Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes

While fibroin isolated from the cocoons of domesticated silkworm Bombyx mori supports growth of human corneal limbal epithelial (HLE) cells, the mechanism of cell attachment remains unclear. In the present study we sought to enhance the attachment of HLE cells to membranes of Bombyx mori silk fibroin (BMSF) through surface functionalization with an arginine-glycine-aspartic acid (RGD)-containing peptide. Moreover, we have examined the response of HLE cells to BMSF when blended with the fibroin produced by a wild silkworm, Antheraea pernyi, which is known to contain RGD sequences within its primary structure. A procedure to isolate A. pernyi silk fibroin (APSF) from the cocoons was established, and blends of the two fibroins were prepared at five different BMSF/APSF ratios. In another experiment, BMSF surface was modified by binding chemically the GRGDSPC peptide using a water-soluble carbodiimide. Primary HLE were grown in the absence of serum on membranes made of BMSF, APSF, and their blends, as well as on RGD-modified BMSF. There was no statistically significant enhancing effect on the cell attachment due to the RGD presence. This suggests that the adhesion through RGD ligands may have a complex mechanism, and the investigated strategies are of limited value unless the factors contributing to this mechanism become better known.

Biomechanics of synthetic elastin : insights from Magnetic Resonance microimaging

We used Magnetic Resonance microimaging (μMRI) to study the compressive behaviour of synthetic elastin. Compression-induced changes in the elastin sample were quantified using longitudinal and transverse spin relaxation rates (R1 and R2, respectively). Spatially-resolved maps of each spin relaxation rate were obtained, allowing the heterogeneous texture of the sample to be observed with and without compression. Compression resulted in an increase of both the mean R1 and the mean R2, but most of this increase was due to sub-locations that exhibited relatively low R1 and R2 in the uncompressed state. This behaviour can be described by differential compression, where local domains in the hydrogel with a relatively low biopolymer content compress more than those with a relatively high biopolymer content.

Synthetic reevesite-like material as a visible light photocatalyst for the decontamination of water

A synthetic reevesite-like material has been shown to decolorize selected dyes and degrade phenolic contaminants photocatalytically in water when irradiated with visible light. This material can photoactively decolorize dyes such as bromophenol blue, bromocresol green, bromothymol blue, thymol blue and methyl orange in less than 15 min under visible light radiation in the absence of additional oxidizing agents. Conversely, phenolic compounds suc has phenol, p-chlorophenol and p-nitrophenol are photocat- alytically degraded in approximately 3hwith additional H2O2 when irradiated with visible light. These reactions offer potentially energy effective pathways for the removal of recalcitrant organic waste contaminants.

Effect of reactive bed mineralogy on arsenic retention and permeability of synthetic arsenic-containing acid mine drainage

Successive alkalinity producing systems (SAPSs) are widely used for treating acid mine drainage (AMD) and alleviating clogging commonly occurring in limestone systems due to an amorphous ferric precipitate. In this study, iron dust, bone char, micrite and their admixtures were used to treat arseniccontaining AMD. A particular interest was devoted to arsenic removal performance, mineralogical constraints on arsenic retention ability and permeability variation during column experiment for 140 days. The results showed that the sequence of the arsenic removal capacity was as follows: bone char > micrite > iron dust. The combination of 20% v/v iron dust and 80% v/v bone char/micrite columns can achieve better hydraulic conductivity and phosphorus-retention capacity than single micrite and bone char columns. The addition of iron dust created reductive environment and resulted in the transformation of coating material from colloidal phase to secondary mineral phase, such as green rust and phosphoerrite, which obviously ameliorates hydraulic conductivity of systems. The sequential extraction experiments indicated that the stable fractions of arsenic in columns were enhanced with help of iron dust compared to single bone char and micrite columns. A combination of iron dust and micrite/bone char represented a potential SAPS for treating As-containing AMD.

Poly(2-oxazoline) hydrogels for controlled fibroblast attachment

Currently there is a lack of choice when selecting synthetic materials with the cell-instructive properties demanded by modern biomaterials. The purpose of this study was to investigate the attachment of cells onto hydrogels prepared from poly(2-oxazoline)s selectively-functionalized with cell adhesion motifs. A water-soluble macromer based on the microwave-assisted cationic ring-opening polymerization of 2-methyl-2-oxazoline and 2-(dec-9-enyl)-2-oxazoline was functionalized with the peptide CRGDSG or controls using thiol-ene photochemistry followed by facile crosslinking in the presence of a dithiol crosslinker. The growth of human fibroblasts on the hydrogel surfaces was dictated by the structure and amount of incorporated peptide. Controls without any peptide showed resistance to cellular attachment. The benignity of the crosslinking conditions was demonstrated by the incorporation of fibroblasts within the hydrogels to produce three-dimensional cell-polymer constructs.

