Purification and biodistribution of extracellular vesicles

Extracellular vesicles (EVs) are nano-sized vesicles that contain bioactive lipids, RNAs and proteins, which can be transferred to recipient cells. EVs are important for physiological as well as pathological processes, such as coagulation and immune homeostasis, aiding cancer metastasis and spread of infectious diseases. Owing to their relatively small size the purification of EVs is a challenge, hence we have established and optimised workflows consisting of ultrafiltration with subsequent size exclusion liquid chromatography (UFLC)( Paper I) and bind-elute combined with size exclusion (BE-SEC) columns (Paper III) for EV purification. UF-LC allowed for purification of biophysically intact EVs with better yield and purity compared to ultracentrifugation (UC), which is the gold standard purification method in the field. The biodistribution of UF-LC EVs was different compared to vesicles isolated using UC, despite having highly similar protein composition according to proteomics analysis. We found that UF-LC vesicles accumulated less in lung, possibly owing to their higher integrity. Indeed, fluorescence correlation spectroscopy and transmission electron microscopy indicated that the high gravitational forces in UC lead to aggregation and disruption of the vesicles. The BE-SEC method is a similar method to UF-LC, however protein impurities less than 700 kDa in size are bound in the interior of the beads, thus improving simple size-based exclusion. The BE-SEC method is scalable, produces samples with better purity than UC, displaying yields exceeding 70% and demonstrates a good reproducibility between samples. Moreover, vesicles purified by BE-SEC display the same EV surface markers as UC purified EVs, and CD63-eGFP positive vesicles are taken up in recipient cells to the same extent. In summary, the BE-SEC method is a reproducible and fast alternative to UF-LC for large media volumes. Reliable purification methods are important for the implementation of therapeutically active EVs, however knowledge regarding their eventual organotropism and biodistribution is equally important. Thus, in article II we evaluated the biodistribution of EVs specifically labeled with a near-infrared dye. The main sites of accumulation of exogenously injected EVs were liver, spleen and lungs. Biodistribution profile of EVs depended strongly on injection route, and to certain extent, on EV cell type source, as dendritic cell derived EVs exhibited a more pronounced uptake in spleen compared to the other cell sources tested. We further showed that small alterations of EV surface proteins could significantly affect biodistribution as well, since EVs equipped with a brain targeting peptide on their surface increased the uptake of targeted EVs in brain. This study highlights that the biodistribution of EVs follows other nano-sized particles with uptake mainly in liver. Administration route, cell source and a targeting peptide influence the distribution, however the overall distribution is unaltered with the highest signal originating from liver. To summarise, this thesis has resulted in improvements of the EV field by systematically enhancing EV isolation workflows to achieve greater sample purity and at the same time preserving EV biophysical characteristics. Furthermore, it has laid groundwork for studying in vivo effects of exogenous vesicles. Both these aspects are particularly important for understanding EV biology more clearly and with increased detail

Amalan pengurusan keselamatan bengkel kemahiran hidup (KHB) Sekolah Menengah Harian Daerah Batu Pahat

Kajian ini dijalankan untuk mengkaji sejauhmanakah persepsi pelajar terhadap amalan pengurusan keselamatan dari aspek keselamatan alatan dan bahan, diri dan rakan serta keselamatan am di bengkel Kemahiran Hidup sekolah menengah harian daerah Batu Pahat. Kajian berbentuk kuantitatif dilaksanakan di empat buah sekolah menengah harian daerah Batu Pahat iaitu SMK Datin Onn Jaafar, SMK Banang Jaya, SMK Dato Onn dan SMK Seri Gading. Sampel kajian terdiri daripada 350 orang pelajar tingkatan dua yang mengambil matapelajaran KHB dan data dikumpul menggunakan instrumen borang soal selidik. Dapatan kajian dianalisis menggunakan perisian Winsteps Version 3.69.1.11 dengan pendekatan Model Pengukuran Rasch sepenuhnya. Data dianalisis menggunakan teknik analisis diskriptif dan analisis inferensi iaitu ujian-t bagi menguji hipotesis kajian. Dapatan kajian menunjukkan bahawa amalan pengurusan keselamatan di bengkel diamalkan secara sederhana bagi ketiga-tiga aspek tersebut. Terdapat perbezaan yang signifikan di antara pelajar di bandar dan luar bandar iaitu t=-4.08 dan p=0.000 di mana nilai min ukuranya pula ialah -.19. Beberapa cadangan telah dikemukakan bagi penambahbaikkan seperti mengadakan kempen kesedaran dan menyediakan sudut standard operasi amalan pengurusan keselamatan agar pelajar sentiasa mematuhi peraturan semasa berada di bengkel Kemahiran Hidup

