Fibronectin adsorption studied using neutron reflectometry and complementary techniques.

In implantology it is known that fibronectin affects cell-substrate adhesion, consequently, the structure and composition of the initially adsorbed fibronectin layer to a large extent determines the biological response to a biomaterial implanted into the body. In this study we have used neutron reflectometry and quartz-crystal microbalance with dissipation to investigate the amount of fibronectin adsorbed, the layer density, thickness and structure of films adsorbed to polished silicon oxide surfaces. We have cultured MG63 osteoblast-like cells on surfaces coated and uncoated with fibronectin and monitored the cellular response to these surfaces. The results show that at fibronectin concentrations in the range 0.01 to 0.1 mg/ml a single highly hydrated layer of fibronectin approximately 40-50 Å in thickness adsorbs to a polished silicon oxide surface and is likely to correspond to one diffuse monolayer of fibronectin arranged side-on. Cells cultured on this fibronectin layer have dramatically different morphology and growth to those grown on bare surfaces. Using a model silicon oxide surface has enabled us to study the substrate/protein interface, together with the impact of a fibronectin layer on the cellular response using consistent experimental conditions across a unique set of experimental techniques.

Sustainable construction: composite use of tyres and ash in concrete.

An investigation was carried out to establish the physical, mechanical and chemical characteristics of a non-standard (unprocessed) pulverised fuel ash (PFA) and waste tyres from a former landfill site at the Power Station Hill near Church Village, South Wales, United Kingdom. Investigations are on-going to establish the suitability of the fly ash and/or tyres in road construction (embankment and pavement) and also in concrete to be used in the construction of the proposed highway. This paper reports on concrete-based construction where concrete blends (using various levels of PFA as partial replacement for Portland cement (PC), and shredded waste tyres (chips 15-20mm) as aggregate replacement) were subjected to unconfined compressive strength tests to establish performance, hence, optimising mix designs. Strength development up to 180 days for the concrete made with PC-PFA blends as binders (PC-PFA concrete), with and without aggregate replacement with tyre chips, is reported. The binary PC-PFA concrete does not have good early strength but tends to improve at longer curing periods. The low early strength observed means that PC-PFA concrete cannot be used for structures, hence, only as low to medium strength applications such as blinding, low-strength foundations, crash barriers, noise reduction barriers, cycle paths, footpaths and material for pipe bedding.

A CMOS neuroelectronic interface based on two-dimensional transistor arrays with monolithically-integrated circuitry.

The ability to monitor and to elicit neural activity with a high spatiotemporal resolution has grown essential for studying the functionality of neuronal networks. Although a variety of microelectrode arrays (MEAs) has been proposed, very few MEAs are integrated with signal-processing circuitry. As a result, the maximum number of electrodes is limited by routing complexity, and the signal-to-noise ratio is degraded by parasitics and noise interference. This paper presents a single-chip neuroelectronic interface integrating oxide-semiconductor field-effect transistors (OSFETs) with signal-processing circuitry. After the chip was fabricated with the standard complementary-metal-oxide-semiconductor (CMOS) process, polygates of specific transistors were etched at die-level to form OSFETs, while metal layers were retained to connect the OSFETs into two-dimensional arrays. The complete removal of polygates was confirmed by high-resolution image scanners, and the reliability of OSFETs was examined by measuring their electrical characteristics. Through a gate oxide of only 7nm thick, each OSFET can record and stimulate neural activity extracellularly by capacitive coupling. The capability of the full chip in neural recording and stimulation was further experimented using the well-characterised escape circuit of the crayfish. Experimental results indicate that the OSFET-based neuroelectronic interface can be used to study neuronal networks as faithfully as conventional electrophysiological tools. Moreover, the proposed simple, die-level fabrication process of the OSFETs underpins the development of various field-effect biosensors on a large scale with on-chip circuitry.

CMOS-micromachined, two-dimenisional transistor arrays for neural recording and stimulation.

In-plane microelectrode arrays have proven to be useful tools for studying the connectivities and the functions of neural tissues. However, seldom microelectrode arrays are monolithically-integrated with signal-processing circuits, without which the maximum number of electrodes is limited by the compromise with routing complexity and interferences. This paper proposes a CMOS-compatible, two-dimensional array of oxide-semiconductor field-effect transistors(OSFETs), capable of both recording and stimulating neuronal activities. The fabrication of the OSFETs not only requires simply die-level, post-CMOS micromachining process, but also retains metal layers for monolithic integration with signal-processing circuits. A CMOS microsystem containing the OSFET arrays and gain-programmable recording circuits has been fabricated and tested. The preliminary testing results are presented and discussed.

GIRK2 deficient mice. Evidence for hyperactivity and reduced anxiety.