An exploratory study of the potential of resurfacing articular cartilage with synthetic phospholipids

This thesis is aimed at further understanding the uppermost lipid-filled membranous layer (i.e. surface amorphous layer (SAL)) of articular cartilage and to develop a scientific framework for re-introducing lipids onto the surface of lipid-depleted articular cartilage (i.e. "resurfacing"). The outcome will potentially contribute to knowledge that will facilitate the repair of the articular surface of cartilage where degradation is limited to the loss of the lipids of the SAL only. The surface amorphous layer is of utmost importance to the effective load-spreading, lubrication, and semipermeability (which controls its fluid management, nutrient transport and waste removal) of articular cartilage in the mammalian joints. However, because this uppermost layer of cartilage is often in contact during physiological function, it is prone to wear and tear, and thus, is the site for damage initiation that can lead to the early stages of joint condition like osteoarthritis, and related conditions that cause pain and discomfort leading to low quality of life in patients. It is therefore imperative to conduct a study which offers insight into remedying this problem. It is hypothesized that restoration (resurfacing) of the surface amorphous layer can be achieved by re-introducing synthetic surface-active phospholipids (SAPL) into the joint space. This hypothesis was tested in this thesis by exposing cartilage samples whose surface lipids had been depleted to individual and mixtures of synthetic saturated and unsaturated phospholipids. The surfaces of normal, delipidized, and relipidized samples of cartilage were characterized for their structural integrity and functionality using atomic force microscope (AFM), confocal microscope (COFM), Raman spectroscopy, magnetic resonance imaging (MRI) with image processing in the MATLAB® environment and mechanical loading experiments. The results from AFM imaging, confocal microscopy, and Raman spectroscopy revealed a successful deposition of new surface layer on delipidized cartilage when incubated in synthetic phospholipids. The relipidization resulted in a significant improvement in the surface nanostructure of the artificially degraded cartilage, with the complete SAPL mixture providing better outcomes in comparison to those created with the single SAPL components (palmitoyl-oleoyl-phosphatidylcholine, POPC and dipalmitoyl-phosphatidylcholine, DPPC). MRI analysis revealed that the surface created with the complete mixture of synthetic lipids was capable of providing semipermeability to the surface layer of the treated cartilage samples relative to the normal intact surface. Furthermore, deformation energy analysis revealed that the treated samples were capable of delivering the elastic properties required for load bearing and recovery of the tissue relative to the normal intact samples, with this capability closer between the normal and the samples incubated in the complete lipid mixture. In conclusion, this thesis has established that it is possible to deposit/create a potentially viable layer on the surface of cartilage following degradation/lipid loss through incubation in synthetic lipid solutions. However, further studies will be required to advance the ideas developed in this thesis, for the development of synthetic lipid-based injections/drugs for treatment of osteoarthritis and other related joint conditions.

Natural, engineered and synthetic inhibitors of kallikrein-related peptidases

There is a rapidly growing appreciation of the important physiological roles played by kallikreins and kallikrein-related peptidases (KLKs). Recent studies have revealed that these enzymes control key events in processes as diverse as inflammation and skin desquamation. Accordingly, there is considerable interest in developing tools to further dissect kallikrein activity, and a burgeoning effort aimed at producing lead inhibitors for therapeutic development. Indeed, several candidate inhibitors are already in clinical trials. This chapter surveys the naturally occurring kallikrein inhibitors, together with strategies for employing these molecules as bioscaffolds, as well as current progress in the development of small-molecule kallikrein inhibitors.

Proof of concept : A bioinformatic and serological screening method for identifying new peptide antigens for Chlamydia trachomatis related sequelae in women

This study aimed to identify new peptide antigens from Chlamydia (C.) trachomatis in a proof of concept approach which could be used to develop an epitope-based serological diagnostic for C. trachomatis related infertility in women. A bioinformatics analysis was conducted examining several immunodominant proteins from C. trachomatis to identify predicted immunoglobulin epitopes unique to C. trachomatis. A peptide array of these epitopes was screened against participant sera. The participants (all female) were categorized into the following cohorts based on their infection and gynecological history; acute (single treated infection with C. trachomatis), multiple (more than one C. trachomatis infection, all treated), sequelae (PID or tubal infertility with a history of C. trachomatis infection), and infertile (no history of C. trachomatis infection and no detected tubal damage). The bioinformatics strategy identified several promising epitopes. Participants who reacted positively in the peptide 11 ELISA were found to have an increased likelihood of being in the sequelae cohort compared to the infertile cohort with an odds ratio of 16.3 (95% c.i. 1.65 – 160), with 95% specificity and 46% sensitivity (0.19-0.74). The peptide 11 ELISA has the potential to be further developed as a screening tool for use during the early IVF work up and provides proof of concept that there may be further peptide antigens which could be identified using bioinformatics and screening approaches.

Production of transgenic rice with rice ragged stunt virus synthetic resistance genes

Rice ragged stunt virus (RRSV) is an important pathogen of rice affecting its cultivation in South and South East Asia. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV genome segments 7 and 10 (non-structural genes), and 5, 8 and 9 (structural genes) were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, Ubil, Actl and RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-mediated delivery of one or more of these PDR gene constructs. A large number of transgenic lines were produced from calli derived from mature or immature embryos, co-bombarded with the marker gene hph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Mendelian segregations were observed in transgenic progeny, especially with transgenic lines produced by biolistics. Preliminary tests conducted in China on selected transgenic lines indicate that plants with RRSV segment 5 antisense PDR gene confer RRSV resistance.

