Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Natural Preservation Technologies for Tropical Fruits

The project intends to establish a cross DEEDI industry-leveraged project for tropical fruit convenience products.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Strategic banana tissue culture industry development and biosecurity act

Application and development of activities based on in vitro technologies delivering research, industry development and biosecurity activities to sustain and improve the Australian banana industry.

Telomere dynamics in the Sydney rock oyster (Saccostrea glomerata): an investigation into the effects of age, tissue type, location and time of sampling.

Telomere length has been purported as a biomarker for age and could offer a non-lethal method for determining the age of wild-caught individuals. Molluscs, including oysters and abalone, are the basis of important fisheries globally and have been problematic to accurately age. To determine whether telomere length could provide an alternative means of ageing molluscs, we evaluated the relationship between telomere length and age using the commercially important Sydney rock oyster (Saccostrea glomerata). Telomere lengths were estimated from tissues of known age individuals from different age classes, locations and at different sampling times. Telomere length tended to decrease with age only in young oysters less than 18 months old, but no decrease was observed in older oysters aged 2-4 years. Regional and temporal differences in telomere attrition rates were also observed. The relationship between telomere length and age was weak, however, with individuals of identical age varying significantly in their telomere length making it an imprecise age biomarker in oysters.

Properties of intramuscular connective tissue in pork and poultry with reference to weakening of structure

The most common connective tissue research in meat science has been conducted on the properties of intramuscular connective tissue (IMCT) in connection with eating quality of meat. From the chemical and physical properties of meat, researchers have concluded that meat from animals younger than physiological maturity is the most tender. In pork and poultry, different challenges have been raised: the structure of cooked meat has weakened. In extreme cases raw porcine M. semimembranosus (SM) and in most turkey M. pectoralis superficialis (PS) can be peeled off in strips along the perimysium which surrounds the muscle fibre bundles (destructured meat), and when cooked, the slices disintegrate. Raw chicken meat is generally very soft and when cooked, it can even be mushy. The overall aim of this thesis was to study the thermal properties of IMCT in porcine SM in order to see if these properties were in association with destructured meat in pork and to characterise IMCT in poultry PS. First a 'baseline' study to characterise the thermal stability of IMCT in light coloured (SM and M. longissimus dorsi in pigs and PS in poultry) and dark coloured (M. infraspinatus in pigs and a combination of M. quadriceps femoris and M. iliotibialis lateralis in poultry) muscles was necessary. Thereafter, it was investigated whether the properties of muscle fibres differed in destructured and normal porcine muscles. Collagen content and also solubility of dark coloured muscles were higher than in light coloured muscles in pork and poultry. Collagen solubility was especially high in chicken muscles, approx. 30 %, in comparison to porcine and turkey muscles. However, collagen content and solubility were similar in destructured and normal porcine SM muscles. Thermal shrinkage of IMCT occurred at approximately 65 °C in pork and poultry. It occurred at lower temperature in light coloured muscles than in dark coloured muscles, although the difference was not always significant. The onset and peak temperatures of thermal shrinkage of IMCT were lower in destructured than in normal SM muscles, when the IMCT from SM muscles exhibiting ten lowest and ten highest ultimate pH values were investigated (onset: 59.4 °C vs. 60.7 °C, peak: 64.9 °C vs. 65.7 °C). As the destructured meat was paler than normal meat, the PSE (pale, soft, exudative) phenomenon could not be ruled out. The muscle fibre cross sectional area (CSA), the number of capillaries per muscle fibre CSA and per fibre and sarcomere length were similar in destructured and normal SM muscles. Drip loss was clearly higher in destructured than in normal SM muscles. In conclusion, collagen content and solubility and thermal shrinkage temperature vary between porcine and poultry muscles. One feature in the IMCT could not be directly associated with weakening of the meat structure. Poultry breast meat is very homogenous within the species.

Interactions between Mast Cells, Fibroblasts and Connective Tissue Components

It has long been recognized that mast cells occur throughout connective tissues. Histologic studies have revealed that such cells release their granules into the surrounding environment upon exposure to both immunologic and nonimmunologic stimuli. By microscopy these extracellular granules appeared to be phagocytosed by fibroblasts and by blood-borne phagocytic cells as they entered the site of mast cell degranulation. Such in vivo observations led to the suggestion that mast cells both altered connective tissue components and influenced fibroblast function through these discharged granules. Recent in vitro studies using cultured fibroblasts and isolated mast cells and mast cell granules have confirmed both these hypotheses. In addition, such studies have also documented that fibroblasts degrade ingested mast cell granules. Such studies document that a number of critical interactions may occur between mast cells and connective tissue components.

Monitoring healing progression and characterizing the mechanical environment in preclinical models for bone tissue engineering

The treatment of large segmental bone defects remains a significant clinical challenge. Due to limitations surrounding the use of bone grafts, tissue-engineered constructs for the repair of large bone defects could offer an alternative. Before translation of any newly developed tissue engineering (TE) approach to the clinic, efficacy of the treatment must be shown in a validated preclinical large animal model. Currently, biomechanical testing, histology, and microcomputed tomography are performed to assess the quality and quantity of the regenerated bone. However, in vivo monitoring of the progression of healing is seldom performed, which could reveal important information regarding time to restoration of mechanical function and acceleration of regeneration. Furthermore, since the mechanical environment is known to influence bone regeneration, and limb loading of the animals can poorly be controlled, characterizing activity and load history could provide the ability to explain variability in the acquired data sets and potentially outliers based on abnormal loading. Many approaches have been devised to monitor the progression of healing and characterize the mechanical environment in fracture healing studies. In this article, we review previous methods and share results of recent work of our group toward developing and implementing a comprehensive biomechanical monitoring system to study bone regeneration in preclinical TE studies.