Future Community-Based Ecotourism (CBET) development

Ecotourism is an alternative form of tourism and is usually confused with natural and cultural tourism.CBET is fast becoming a popular biodiversity conservation tool that develops and benefits the local community. Based on the context of conservation theory and practice, Community-Based Ecotourism (CBET) is a form of community-based natural resource management. However, a sustainable CBET development through Community Capacity Building (CCB)programs is not something that it easily achievable. Local community’s capacity varies from one culture to another. It takes a high level of community participation, in order for it to come to a level where the community members themselves are motivated to participate and contribute to the development of the program. This fully qualitative research involved 15 respondents from the community of Kg. Selai, Bekok in Johor, Malaysia. The result show there are five factors that sustained the ecotourism development based for Orang Asli Community in Kg. Selai, namely, existing CBET development, past CBET development, local community participation in planning stage of tourism, local participation in implementation stage of tourism and participation in nature conservation

Waste to Valuable By-product: Palm Oil Mill Decanter Cake and its Ability to Remove Cd, Cu and Pb Ions

Palm oil mills generate about 4 - 5 tons of decanter cake for every 100 tons of palm fresh fruit bunch processed. Due to the high organic content, the decanter cake could be converted into adsorbent for the removal of metal ions from waste water. The decanter cake was first dried at 105 oC and then carbonized at various temperatures. The resulting carbonized decanter cake were tested for removing cadmium (II), copper (II), and lead (II) ions. Proximate analysis using thermogravimetry of decanter cake carbonized at 500 oC indicated that the adsorbent contained 4% moisture, 21% volatile,23% fixed carbon, and 52% ash. Adsorption test was carried out by mixing 1.0 g of the decanter cake in 100 mL aqueous solution of the various ions. The concentration of metal ions in the solutions used is in the range of 100 – 1000 mg/L. The results of adsorption studies indicated that the removal of metal ions was highest in the case of Pb when the carbonization temperature was 500 oC and 600 oC in the case of Cd and Cu. Maximum removal of the Cd, Cu and Pb were also observed to take place when the pH of the solution is in the range of 4 – 5. Langmuir and Freundlich isotherm models were used to fit the isotherm experimental data. The maximum uptakes of Cd, Cu and Pb onto the carbonized decanter cake in this study were estimated to be 24, 23, and 97 mg/g respectively. The ability of the carbonized decanter cake to remove the metal ions was found to be comparable to that of other adsorbents derived from agricultural waste

Test-retest reliability of knee kinematics measurement during gait with 3D motion analysis system

Restoration of gait symmetry following anterior cruciate ligament reconstruction (ACLR) is crucial to minimize the risk of joint degeneration. To achieve this, it is essential that the chosen measurement method can accurately assess knee kinematics and detect the changes in multi-planes of motion. However to date, limited study is available on repeatability of the multi-planes knee angle measurements particularly among male patients post ACLR. The purpose of this study was to assess the test-retest reliability of knee kinematic measurements using three-dimensional (3D) motion analysis system during gait in post ACLR patients. Eight patients with mean (SD) age 28.89 (4.0) years, 5.82(4.07) months post ACLR were recruited from a tertiary hospital of Kuala Lumpur. All patients undergone two sessions of knee joint angles measurement during gait at four hours interval, for the injured and the non-injured knees. Angles in the sagittal, frontal and transverse planes during initial contact phase of gait that derived from the two measurement sessions were compared.  The results showed high test-retest reliability of the measurement in sagittal and transverse planes; the intraclass correlation coefficient (ICC) was 0.97(95% CI: 0.84-0.99) and 0.96 (95% CI: 0.83-0.99) respectively, and moderate test-retest reliability for the measurements in frontal planes, with ICC 0.44 (95% CI: -0.32-0.86). The study findings suggest that multi-planes knee angle measurements during initial contact phase of gait could reliably be measured using a 3D motion analysis system. Further research may focus on knee kinematics measurements at other phases of gait. Keywords: biomechanics, reliability, walking, knee, anterior cruciate ligament reconstructio