Effectiveness of an acellular synthetic matrix in the treatment of hard-to-heal leg ulcers

Hard-to-heal leg ulcers are a major cause of morbidity in the elderly population. Despite improvements in wound care, some wounds will not heal and they present a significant challenge for patients and health care providers. A multi-centre cohort study was conducted to evaluate the effectiveness and safety of a synthetic, extracellular matrix protein as an adjunct to standard care in the treatment of hard-to-heal venous or mixed leg ulcers. Primary effectiveness criteria were (i) reduction in wound size evaluated by percentage change in wound area and (ii) healing assessed by number of patients healed by end of the 12 week study. Pain reduction was assessed as a secondary effectiveness criteria using VAS. A total of 45 patients completed the study and no difference was observed between cohorts for treatment frequency. Healing was achieved in 35·6% and wound size decreased in 93·3% of patients. Median wound area percentage reduction was 70·8%. Over 50% of patients reported pain on first visit and 87·0% of these reported no pain at the end of the study. Median time to first reporting of no pain was 14 days after treatment initiation. The authors consider the extracellular synthetic matrix protein an effective and safe adjunct to standard care in the treatment of hard-to-heal leg ulcers.

DrsG from Streptococcus dysgalactiae subsp equisimilis inhibits the antimicrobial peptide LL- 37

SIC and DRS are related proteins present in only four of the more than 200 Streptococcus pyogenes emm-types. These proteins inhibit complement mediated lysis and/or the activity of certain antimicrobial peptides. A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp equisimilis (SDSE). Here we show that geographically dispersed isolates representing 14 of 50 emm-types examined possess variants of drsG. However not all isolates within the drsG-positive emm-types possess the gene. Sequence comparisons also reveal a high degree of conservation in different SDSE emm-types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatant. Unlike SIC, but similar to DRS, DrsG does not inhibit complement mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelcidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. The greatest similarity between DrsG and DRS/SIC is found in the signal sequence at the amino terminus and proline rich domains in the C-terminal half of the protein. Conservation of prolines in this latter region also suggests these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP inhibitory protein in SDSE. These results also suggest that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of complement inhibitory activity of SIC may reflect its continuing evolution.

Synthetic rating procedures for railway bridges

One of the most important parts of any Bridge Management System (BMS) is the condition assessment and rating of bridges. This paper, introduces a procedure for condition assessment, based on criticality and vulnerability analysis. According to this procedure, new rating equations are developed. The inventory data is used to determine the contribution of different critical factors such as environmental effects, flood, earthquake, wind, and vehicle impacts. The criticality of the components to live load and vulnerability of the components to the above critical factors are identified. Based on the criticality and the vulnerability of the components and criticality of factors, and by using the new rating equations, the condition assessment and the rating of the railway bridges and their components at the network level will be conducted. This method for the first time incorporates structural analysis, available knowledge of risk assessment in structural engineering standards, and the experience of structural engineers in a practical way to enhance the reliability of the condition assessment and rating a network of bridges.

Dosimetry of cone-defined stereotactic radiosurgery fields with a commercial synthetic diamond detector

Purpose Small field x-ray beam dosimetry is difficult due to a lack of lateral electronic equilibrium, source occlusion, high dose gradients and detector volume averaging. Currently there is no single definitive detector recommended for small field dosimetry. The objective of this work was to evaluate the performance of a new commercial synthetic diamond detector, namely the PTW 60019 microDiamond, for the dosimetry of small x-ray fields as used in stereotactic radiosurgery (SRS). Methods Small field sizes were defined by BrainLAB circular cones (4 – 30 mm diameter) on a Novalis Trilogy linear accelerator and using the 6 MV SRS x-ray beam mode for all measurements. Percentage depth doses were measured and compared to an IBA SFD and a PTW 60012 E diode. Cross profiles were measured and compared to an IBA SFD diode. Field factors, Ω_(Q_clin,Q_msr)^(f_clin,f_msr ), were calculated by Monte Carlo methods using BEAMnrc and correction factors, k_(Q_clin,Q_msr)^(f_clin,f_msr ), were derived for the PTW 60019 microDiamond detector. Results For the small fields of 4 to 30 mm diameter, there were dose differences in the PDDs of up to 1.5% when compared to an IBA SFD and PTW 60012 E diode detector. For the cross profile measurements the penumbra values varied, depending upon the orientation of the detector. The field factors, Ω_(Q_clin,Q_msr)^(f_clin,f_msr ), were calculated for these field diameters at a depth of 1.4 cm in water and they were within 2.7% of published values for a similar linear accelerator. The corrections factors, k_(Q_clin,Q_msr)^(f_clin,f_msr ), were derived for the PTW 60019 microDiamond detector. Conclusions We conclude that the new PTW 60019 microDiamond detector is generally suitable for relative dosimetry in small 6 MV SRS beams for a Novalis Trilogy linear equipped with circular cones.