Postharvest physiology and volatile production by flowers of Ptilotus nobilis

Ptilotus nobilis (Lindl.) F. Muell. has potential in the floriculture industries as a cut flower crop. Ethylene production and respiration rates, fresh weight changes and volatile scent production from cut inflorescences of P. nobilis cultivars Passion (dark pink flowers) and Purity (white-green flowers) were measured during vase life. Inflorescence weight loss was significant (P < 0.001) during vase life with wilting and colour loss being the primary reasons for loss of vase life. Inflorescences ready for the cut market stored and at 22 °C had vase lives of >12 d. Ethylene production by inflorescences was low to negligible. Treatment with silverthiosulphate (STS) and ethylene had no effects on vase life. Evidently, ethylene did not play a role in determining the postharvest longevity of cut P. nobilis flowers. Respiration rates of inflorescences were high at harvest (>700 mg CO2 kg−1 FW h−1) and declined gradually thereafter during vase life. Total volatile emissions followed a similar pattern. For Passion, respiration rates of immature florets were significantly greater (P = 0.02) than florets from other developmental stages while the calyx produced the most CO2. For Purity, respiration rates of florets of different maturities did not differ and the reproductive tissue produced the most CO2. Only fully opened mature florets with their stigma and anthers revealed, emitted significant quantities of volatiles (P < 0.001) and primarily from the calyx tissue for both cultivars. The individual volatiles differed somewhat for the two cultivars. However, both produced significant quantities of benzaldehyde, 3,5-dimethoxytoluene and benzyl alcohol. These compounds have previously been associated with desirable floral scent.

A transcriptomic analysis of the kidney tissue of Tra catfish (pangasianodon hypophthalmus) reared in saline condition: De novo assembly, annotation, SNP discovery

Pangasianodon hypophthalmus is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The current study using Ion Torrent technology generated EST resources from the kidney for Tra catfish reared at a salinity level of 9 ppt. We obtained 2,623,929 reads after trimming and processing with an average length of 104 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 29,940 contigs, and allowing identification of 5,710 putative genes when comppared with NCBI non-redundant database. A large number of single nucleotide polymorphisms (SNPs) were also detected. The sequence collection generated in our study represents the most comprehensive transcriptomic resource for P. hypophthalmus available to date.

Postharvest physiology and volatile production by flowers of Ptilotus nobilis

Ptilotus nobilis (Lindl.) F. Muell. has potential in the floriculture industries as a cut flower crop. Ethylene production and respiration rates, fresh weight changes and volatile scent production from cut inflorescences of P. nobilis cultivars Passion (dark pink flowers) and Purity (white-green flowers) were measured during vase life. Inflorescence weight loss was significant (P < 0.001) during vase life with wilting and colour loss being the primary reasons for loss of vase life. Inflorescences ready for the cut market stored and at 22 °C had vase lives of >12 d. Ethylene production by inflorescences was low to negligible. Treatment with silverthiosulphate (STS) and ethylene had no effects on vase life. Evidently, ethylene did not play a role in determining the postharvest longevity of cut P. nobilis flowers. Respiration rates of inflorescences were high at harvest (>700 mg CO2 kg−1 FW h−1) and declined gradually thereafter during vase life. Total volatile emissions followed a similar pattern. For Passion, respiration rates of immature florets were significantly greater (P = 0.02) than florets from other developmental stages while the calyx produced the most CO2. For Purity, respiration rates of florets of different maturities did not differ and the reproductive tissue produced the most CO2. Only fully opened mature florets with their stigma and anthers revealed, emitted significant quantities of volatiles (P < 0.001) and primarily from the calyx tissue for both cultivars. The individual volatiles differed somewhat for the two cultivars. However, both produced significant quantities of benzaldehyde, 3,5-dimethoxytoluene and benzyl alcohol. These compounds have previously been associated with desirable floral scent.

Benzyl isothiocyanate: maximising production in papaya tissue extracts

Abstract: Although mainly grown for its sweet flavoured fruit, papaya (Carica papaya) has also been used for pharmacological purposes for many years. The reasons for use are varied with one of the best known being its anti-fungal action. Benzyl isothiocyanate (BITC) is the constituent most often implicated in this activity. Isothiocyanates are formed when the enzyme myrosinase catalyses the hydrolysis of the non-bioactive glucosinolates. This occurs when cellular contents come into contact through chewing, cutting or during extraction processes in the laboratory. While this is common in Brassica vegetables, the glucosinolate-myrosinase system is rare in fruit, papaya being a notable exception. It contains benzyl glucosinolate (BG), the glucosinolate precursor of BITC, in significant quantities. Parameters that determine the amount of BITC formed are duration of hydrolysis, presence/absence of nitrile-specifier proteins and BG content of different cultivars and tissues. We experimented with differing BITC extraction solvents, with the intention of developing a low cost, natural anti-fungal extract based on under-utilised papaya tissues. The findings suggest that papaya seeds, particularly from quarter-ripe fruit, have the potential to produce the highest levels of BITC necessary. Furthermore, they compare well with the nitrile-specifier protein-containing garden cress seeds (Lepidium sativum). To utilise the papaya seeds as a BITC source, an organic solvent such as ethanol is required to extract the largely water-insoluble BITC from the hydrolysed papaya seed mixture.