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Casein is a naturally derived amino group (-NH2) rich protein, that enables surface functionalization leading to hydrophilicity, which in turn facilitates better cell adhesion. Casein obtained from either commercial β-casein rich skim milk (A2 milk) or dissolved air flotation (DAF) technology was tested for its potential for tissue engineering applications in a comparative study. A novel biodegradable biomaterial was synthesized from casein by chemically modifying with methacrylic anhydride (MA) and combined with polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blend. The resulting methacrylated casein (CasMA) with the two polymers was processed into porous scaffolds with low and high MA concentrations to demonstrate CasMA's ease of modification and reproducibility. Fourier Transform Infrared Microscopy (FTIR) and Proton Nuclear Magnetic Resonance (1H NMR) revealed the presence of all the components and the successful modification of casein. The rheological and morphological analysis presented viscous behaviour and columnar hollow tube-like microstructures in agreement with the biomaterials' swelling and biodegradation behaviour. The live/dead in vitro assay showed high cell viability that agreed with the cell proliferation (MTT) assay in vitro, which indicated increased proliferation upon casein modification at appropriate biomaterial concentrations and volumes. This study not only showed a possible mechanism of casein methacrylation but also presented the potential use of waste materials like DAF-casein as a value-added product for tissue engineering applications.

Dental pathologies such as caries is one of the most prevalent diseases worldwide. Dental pulp contains stem cells capable of regenerating the dentine in the tooth, consequently, healthy dental pulp is essential for long term tooth survival. The aim of this study was to incorporate a variety of polymers that provide strength, an antibacterial substance and a protein-based polymer to provide cell support. These components were combined into a triphasic hybrid dental biocomposite (3HB), that together could provide regenerative properties for the pulp tissue. The 3HB biocomposite was incorporated into Organic–inorganic nanostructured materials such as Mineral Trioxide Aggregate (MTA) as a base to assemble a hybrid dental biocomposite. The effects of the 3HB on cytotoxicity was examined in mouse dental pulp cells, MDPC-23. In vitro studies showed that 3HB supported the proliferative growth of the cells significantly more than the no treatment control. 3HB also caused little stress to the cells and supported cell viability. Fourier transform infrared (FTIR) spectra confirmed the presence of polymer functional groups within the 3HB biocomposite. Therefore, 3HB compound has the potential to be applied as a pulp wound dressing providing superior cytocompatibility than the present options but also may be indispensable for the regeneration of dental pulp.

Wound healing is a complex process that is critical in restoring the skin's barrier function. This process can be interrupted by numerous diseases resulting in chronic wounds that represent a major medical burden. Such wounds fail to follow the stages of healing and are often complicated by a pro-inflammatory milieu attributed to increased proteinases, hypoxia, and bacterial accumulation. The comprehensive treatment of chronic wounds is still regarded as a significant unmet medical need due to the complex symptoms caused by the metabolic disorder of the wound microenvironment. As a result, several advanced medical devices, such as wound dressings, wearable wound monitors, negative pressure wound therapy devices, and surgical sutures, have been developed to correct the chronic wound environment and achieve skin tissue regeneration. Most medical devices encompass a wide range of products containing natural (e.g., chitosan, keratin, casein, collagen, hyaluronic acid, alginate, and silk fibroin) and synthetic (e.g., polyvinyl alcohol, polyethylene glycol, poly[lactic-co-glycolic acid], polycaprolactone, polylactic acid) polymers, as well as bioactive molecules (e.g., chemical drugs, silver, growth factors, stem cells, and plant compounds). This review addresses these medical devices with a focus on biomaterials and applications, aiming to deliver a critical theoretical reference for further research on chronic wound healing.

