concentration of electrolyte, surface area ratio between anode and cathode, type of coupling material, and coupling manner. PURPOSE: The purpose of this study was to investigate and compare the galvanic corrosion behavior of commercially pure titanium when coupled with type IV Au alloy, Au-Ag-Pt alloy, and Ag-Au-Pd alloy by different coupling methods. MATERIALS AND METHODS: Couples were prepared by a laser welding or a mechanical adhering method. Electrochemical corrosion studies were conducted in a Ringer's solution at a scanning rate of 0.1 mV/sec in a range from -250 mV to +250 mV with respect to E(OCP). Corrosion parameters (E(OCP), I(CORR), E(CORR)) were obtained. RESULTS: It was found that (i) there was a significant difference between LWC and AJC for three couples (p<0.05), (ii) the crevice line caused all three couples more corrosive than weld joint line, (iii) for both joint, it was found that type (IV) Au alloy exhibited discoloration to some extent. CONCLUSIONS: It is concluded that among the three couples with two different coupling methods, Ti/Ag-Au-Pd couple exhibited best corrosion resistance in a room temperature Ringer's solution.

Topological organization of Sindbis virus capsid protein in isolated nucleocapsids.

Sindbis virus nucleocapsids were isolated from mature virions by a two-step purification method. Detergent-treated virions were sedimented in sucrose gradients and the nucleocapsid peaks chromatographed on RNase-free Sephadex G-200. The purified nucleocapsids displayed several morphologies when examined in the electron microscope. These morphologies, and the results of double-angle shadowing, suggest that the core of this enveloped virus has the shape of a regular icosahedron with a triangulation number of 4. Peptide mapping of capsid protein obtained from nucleocapsids that had been radioiodinated by a variety of means, indicated that of the four tyrosine residues in the protein, only Tyr180 was exposed at the surface of the icosahedral structure. The other three residues were not exposed on the outer surface of the nucleocapsid shell, nor on the surface of capsid protein itself, implying that they were buried within the folded protein.

Evidence for a change in capsid morphology during Sindbis virus envelopment.

Sindbis virus nucleocapsids were isolated from intact virus particles and from the cytoplasms of infected cells. These two isolates of purified nucleocapsids differed from one another in ribonuclease sensitivity and in sedimentation velocity. These findings suggest that a conformational change takes place in the Sindbis virus nucleocapsid during envelopment.

Characterization of the infection of Aedes albopictus cell clones by Sindbis virus.

We have investigated the infection of Aedes albopictus (mosquito) cell clones by Sindbis virus. Variation in the multiplicity of infection (MOI) from ranges of 50-0.00005 pfu/cell was determined to have no effect on the progression of the infection to high acute phase titer, suggesting that intracellular factors alone are responsible for the restriction of virus production seen as the infection enters the persistent phase: While persistently infected (over 1 year post infection) cell clones are morphologically indistinct from uninfected cells, they do display a uniform 30% reduction in growth rate compared with uninfected cells of the same clone. Using flow cytometry-based DNA content analysis, we found that persistent Sindbis virus infection induces distinct cytological effects on these cells, including an increase in apoptosis and polyploidy in one clone and cell cycle phase effects in another. Finally, the observation that the number of cells in persistently infected cell cultures which are productively infected closely approximates the number of cells dying by apoptosis prompted us to investigate the role that cell death may play in the maintenance of the persistent infection. Persistently infected cell cultures which were artificially induced into apoptosis by short 45 degrees C heat treatments do not display increased Sindbis virus production. This result does not support the hypothesis that infection sensitivity induced by random apoptosis in persistently infected cell cultures is responsible for the long-term maintenance of the persistent infection.

The effect of monoclonal antibodies to calcitonin gene-related peptide (CGRP) on CGRP-induced vasodilatation in pig coronary artery rings.

1. The modification of the vasodilator effect of calcitonin gene-related peptide (CGRP) by a panel of monoclonal antibodies (MAbs), which map to discrete epitopes on the CGRP molecule, was investigated in pig coronary artery rings (PCA). The preparations were pre-constricted with acetylcholine (3 x 10(-7) M) and concentration-response curves to CGRP (2 x 10(-10)-2.56 x 10(-8) M) were obtained in the presence or absence of each MAb. 2. CGRP caused a concentration-dependent relaxation of PCAs which reached a maximum (98.2 +/- 4.8%, n = 25) at 1.28 x 10(-8) M and gave an EC50 of 3.8 +/- 0.8 x 10(-9) M. 3. Two MAbs which map to the N-terminal, CN1 and CRA3, did not affect the CGRP response whilst a third, CRA5, significantly inhibited its effect. 4. The C-terminal MAb, CRA2, did not modify the CGRP response whilst, in contrast, CB3 (C-terminal) potentiated its effect. A similar augmentation of the CGRP-induced vasodilatation was seen in the presence of the middle-region MAb, CRA8. 5. These results suggest that regional specific MAbs can modify the vasodilator effect of CGRP causing either inhibition (CRA5, N-terminal) or potentiation (CB3, C-terminal; CRA8, middle region).

Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue.