G-protein activated inwardly rectifying potassium channel (GIRK2)-deficient (null mutant) mice were examined in three tests for anxiety: the elevated plus-maze, light/dark box and "canopy" test. In the elevated plus-maze test, GIRK2 null mutant mice spent a higher percentage of time in the open arms and showed a higher number of total entries. A short (6 days) period of social isolation decreased anxiety and also increased the total activity in GIRK2 mutant mice. However, the increase of total activity in GIRK2 null mutant mice was mostly due to an increase in the number of entries into the open arms. The behavior of the wild-type animals was not substantially changed after social isolation. In the light/dark box, GIRK2 homozygous (-/-) mice demonstrated a higher level of locomotion and a higher number of rearings in the light area. In the "canopy" test, GIRK2 mutant mice displayed an increased locomotion in the exposed area and a strong trend to decrease in the number of stretched attend postures (SAP) in the most secure "canopy" area. GIRK2 heterozygous (+/-) animals showed behavioral changes intermediate between wild-type and null mutants only in the elevated plus-maze test after social isolation. In all other tests, GIRK2 heterozygous (+/-) animals did not differ from wild-type mice. Taken together, this data demonstrates that GIRK2 null mutant mice have reduced anxiety with signs of hyperactivity. We suggest that the functional block of dopamine D3 receptors may be a reason for this phenotype.

Potassium channels as targets for ethanol: studies of G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2) null mutant mice.

G-Protein-coupled inwardly rectifying potassium channels (GIRKs) regulate synaptic transmission and neuronal firing rates. Selective enhancement of GIRK2 function by intoxicating concentrations of ethanol was recently shown for recombinant homomeric and heteromeric channels. We proposed that specific behavioral actions of ethanol are due to activation of GIRK channels and that these behaviors would be reduced or eliminated in GIRK2 null mutant ("knockout") mice. Three behavioral effects of ethanol were absent in mutant mice as compared with wild-type littermates: stimulation of home cage (habituated) motor activity, anxiolytic action in elevated-plus maze test, and handling-induced convulsions (HIC) after an acute injection of ethanol. In contrast to these reductions of ethanol action, mutant mice displayed greater ethanol-stimulated activity in peripheral regions of an open field. There were no differences between mutant and wild-type mice for ethanol-induced sleep time, acute functional tolerance, or HIC following chronic matched consumption of a liquid diet. Ethanol preference and consumption were equal for wild-type and mutant mice using the standard two-bottle choice test with alternation of the bottles. However, this test was complicated by the strong side preference of the mice. When ethanol was presented constantly in their favored location, the consumption of ethanol was substantially higher for mutant than for wild-type mice. In the absence of ethanol, GIRK2 knockout mice showed more motor activity, less anxiety, and higher HIC. These results provide evidence that GIRK2 channels mediate specific behaviors, including anxiety and convulsions, and may influence effects of ethanol on these behaviors.

Communicating outpatient perception to improve quality management.

The authors report a study of outpatients' perceptions of clinic waiting times in relation to their expectations and to waiting time norms. The waiting time norm for online registration was 10 minutes, and for blood sampling was 20 minutes. Satisfaction with the behavior of clinic nurses also is reported and analyzed.

Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices.

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.

Human dentin phosphophoryn nucleotide and amino acid sequence.

Dentin sialoprotein (DSP) and phosphophoryns (DPP) are major dentin-specific non-collagenous proteins and are synthesized by odontoblasts. DPP are extremely acidic, rich in aspartic acid and serine, possess a high affinity for calcium and collagen, and are believed to function in dentin mineralization. Whereas DSP and DPP are the products of a single gene in mouse and rat, an analogous human gene has not been described. Using RT-PCR based cloning strategies, we have cloned human DPP cDNA from immature molar root total RNA. The open reading frame of this human DPP cDNA comprises 2364 bp encoding 788 amino acids rich in serine (58%), aspartic acid (26%) and asparagine (9%). These are mostly arranged as (DSS)n (n = 1-16), DS and NSS motifs. The N-terminal sequence (DDP) matches that obtained from human DPP extracted from the roots of immature teeth. The core protein of this human DPP was calculated to have a molecular weight of 76,906 Da and a net charge of -206 with an isoelectric point of 2.65. Of the serine residues, 53% can potentially be phosphorylated by casein kinases I and II. Thus, this newly cloned human cDNA, which encodes a protein with characteristics similar to rat and mouse DPP, is identified as a human DPP.

Characterization and identification of a human dentin phosphophoryn.

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with "Stains-All." Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0-0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.

Pregnancy following in vitro fertilization and embryo transfer by microsurgical epididymal sperm aspiration from a patient with congenital absence of the vas deferens: a case report.