Skeletal muscle has the remarkable capability to regenerate itself following injury. Adult myogenic stem cells (MSCs) are responsible for the repair and regeneration, and their activity is controlled by intrinsic and extrinsic factors. The aim of this study was to examine and compare the expression levels of Pax3, Pax7, MRF and p38 proteins during the course of regeneration and in different areas of the focal freeze‐lesion damaged adult rat TA muscle. Using the focal freeze injury model, immunohistochemistry, laser‐capture micro‐dissection and Western blot analysis were performed. The results show that (1) in the severely damaged area, the focal freeze‐lesion injury significantly activated Pax7 and myogenin expression within 7 days and down‐regulated Pax3, MyoD and Myf‐5 within 1 or 3 days, and (2) the level of the p38 protein was strongly and transiently up‐regulated in the whole muscle on day 7 following injury, whereas the level of the pp38 protein was down‐regulated within 3 days in the severely damaged and non‐damaged areas. These findings indicate that the temporal (e.g. the time course of regeneration) and spatial (e.g. three zones created by the focal freeze‐lesion) cues in a regenerating muscle have a significant impact on the activity of the adult MSCs.

BackgroundSevere acute respiratory syndrome coronaviruses -2 (SARS-COV2) named as COVID-19 had spread worldwide and leading to 1,210,956 confirmed cases and 67,594 deathsMethodsA data of 1192 confirmed cases and 43 deaths due to COVID-19 in Japan collected from the Ministry of Health, Labour and Welfare of Japan and analysed for different epidemiological parameters and their clinical manifestations. We used Clauset-Newman-Moore (CNM) clustering algorithm to develop web-network of confirmed cases to identified clusters of community transmission.ResultsOut of 1192 confirmed cases, 90.60% were symptomatic and 9.39% were asymptomatic. The prevalence of COVID19 in males was 56.29% and 43.20 % in females. The mean interval (±SD) from symptom onset to diagnosis was 6±22.6 days while mean interval (±SD) from contact to onset of symptoms was 5±19.5 days. People of age range 40-79 were more infected and deaths median age was 80. The main symptoms were fever, dry cough, fatigue and pneumonia. The main infected cities were Tokyo (195/1192, 16.35%), Hokkaido (160/1192 13.42%), Aichi (150/1192, 12.58%) and Osaka (145/1192, 12.16%). Only 2.34% cases had travel history from Wuhan China and Osaka music concert was identify as main cluster for community transmission. While 556 (46.64%) cases were clinically diagnosed and 557 (46.72%) were confirmed by using RT-PCR.ConclusionsOther than, declare emergency Japan need to change their approach of diagnosing COVID-19, as asymptomatic cases prevalence is high and maybe it is reason for current sudden increase of cases. Screening centre should be establish away from hospitals, which are treating positive cases.

ObjectiveThe Coronavirus (COVID-19) has advanced into 197 countries and territories leaving behind a total of 372,757 confirmed cases and 16231 deaths.MethodsOne the basis of WHO situation reports data of COVID-19 along with daily official reports from the Japan, China and the Kore we modelled the spread of COVID19 by using the Successive Approximation Method. We defined the two state of data to find the mean ratio (η) of the present cases count to the sum of previous and present cases. This ratio further predicts the future state of COVID-19 pandemic.ResultsThe mean ratio (η) of expected cases were found 0.485, while the mean ratio for deaths was found to be 0.49. We calculated worldwide expected lower bound value for confirmed cases 247007 cases with maximum limit of 1667719 cases and minimum deaths count 8660 with upper limit of 117397 deaths in next 30 days. While in the case of Iran, a large increase in the number of deaths are expected in the upcoming 30 days with lower bound value of 1140 deaths and maximum value of 598478 deaths.InterpretationIran whole population is on risk.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location. Agency for Science, Technology and Research (A*STAR) Published version We thank the Cancer Society of New Zealand (11/07), the Maurice Wilkins Centre of New Zealand and the Dean’s Bequest Fund (Dunedin School of Medicine) for their funding support of this project. We also acknowledge the Health Research Council (HRC) of New Zealand and the Royal Society James Cook Fellowship for their ongoing support of Antony Braithwaite and his lab group. The Westmead Institute of Medical Research (WIMR) Flow Cytometry Centre, supported by the Australian NHMRC and the Cancer Institute NSW are thanked for cell sorting. Anthony Cesare and his lab are supported by grants from the NHMRC (1053195, 1106241). Srinivasaraghavan Kannan and Chandra Verma would like to thank A*STAR and National Supercomputer Centre Singapore for support. Srinivasaraghavan Kannan and Chandra Verma are founders/scientific consultants of Sinopsee Therapeutics, a biotechnology company developing molecules for therapeutic purposes; the current work does not conflict with the company.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, &beta, actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

