Flaxseed (linum usitatissimum) ethanolic extract affects WNT signalling pathway-associated molecules; β-catenin and DKK1 expressions, during osteoblast differentiation of SHED

WNT signalling is important in regulating developmental process including bone development. Additionally, WNT signalling also involves in lineage differentiation of mesenchymal stem cells (MSC), including osteogenic differentiation, through canonical WNT pathway. Flaxseed (linum usitatissimum) is a plant with many health benefits including promoting bone health. Our previous study demonstrated that Flaxseed ethanolic extract (FEE) reduced the osteoblast differentiation potential of stem cells from human exfoliated deciduous teeth (SHED), a type of MSC. Hence, we aimed to analyse the effect of FEE on WNT signalling pathway-associated molecules; β-catenin, and DKK1 expressions, during the osteoblast differentiation of SHED. SHED cultured in osteoblast induction media (OIM) was treated with FEE at 4 mg/ml. RNA extracted from cells cultured at day 7, 14 and 21 was subjected to reverse-transcriptase PCR for β-catenin, and DKK1 gene expression analysis. FEE at 4 mg/ml significantly reduced β-catenin and DKK1 expression of SHED at day 7 (0.5293 ± 0.01, 1.0792 ± 0.02 respectively, p < 0.01) but induced their expression at day 14 (0.7675 ± 0.05, 1.7176 ± 0.07 respectively, p < 0.01). The expression was later reduced at day 21 (0.2592 ± 0.01, 1.0653 ± 0.04 respectively, p < 0.01). Changes in the β-catenin, and DKK1 expressions levels at different time frame might explain how FEE reduced the osteoblast differentiation potential of SHED. Overall, FEE modulates the expressions of WNT signalling pathway-associated molecules: β-catenin, and DKK1, during the process of osteoblast differentiation of SHED which could possibly interrupt the process of osteogenesis in the current environment

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Mental health mobile apps during Covid-19 Pandemic to evaluate stress level in Selangor

The COVID-19 pandemic has impacted negatively on public mental health. As a result, monitoring the level of the population mental health is a priority during crises. This study aims to measure stress during the COVID-19 pandemic in Selangor. Cross-sectional study was done using SELANGKAH apps, where users are Selangor citizens. Data was collected from September 2021 until March 2022. This app was initially used as contact tracing and mental health modules (SEHAT) were added, consisting of a validated Perceived Stress Scale (PSS-10) questionnaire. Out of 42072 SEHAT users, 6411 people had completed the questionnaire. Majority were female (53.6%), Muslims (79.6%), had formal education up to secondary (49.0%), low income (89.9%), and young and middle- aged adults (59.7%). Majority have a moderate stress (66.8%), while 23.3% and 9.9% are low and high levels, respectively. High stress is significantly associated with females, high education, younger age groups, and low monthly income. Several factors could have contributed to this throughout the COVID-19 pandemic, such as online learning, uncertainty on study duration, financial constraints and limited social interactions. Moreover, as an effect of prolonged pandemic and MCO, a surge in the number of job terminations has also affected the source of income, which contributed to high levels of stress among the general population. The level of stress in Selangor was high during the pandemic as an effect of MCO

Advanced Therapy Medicinal Products for Eye Diseases: Goals and Challenges

The concept of advanced therapy medicinal products (ATMPs) encompasses novel kinds of medicines for human use that are based on genes, cells or tissues. These intend to offer not only regeneration, but complete functional recovery of diseased tissues and organs using different strategies. Gene therapy, cell therapy and tissue engineering are the main areas in which promising advanced therapies are emerging. The eye is a very complex organ whose main structures, the cornea and the retina, play a pivotal role in maintaining normal vision, as severe alterations in these tissues can lead to blindness. Ocular tissues are starting to benefit from ATMPs by fighting against the enormous complexity and devastating potential of many ocular diseases. However, developments arising from this field of work face important challenges related to vectors to deliver drugs and genetic material to target tissues, suitable biomaterials to prepare cell scaffolds and cell stemness, among others—not to mention the complicated legislation around ATMPs, the complexity in production and quality control and the absence of standardized protocols.The purpose of this Special Issue is to serve as an overview of the current progress in the application of cell and gene therapies, as well as tissue engineering to restore functionality in diseased ocular structures, and the challenges linked to reaching patients