Selective amplification of bacterial 16S rDNA sequence from clinical samples

Background: Conventional blood culture method is time consuming and less sensitive; when fastidious or un-culturalable organisms are involved. The use of PCR targeting the 16S rRNA allows detection of bacteria; however, these primers have ability to co-amplify human DNA. This Polymerase Chain Reaction (PCR) method is based on nucleic acid amplification test. Objective of the study: This study determined a method for selective amplification of bacterial DNA from clinical samples without co-amplification of human DNA. Materials and methods: Seventy one blood samples from clinically suspected cases of early onset neonatal sepsis were collected and analysed in parallel by culture and 16S rRNA amplification. DNA was extracted using commercial extraction QiAmp mini DNA kit and subjected to 16S rRNA amplification. The products were sequenced, analysed and compared with blood culture results. Positive and negative controls were used for extraction and amplification respectively. Results: Out of 71 samples analysed, 5 (7.0%) samples by blood culture were equally positive for 16S rRNA PCR; the PCR was also able to identified 16 (22.5%) more positive samples which blood culture could not identify, but only 1 (1.4%) sample was identified positive using blood culture while PCR identified it as negative. During the study, 7 (9.9%) samples were identified positive by conventional blood culture but later found to be contaminants. Conclusion: This study confirmed the presence of 16S rRNA among bacterial isolates and modification of PCR protocol with shorter denaturation temperature and time, leading to selective amplification of bacterial DNA. Therefore, there is need to carry-out this investigation on both culturable and unculturable specimens. Keywords:16SrRNA amplification; bacterial DNA; human DNA and Polymerase chai

Elements of Community Capacity Building (CCB)for CBET development

Ecotourism is an alternative form of tourism and is usually confused with natural and cultural tourism.CBET is fast becoming a popular biodiversity conservation tool that develops and benefits the local community. Based on the context of conservation theory and practice, Community-Based Ecotourism (CBET) is a form of community-based natural resource management. However, a sustainable CBET development through Community Capacity Building (CCB)programs is not something that it easily achievable. Local community’s capacity varies from one culture to another. It takes a high level of community participation, in order for it to come to a level where the community members themselves are motivated to participate and contribute to the development of the program. This fully qualitative research involved 15 respondents from the community of Kg. Selai, Bekok in Johor, Malaysia. The result show there are five factors that sustained the ecotourism development based for Orang Asli Community in Kg. Selai, namely, existing CBET development, past CBET development, local community participation in planning stage of tourism, local participation in implementation stage of tourism and participation in nature conservation

Genetic diversity and geographical differentiation of nipa (Nypa fruticans Wurmb.) populations in peninsular Malaysia based on AFLP

Nipa (Nypa fruticans Wurmb.) is a useful and widely distributed palm in Southeast Asia, but its habitats are declining due to industrial development. Information on genetic diversity, geographical differentiation, and phylogenetic relationships among the populations is important in order to develop conservation strategies for this species. In this study, the genetic diversity within and among 29 natural populations in Peninsular Malaysia was analyzed using the AFLP molecular technique. Polymorphism within a population was detected for 27 populations and three geographically distinct groups of nipa. The results showed that the highest frequency of heterozygosity was found in the west coast southern region, followed by the west coast northern region, and with the lowest frequency in the east coast region. In the phylogenetic tree, the three geographical regions that formed distinct groups with the west coast southern region group were located between the west coast northern region group and the east coast region. These results may suggest that nipa in Peninsular Malaysia has been diffused from or along the west coast southern region to the other regions. The three regional groups are differentiated genetically and it is recommended that all three regions should be targets for the conservation of nipa habitats, in order to reduce genetic erosion within the nipa in Peninsular Malaysia