The First Industrial Revolution began when manual labour transitioned to machines. Fossil fuels and steam eventually replaced wood and water as an energy source used predominantly for the mechanized production of textiles and iron. The emergence of the required numerous enormous factories gave rise to smoke pollution due to the immense growth in coal consumption. The manufactured gas industry produced highly toxic effluent that was released into sewers and rivers polluting the water. Many pieces of legislation were introduced to overcome this issue, but with varying degrees of effectiveness. Alongside our growth in world population, the problems that we had with waste remained, but together with our increase in number the waste produced has also increased additionally. The immense volume of waste materials generated from human activity and the potentially detrimental effects on the environment and on public health have awakened in ourselves a critical need to embrace current scientific methods for the safe disposal of wastes. We are informed daily that our food waste must be better utilized to ensure enough food is available to feed the world's growing population in a sustainable way (Thyberg and Tonjes, 2016). Some things are easy, like waste food and cellulose products can be turned into compost, but how do we recycle sheep’s wool? Or shrimp shells? Despite the fact that both these substances are hazardous, and have caused environmental and economic impact from being incinerated; but we anticipate that those substances may have the potential to convert into added value applications.We have been working in this area for over 15 years, working towards managing them and seeking their added value applications. We take the biological products, process (reconstitute) and engineer them into added value products such as functional and nanostructure materials including edible films, foams and composites including medical devices useful in the human body. Anything that we can ingest, should not cause an immune response in the human system. Natural biomacromolecules display the inherent ability to perform very specific chemical, mechanical or structural roles. Specifically, protein- and polysaccharide-based biomaterials have come to light as the most promising candidates for many biomedical applications due their biomimetic and nanostructured arrangements, their multi-functional features, and their capability to function as matrices that are capable of facilitating cell-cell and cell-matrix interactions.

A xenograft (bovine hydroxyapatite [BHA]) was developed from New Zealand sourced bovine cancellous bone by a successful defatting and deproteinizing procedure. The BHA was chemically, compositionally and structurally characterized. Fourier transform infrared spectroscopy confirmed the removal of organic matter from the bone matrix and the presence of carbonate ( CO32-), hydroxyl (OH- ), and phosphate ( PO43-) functional groups. X-ray diffraction analysis suggested that the processed bone corresponds characteristically to hydroxyapatite (HA). SEM analysis showed that the BHA has an interconnected porous architecture with a pore diameter ranging from 100 to 700 μm while µCT analysis calculated the total porosity as 73.46% ± 1.08. Furthermore, the BHA was stable up to 1000°C and lost only 1.8% of its weight. The Ca/P molar ratio of the BHA was 1.58, which is comparable with commercially available natural HA-Endobon® . After 28 days of incubation in simulated body fluid (SBF), the pH value only fluctuated between 7.1 and 7.5 and the BHA scaffold did not degrade significantly by weight indicating the scaffold had excellent chemical and structural stability. In vitro studies showed the BHA was cytocompatible and supported the proliferative growth of Saos-2 osteoblast cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1054-1062, 2017.

Diclofenac potassium loaded sutures based upon PEG/PCL/chitosan-keratin blends were fabricated using the hot-melt extrusion technique. Polymer sutures were evaluated based on their physical, thermal and mechanical properties, while the drug-eluting sutures were evaluated for drug release properties. Lastly, the performance of the drug-loaded sutures in the contact with the human keratinocyte cell line HaCat were assessed. Results showed that the sutures extruded homogeneously at a temperature of 63 ± 1 °C providing a uniform thickness of fibres. Analysis by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) showed that completely amorphous and miscible solid dispersions were created. Fourier transform infrared (FTIR) spectroscopy indicated that the presence of hydrogen bonds between the polymers improved material miscibility. Tensile properties of the sutures were clearly affected by the PEG, chitosan and keratin additions. The optimal formulation of tensile strength was obtained when PCL/PEG/chitosan-keratin were combined at a ratio of 80/19/1 w/w. Rapid and sustained drug release rates were achieved with the PEG/PCL/chitosan/keratin blends at various combinations. The composite of PCL/PEG/chitosan-keratin with 30 wt% of diclofenac potassium also exhibited high cell viability and wound healing rates in vitro cytotoxicity testing. The anti-inflammatory properties imparted by the PCL/PEG/chitosan/keratin/drug sutures may further the use of composite sutures for wound healing in clinical settings.