Exploring the Multifaceted Roles of Glycosaminoglycans (GAGs) - New Advances and Further Challenges

Glycosaminoglycans are linear, anionic polysaccharides (GAGs) consisting of repeating disaccharides. GAGs are ubiquitously localized throughout the extracellular matrix (ECM) and to the cell membranes of cells in all tissues. They are either conjugated to protein cores in the form of proteoglycans, e.g., chondroitin/dermatan sulfate (CS/DS), heparin/heparan sulfate (Hep/HS) and keratan sulfate (KS), as well as non-sulfated hyaluronan (HA). By modulating biological signaling GAGs participate in the regulation of homeostasis and also participate in disease progression. The book, entitled “Exploring the multifaceted roles of glycosaminoglycans (GAGs)—new advances and further challenges”, features original research and review articles. These articles cover several GAG-related timely topics in structural biology and imaging; morphogenesis, cancer, and other disease therapy and drug developments; tissue engineering; and metabolic engineering. This book also includes an article illustrating how metabolic engineering can be used to create the novel chondroitin-like polysaccharide.A prerequisite for communicating in any discipline and across disciplines is familiarity with the appropriate terminology. Several nomenclature rules exist in the field of biochemistry. The historical description of GAGs follows IUPAC and IUB nomenclature. New structural depictions such as the structural nomenclature for glycan and their translation into machine-readable formats have opened the route for cross-references with popular bioinformatics resources and further connections with other exciting “omics” fields

Characterisation of novel matrix-binding interactions for latent transforming growth factor-β-binding protein-2 (LTBP-2), with emphasis on heparin and heparan sulphate proteoglycans.