Congenital absence of the vas deferens (CAVD) has been considered a virtually untreatable cause of male infertility. Men with this condition have been shown on testicular biopsy to have adequate spermatogenesis, and are theoretically producing sperm capable of fertilizing an oocyte. Yet epididymal transit was thought to be essential for the maturation of spermatozoa and development of their fertilizing ability since the characteristics of sperm motility improve as the sperm passes through the cauda. However recent studies in man have shown that spermatozoa aspirated from the obstructed caput epididymis and ductuli efferentia are, in fact, capable of fertilization in vitro. Microsurgical epididymal sperm aspiration (MESA) from the proximal region (caput) of the epididymis, obtained 0.5 x 10(6) sperm per ml, following washing and direct swim-up. Twelve oocytes were inseminated and three embryos were generated for transfer. The patient conceived and delivered a healthy female baby weighting 2838 gm, on March 3, 1994. This is the first documentation in Taiwan of live birth resulting from MESA from a patient with CAVD combined with in vitro fertilization and embryo transfer.

The correlation of the embryo implantation rate with uterine arterial impedance in in vitro fertilization and embryo transfer.

The influence of uterine blood flow impedance on embryo implantation rate was investigated by transvaginal color Doppler sonography examination before embryo transfer. A total of 108 women undergoing in vitro fertilization (IVF) procedures and who had at least one good quality embryo for transfer to the uterus received Doppler evaluation before embryo transfer. Color flow imaging with blood flow waveform analysis from bilateral uterine arteries was obtained to calculate the mean pulsatility index (PI). The correlations between mean PI with the pregnancy rate and the embryo implantation rate (number of embryos implanted/number of embryos transferred) were analyzed. Patients were grouped according to the mean PI value, and the pregnancy rate and embryo implantation rate were 25% (5/20) and 10.7% (9/84), respectively, with a PI < 2.0 (n = 20); 27.5% (14/51) and 12.2% (12/109), respectively, with a PI = 2.00-2.49 (n = 51); 9.5% (2/21) and 3.5% (2/57), respectively, with a PI = 2.50-2.99 (n = 21); and 6.3% (1/16) and 4.3% (2/47), respectively, with a PI [symbol: see text] 3.0 (n = 16). There were no significant differences in either pregnancy rate or embryo implantation rate between the groups with mean PI values less than 2.00 and between 2.00 and 2.49. If a mean PI value of 2.50 was used as the cut-off value, both the pregnancy rate and embryo implantation rate were significantly higher in patients with a mean PI less than 2.50 (p < 0.05). The uterine arterial impedance measured by the Doppler sonographic examination is a non-invasive method for evaluating the endometrial response and a mean uterine PI value of 2.5 can be used as a cut-off value to identify optimal uterine receptivity before embryo transfer.

Effect of peritoneal fluid and serum from patients with endometriosis on mouse embryo in vitro development.

BACKGROUND: The adverse effects on early embryo development as caused by peritoneal fluid exudate and serum from endometriosis patients have been shown, but the underlying mechanism and clinical significance remain unknown. METHODS: Peritoneal fluid (PF) and serum (S) from patients with minimal to mild endometriosis (Group A, n = 12), moderate to severe endometriosis (Group B, n = 6), and others including tubal ligation and uterine myoma (Group C, controls n = 6) were obtained during laparoscopy. Two-cell mouse embryos were cultured at 37 degrees C in 5% CO2, 95% air with supplementation of 10%PF + 1%BSA, 10%S and 10%S + 10%PF in HTF medium. The percentage of progression to the blastocyst stage at 72 and 96 hours was observed and compared among the three groups. RESULTS: Serum and peritoneal fluid from infertile patients with moderate to severe endometriosis appeared to be embryotoxic to the in vitro development of two-cell mouse embryos, but no significant differences were found between minimal to mild endometriosis and group C patients. CONCLUSIONS: These data suggest that the production of embryotoxic factor(s) is related to the clinical stage, and may be derived from endometriotic implants. The correlation of the embryotoxic effect of the peritoneal fluid with that of the serum indicates that embryotoxic factor(s) may enter the systemic circulation and impede early embryogenesis in the reproductive tract. The nature and mechanism of this result demand further study.

Independent control of fiber development and nitrate reduction in cultured cotton ovules.

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA(3)) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.In vivo, where ovules received mostly reduced nitrogen and very little nitrate, they did not display appreciable nitrate reductase activity even when nitrate was forced into the ovary wall by transpiration. After initiation of culture, nitrate became freely available to ovules and their nitrate reductase activity increased rapidly. Treatment with ammonium, GA(3), IAA, or increased temperature had no effect upon this induction. It is concluded that ammonium, hormone, and temperature effects on fiber development are independent of the availability of reduced nitrogen as a general substrate for growth.