A bovine dentine derived hydroxyapatite (BDHA) bone graft was developed from waste bovine teeth using a successful defatting and deproteinizing procedure. The resulting BDHA material was characterised using several analytical techniques to investigate its chemical, physical, thermal and structural properties. Fourier-transform-infrared-spectroscopy spectra confirmed the presence of carbonate, hydroxyl and phosphate functional groups which is associated with hydroxyapatite. X-ray diffraction analysis suggested that the processed BDHA corresponds characteristically to hydroxyapatite. Scanning electron microscopy showed the presence of dentinal tubules with an interconnected porous architecture in conjunction with a pore diameter of ∼3-4 μm. Thermogravimetric analysis showed that the BDHA was stable up to 1000 °C and lost only 1.5% of its weight. Both Inductively-coupled-plasma-mass spectrometry and Energy-dispersive-X-ray analysis revealed that the inorganic phases of BDHA were mainly composed of calcium and phosphorous with trace amounts of sodium and magnesium. The BDHA was chemically stable and did not degrade significantly after 28 days of incubation in simulated body fluid (SBF). The BDHA material showed excellent mechanical properties in terms of yield strength and young’s modulus. In-vitro studies showed that the BDHA was non-toxic, supported the proliferative growth of Saos-2 cells and expressed the bone marker “osteonectin” after 14 days.

Calcium phosphate ceramics that mimic bone composition provide interesting possibilities for the advancement in bone tissue engineering. The present study reports on a chitosan composite reinforced by hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) obtained from waste mussel shells and cross-linked using tripolyphosphate (TPP). The ratios of the ceramic components in composites were 20/10/70, 30/20/50 and 40/30/30 (HA/β-TCP/CH, w/w %). Biodegradation rate, structural properties and in-vitro degradation of the bone-like composite scaffolds were investigated. The optimum amount of TPP required for composite was 2.5% and glycerol was used as plasticizer at an optimized concentration of 1%. Tripolyphosphate cross-linked chitosan composites were developed by freezing and lyophilisation. The Young's modulus of the scaffolds was increased from 4kPa to 17kPa and the porosity of composites dropped from 85 to 68% by increasing the HA/β-TCP ratio. After 28days in physiological solution, bone-like composite scaffolds with a higher ratio of HA/β-TCP (e.g. 40/30/30) showed about 2% lower biodegradation in comparison to scaffolds with a lower ratio of HA/β-TCP (i.e. 20/10/70). The obtained data suggest that the chitosan based bone-like composites could be potential candidates for biomedical applications.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli ( Lactobacillus -free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus -free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1 , angptl4 , and hspa1b , associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus -free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.

In normal prostate cells, cell membrane receptors are located within signalling microdomains called caveolae. During cancer progression, caveolae are lost and sequestered receptors move out onto lipid rafts. The aim of this study was to investigate whether a change in the localisation of receptors out of caveolae and onto the cell membrane increased cell proliferation invitro, and to determine whether this is related to changes in the cell signalling pathways. Normal human prostate epithelial cells (PrEC) and androgen-independent (PC3) cancer cells were cultured with 10nM dihydrotestosterone (DHT). The effects of oxytocin (OT) and gonadal steroids on proliferation were assessed using the MTS assay. Androgen receptor (AR) and oxytocin receptor (OTR) expression was identified by immunofluorescence and quantified by western blot. OTR and lipid raft staining was determined using Pearson’s correlation coefficient. Protein–protein interactions were detected and the cell signalling pathways identified. Treatment with OT did not affect the proliferation of PrEC. In PC3 cells, OT or androgen alone increased cell proliferation, but together had no effect. In normal cells, OTR localised to the membrane and AR localised to the nucleus, whereas in malignant cells both OTR and AR were identified in the cell membrane. Colocalisation of OTR and AR increased following treatment with androgens. Significantly fewer OTR/AR protein–protein interactions were seen in PrEC. With OT treatment, several cell signalling pathways were activated. Movement of OTR out of caveolae onto lipid rafts is accompanied by activation of alternative signal transduction pathways involved in stimulating increased cell proliferation.