The filamentous fungus Fusarium lateritium is cyanide tolerant, due partly to the induction of the enzyme cyanide hydratase in the presence of cyanide. This enzyme catalyses the hydration of cyanide to formamide. The expression in Escherichia coli of a cDNA clone encoding cyanide hydratase is described. The cDNA cloned was expressed as a transcriptional fusion in the expression vector pKK233-2 and a high level of activity of cyanide hydratase was detected in E. coli. Site-directed mutagenesis of the cys-163 residue inactivated the enzyme.

Laser assisted soldering: effects of hydration on solder-tissue adhesion.

Wound stabilization is critical in early wound healing. Other than superficial skin wounds, most tissue repair is exposed to a hydrated environment postoperatively. To simulate the stability of laser-soldered tissue in a wet environment, we studied the effects of hydration on laser soldered rat dermis and baboon articular cartilage. In this in vitro study, we used a solder composed of human serum albumin, sodium hyaluronate, and Indocyanine Green. A 2 µL solder droplet was deposited on each tissue specimen and then the solder was irradiated with a scanning laser beam (808 nm and 27 W/cm2). After photocoagulation, each tissue specimen was cut into two halves dividing the solder. One half was reserved as control while the other half was soaked in saline for a designated period before fixation (1 h, 1, 2, and 7 days). All tissue specimens were prepared for scanning electron microscopy (SEM). SEM examinations revealed nonuniform coagulation across the solder thickness for most of the specimens, likely a result of the temperature gradient generated by laser heating. Closer to the laser beam, the uppermost region of the solder formed a dense coagulum. The solder aggregated into small globules in the region anterior to the solder-tissue interface. All cartilage specimens soaked in saline suffered coagulum detachment from tissue surface. We noted a high concentration of the protein globules in the detached coagulum. These globules were likely responsible for solder detachment from the cartilage surface. Solder adhered better to the dermis than to cartilage. The dermal layer of the skin, composed of collagen matrix, provided a better entrapment of the solder than the smooth surface of articular cartilage. Insufficient laser heating of solder formed protein globules. Unstable solder-tissue fusion was likely a result of these globules being detached from tissue substrate when the specimen was submerged in a hydrated environment. The solder-tissue bonding was compromised as a result of this phenomenon. © 1998 Society of Photo-Optical Instrumentation Engineers.

Validation of continuous thermodilution cardiac output in critically ill patients with analysis of systematic errors.

PURPOSE: Bolus thermodilution cardiac output (BCO) measurements are affected by variations in injectate volume, rate, and temperature. These variations are eliminated when CO is measured by a continuous automated thermal technique (CCO). Further, CCO eliminates the need for fluid boluses, reduces contamination risk, requires no operator, and provides a continuous CO trend. We prospectively evaluated CCO versus BCO in a population of critically ill adults with low, normal, and high CO states. We sought to discern any systematic effects of temperature fluctuations or signal-to-noise-ratios (SNR) on disparities between BCO and CCO measurements and also sought to assess the relative cost effectiveness of the CCO system. MATERIALS AND METHODS: Pulmonary artery catheterizations were performed in a convenience sample of 20 patients over 6 months. BCO data were obtained using a standardized protocol. Three bolus injections of 5% dextrose were given when each CO was within 10% of the median before averaging; otherwise five boluses were given, with the high and low values eliminated before averaging. Injectates were administered randomly through the respiratory cycle and at 1-minute intervals. CCO measurements were recorded from a Vigilance monitor pre and post BCO measurements, yielding an average CCO value. Also recorded were pre- and post-core temperatures and SNR during the first CCO measurement. Cost data included estimates of operator time for BCO determinations as well as costs of Intellicath (Baxter-Edwards, Irvine, CA) pulmonary artery catheters, Vigilance (Baxter-Edwards, Irvine, CA) monitors, conventional catheters, and injectates. RESULTS: Of the 20 patients, 15 were mechanically ventilated. A total of 306 paired CO values were obtained for analysis. CCO ranged from 2.5 to 14.4 L/min and BCO from 2.4 to 13.3 L/min. Absolute differences between CCO and BCO measurements increased with increasing CO, but percentage differences did not. Of the paired values, 77% were within 1 L/min of one another. Temperature instability and SNR independently had weak correlations with CCO/BCO disparities. The Vigilance system had a slightly higher net cost than conventional BCO, although no economical value was assigned to the clinical usefulness of continuous, as opposed to intermittent, CO monitoring. CONCLUSIONS: Continuous CO is a reliable and cost-effective alternative to bolus thermodilution CO for critically ill patients in low, normal, and high CO states.

Human tracheal mucin: a preliminary study of physicochemical properties and mucociliary transport.

Human tracheal secretions were collected from patients with chronic tracheostomies in the intensive care unit and through an endotracheal tube from pediatric surgical patients under general anesthesia. These secretions were dialyzed, fractionated on an agarose column, and lyophilized to recover the tracheal mucin fraction. Analysis of pooled mucin samples included determination of amino acids, carbohydrates, and sulfates. Intrinsic viscosity, microrheometry, and mucociliary transport studies were also performed. Structural and functional differences between several pools of specimens are cited with an emphasis on possible clinical correlations.