Elastic fibres are important components of the extracellular matrices, being composed of an elastin core and fibrillin-microfibrils around the periphery. Elastic fibre formation is a complex developmentally regulated process whereby fibrillin-microfibrils act as templates for the deposition of elastin. Additional matrix macromolecules, including fibulin-4, fibulin-5 and as yet unidentified heparan sulphate proteoglycans (HSPGs), have also been identified as playing important roles in this process. Fibrillins-1, -2, -3 and latent transforming growth factor-β binding protein (LTBP)-1, -2, -3, -4, associated components of fibrillin-microfibrils, make up a superfamily of extracellular matrix proteins. Fibrillins and LTBPs share a high degree of structural similarity since they both have rod-like structures of tandem EGF-like 6-cysteine repeats interspersed with unique 8-cysteine motifs. LTBP-1, -3 and -4 covalently bind TGF-β and target and store the latent growth factor in the matrix. Unlike the other LTBPs, LTBP-2 does not bind latent TGF-β and its function is poorly understood. LTBP-2 has been shown to bind fibulin-5, an elastin-binding protein and through this interaction it may target tropoelastin-fibulin-5 complexes on to fibrillin-1-microfibrils during elastic fibre assembly. In order to understand more about the role of LTBP-2 in the assembly of elastic fibres and to identify other novel functions, this study involved screening for potential molecular interactions of LTBP-2 with other matrix components, particularly heparin/HSPGs. In elastic tissues HSPGs are found on cell surfaces as syndecans and glypicans and in basement membranes as perlecan. Full length human recombinant LTBP-2 (rLTBP-2) was expressed in 293 EBNA cells using a modified pCEP-4 vector and purified by nickel affinity chromatography. Upon validation of the purified protein using western blots, solid phase binding assays were used to screen for interaction between rLTBP-2 and heparin. Heparin serves as a useful model for heparan sulphate; due to the lack of adherence of heparin to microtitre plates heparin-BSA conjugate was synthesised and purified for the binding assays. Recombinant LTBP-2 was found to interact with heparin-BSA conjugates using an established solid phase binding assay. The binding was blocked by the addition of heparin (but not chondroitin sulphate) to the liquid phase, confirming the specificity of the interaction. Furthermore, the binding was blocked by the addition of 5mM EDTA and 5mM EGTA, showing that the interaction was cation (calcium) dependent. An apparent Kd of 14.5±3.7nM was calculated from non-linear regression analysis of the LTBP-2-heparin binding curve, indicating a strong affinity. To identify the location of the heparin binding site(s) on LTBP-2, expression constructs were produced encoding three fragments of LTBP-2, i.e. rLTBP-2NT(H), rLTBP-2C(H) and rLTBP-2CT(H), corresponding to the N-terminal, central and C-terminal regions of the molecule. Good yields of rLTBP-2C(H) were obtained using the pCEP4-293-EBNA system, and rLTBP-2CT(H) was previously expressed and purified by members of the Gibson laboratory. However, difficulties were encountered with the rLTBP-2NT(H) expression construct and no LTBP-2NT(H) was available during the candidature. The central fragment LTBP-2C(H), (but not the C-terminal fragment LTBP-2CT(H)), was found to bind heparin. However, the apparent Kd of 52.2±6.9nM was significantly higher than that for full length LTBP-2, indicating that LTBP-2C(H) had relatively lower heparin-binding affinity. This result suggested that an additional heparin binding site(s) is present in the N-terminal region of the molecule. It was considered that the true tissue ligand(s) for LTBP-2 would be a HSPG rather than heparin. Therefore, LTBP-2 was screened for interaction with HSPGs, recombinant syndecans-2 and -4, and endothelial cell-derived perlecan. Interestingly, LTBP-2 bound strongly to r-syndecan-4 but not r-syndecan-2 even though both molecules were produced in the same mammalian cell system and had been screened for binding to HS-binding growth factor, fibroblast growth factor-2. This finding indicates that LTBP-2 does not interact with all HS and must recognise specific microstructures within the heparan sulphate chains. It appears that syndecan-4 is now a strong candidate as mediator of LTBP-2-cell signalling. LTBP-2 was also found to specifically interact with perlecan in a cation-dependent, heparininhibitable manner. Confocal immunohistochemical studies using foetal human aorta showed that LTBP-2 and perlecan generally had distinct distribution patterns within the medial layer, located on fibrillin-microfibrils and basement membranes respectively. However, there were small but widespread regions of LTBP-2-perlecan colocalisation which showed a similar pattern to the fibrillin-1-perlecan colocalisation. Thus it would appear that LTBP-2 is present at microfibril-basement membrane interfaces and may be involved in stabilising the interaction between these two structural elements of the matrix. This concept needs to be confirmed at the ultrastructural level. The interaction of LTBP-2 with perlecan during embryonic development is also worthy of investigation. In parallel studies, a proteomic approach was used to identify other matrix binding proteins for LTBP-2. This was carried out in parallel with another matrix protein of poorly defined function, transforming growth factor-beta-inducible gene-h3 (βig-h3). Recombinant LTBP-2 and βig-h3 coupled to sepharose were used as bait proteins to screen complex mixtures of matrix proteins from the elastic tissue nuchal ligament and basement membrane preparation Matrigel. Initial studies showed that non-specific background binding to these proteins was a major problem. In efforts to overcome this difficulty, binding conditions were varied with limited success. A two-dimensional gel approach was used to fractionate and compare proteins binding to LTBP-2 with those binding to βig-h3. The differentially displayed protein spots were to be identified by mass spectrometry. However, complications in comparing the patterns on two separate gels made identification of candidate spots challenging. Finally, CyDye DIGE fluor dyes were used to fractionate proteins binding to LTBP-2 and βig-h3 on the same gel, but unfortunately this work could not be completed in the time frame of the candidature.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences School, 201