BACKGROUND Caveolae are specialized invaginations in the cell membrane involved in the regulation of cell transport and signal transduction. The aims of this study were to investigate the number of caveolae and expression of caveolae-associated proteins, caveolin-1 and -2, and polymerase 1 and transcript release factor (PTRF) with development of prostate cancer. METHODS Transmission electron microscopy was used to investigate the number of caveolae in normal human prostate stromal, epithelial cells, and androgen-dependent (LNCaP) and androgen-independent (PC3) cancer cell lines. Surgical tissue was obtained from patients with benign prostatic hyperplasia (BPH), well-differentiated and poorly differentiated prostate cancer. Caveolin-1, caveolin-2, and PTRF expression was identified by immunohistochemistry in tissue samples and quantified by Western blot analysis in cell lines. RESULTS Caveolae were identified in normal epithelial and stromal prostate cells. The number of caveolae was significantly reduced in LNCaP and PC3 cells (P

There are significant problems in obtaining normal human material for histology for teaching or research purposes. This study shows that tissue from cadavers embalmed for teaching can be used for routine histology. Twelve cadavers embalmed with four different formalin-containing embalming fluids were used (n = 3 per fluid): (1) formalin mix (10% formalin); (2) Dunedin mix (an alcohol-based fluid containing phenol); (3) Michigan mix (a water-based fluid); and (4) phenoxyethanol mix (an alcohol-based fluid containing phenoxyethanol). Tissue blocks of liver, heart, kidney, skin and skeletal muscle were taken from each cadaver, paraffin embedded, sectioned and stained with haematoxylin and eosin (H & E), Periodic Acid Schiff (PAS), or Mallory trichrome (Malt). Each section was assigned an overall score based on the histological quality of the cellular components of the tissue. Sections were scored from 1 to 3 (1 = poor, 2 = satisfactory, 3 = good). Satisfactory sections were obtained from all cadavers except those embalmed with the Dunedin mix. The Michigan and phenoxyethanol fluids resulted in consistently good quality sections. No significant differences in tissue morphology were observed between the different stains. The clearest morphology was observed in the skin and skeletal muscle sections, and in tissues embalmed with fluids which do not contain phenol.

Squid pen chitosan was used in the fabrication of biocomposite scaffolds for bone tissue engineering. Hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) obtained from waste mussel shells were used as the calcium phosphate source. The composite was prepared using 2.5% tripolyphosphate (TPP) and 1% glycerol as a cross-linker and plasticizer, respectively. The weight percent (wt.%) ratios of the ceramic components in the composite were 20/10/70, 30/20/50 and 40/30/30 (HA/β-TCP/Chi). The biodegradation rate and structural properties of the scaffolds were investigated. Scanning electron microscopy (SEM) and microCT(μCT) results indicated that the composites have a well defined lamellar structure with an average pore size of 200 μm. The porosity of the composites decreased from 88 to 56% by increasing the ratio of HA/β-TCP from 30 to 70%. After 28 days of incubation in a physiological solution, the scaffolds were degraded by approximately 30%. In vitro investigations showed that the composites were cytocompatible and supported the growth of L929 and Saos-2 cells. The obtained data suggests that the squid pen chitosan composites are potential candidates for bone regeneration.

Background and Aims: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. Methods: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post-mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin-embedded sections (3 µm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c-kit antibodies and mast cell tryptase was used to distinguish c-kit-positive cells with typical ICC morphology from c-kit-positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. Results: c-kit-positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. Conclusion: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.

Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor alpha (ER alpha) or beta (ER beta) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ER alpha knockout mice (alpha ERKO) and ER beta knockout mice (beta ERKO) and wild-type littermates (n=5-8) were killed at 11 weeks post partum. One testis was fixed in Bouin's fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in beta ERKO mice but not in alpha ERKO mice. Plasma and testicular testosterone concentrations were increased in alpha ERKO mice but no changes were observed in beta ERKO mice. Hypertrophic Leydig cell changes were observed in alpha ERKO mice, and a decreased mean cell volume was seen in beta ERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in beta ERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in alpha ERKO mice but not beta ERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in alpha ERKO mice. This study suggests that ER beta may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.

Oxytocin has been implicated in the regulation of prostate growth. However, the cellular localisation of oxytocin in the normal and diseased human prostate is not known. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were detected by immunohistochemistry in tissues from patients undergoing routine prostatectomy and in normal human prostate epithelial and stromal cell lines. Western blot analysis detected a single band at 14 kDa with neurophysin antiserum and a 66-kDa band with oxytocin receptor antiserum in epithelial and stromal cell lines. Similar sized bands were also detected in extracts of hyperplastic and adenocarcinomic prostate tissues. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were present in stromal and epithelial cell lines and in tissue from patients with benign prostatic hyperplasia. The peptides were localised predominantly to the epithelial cells, although discrete areas of stromal staining were also observed. There was a significant difference in the intensity of oxytocin-staining between tissue displaying benign prostatic hyperplasia and invasive carcinoma, with less immunoreactivity being present in the malignant epithelial cells. Thus, oxytocin and its neurophysin and receptor are present in epithelial and stromal cells of the human prostate. Oxytocin expression is reduced with tumour progression and may provide a marker for invasive disease.

The prostate of the brushtail possum undergoes growth and regression during the year. The present study investigated the morphological changes and expression of androgen and oestrogen receptors during the breeding and non-breeding seasons. Prostate tissue was collected from adult possums at 2-monthly intervals. The periurethral and outer glandular areas were separated and the volume of stromal, epithelial and luminal tissues measured in each area. Immunohistochemistry was used to investigate cell proliferation with proliferating cell nuclear antigen (PCNA) and to localise androgen receptor (AR) and oestrogen receptors α and β (ERα, ERβ). Seasonal changes in expression of the three receptors were investigated using quantitative PCR and western blot analysis. During the breeding season the volume of stromal tissue in the periurethral area and the luminal volume in the glandular area significantly increased. The change in periurethral volume was associated with increased PCNA-immunopositive cells. While the localisation of AR to the stromal and epithelial cells did not change, there was a significant increase in receptor expression before the main breeding season. ERα and ERβ expression and localisation did not alter during the year. Similarities in receptor expression and localisation suggest that the possum may be a suitable animal model for the study of human prostate growth.

Recent evidence suggests that oestrogen plays a physiological role in the testis. Both oestrogen receptor alpha and oestrogen receptor beta (ERb) are present in the testis and administration of oestrogen has been shown to inhibit the development of Sertoli, Leydig and germ cells. This study investigates the effect of ERb on the testis using ERb knockout mice (bERKO). Adult male bERKO mice (n=8) and their wild-type littermates (n=7) were killed at 11 weeks postpartum. One testis from each animal was fixed in Bouin’s fluid and embedded. Each testis was fractionated and thick sections cut and stained with PAS. The optical disector method was used to count the number of Leydig cells, Sertoli cells, spermatogonia, spermatocytes and spermatids in each testis. Trunk blood was collected and plasma testosterone concentrations measured by radioimmunoassay. No significant differences in body or testis weight were seen between the bERKO or wild-type mice. Similar numbers of Sertoli cells, spermatogonia, spermatocytes and spermatids were also observed between the two groups. The number of Leydig cells was significantly increased in bERKO mice compared with their wild-type littermates (P < 0.05). Despite the increased number of Leydig cells in the bERKO mice there was no significant difference in plasma testosterone concentrations in this group compared to the wild-type mice. Oestrogen has been reported to inhibit proliferation of adult-type Leydig cells and to inhibit steroidogenesis. This study suggests that the regulation of Leydig cell proliferation may be mediated by ERb. The presence of normal circulating testosterone concentrations in bERKO mice suggests that the effects of oestrogen on steroidogenesis are not brought about by ERbeta.