Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection

In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van’t Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases

In vitro somatic embryos multiplication of Eurycoma longifolia Jack using Temporary Immersion System RITA®

Temporary immersion system, Recipient for Automated Temporary Immersion® (RITA®), is one of the innovative systems that allow the production of large number of somatic embryos or plantlets via in vitro plant propagation. It has been widely used to avoid associated problems such as low multiplication rate of somatic embryos and hyperhydricity during plant propagation. Thus, an attempt was made to investigate optimal parameters such as immersion time and immersion frequency, for the multiplication of direct somatic embryogenesis from cotyledon culture of Eurycoma longifolia Jack using RITA®. Four periods of immersion time (1, 5, 10 and 15 min every 4 h) were evaluated for the efficiency in somatic embryos multiplication. In order to optimize repetitive somatic embryogenesis, three different immersion frequencies (5 min immersion every 2, 4 and 8 h) were applied. The highest number of secondary embryos (69.67 ± 9.73) was found significant when immersing the globular, primary embryos for 5 min every 4 h as compared to other immersion time tested. The secondary somatic embryos obtained in this study could be further used for the development of plantlet regeneration of E. longifolia

Effect of pH on Different Strains of Phytase Producing Bacteria from Malaysia’s Hot Spring

In the recent research, the optimisation of culture condition for phytase production rarely done for Acetinobacter baumanii. The optimisation of the phytase production from the bacterial strains largely contributed by Bacillus sp. The study on the phytase originated from hot spring are limited and the species that identified from the hot spring samples are not in the same species from the previous study and mainly the species isolated from Bacillus sp. In this study, four potential strains of bacteria producing phytase isolated from hot spring in several regions in Malaysia. For enrichment of the bacterial, Nutrient Agar was used, meanwhile for batch culture optimisation, the bacteria producing phytase grown in modified liquid Phytase Screening Media with soy extract as agro residual substrate as a replacement for sodium phytate, the chemical substrate. The bacteria were screened for their ability to produce clear zone in solid PSM with sodium phytate as substrate. Optimisation of media through its physical factor that is pH of the media carried out using shake flask scale in laboratory. The growth of the bacterial strains and phytase activity measured quantitatively through the two different pH of media at pH 5.5 and pH 7. The analysis of colony-forming unit and pH determination after fermentation was carried out in this study. From the study, bacterial strain L3 from Labis, Johor has the highest phytase activity in the two parameters studied where the inorganic phosphate released at pH 5.5 (0.21953 U/mL) and pH 7 (0.2047 U/mL). Optimisation carried out through manipulating the culture condition that is pH of the media to determine at which condition has the highest phytase production. Several effects on enzyme activity caused by culture conditions identified. The optimisation of the fermentation medium able to contribute to the less cost production of the industrial enzyme as phytase has potential production for feed additives for poultry feeding. In the future research, molecular identification of the bacterial strains from the better-quality bacteria producing phytase grown in optimised culture media to investigate the molecular identity of the bacterial

Comparison on Effects of Temperature on Different Strains of Phytase Producing Bacteria Isolated from Malaysia’s Hot Spring

The main purpose of this research was to find the best growth curve for bacterial growth and the optimum temperature for the production of phytase from different potential phytase producing bacterial strains. A total of four strains used were originally isolated from hot springs in Malaysia, which were in Labis, Johor (L3), Dusun Tua, Selangor (RT), Ulu Legong, Kedah (A) and Ranau, Sabah (B9). Nutrient Agar (NA) and modified Phytase Screening Medium (PSM) liquid media were used for the culture enrichment while optimisation was carried out through batch culture method using a shake-flask scale. Strains growth and enzyme activity were quantitatively measured at different temperatures at (30°C and 37°C) values. Enzyme activity was determined according to the reaction of the phytase with its substrate (sodium phytate) and expressed in units of phytase activity (U/ mL). As for the overall, strain L3 (from Labis, Johor) exhibit promising rate of Pi released in the media at 30°C and 37°C, with optimum phytase activity values of 0.2047 U/mL and 0.2195 U/mL, respectively. The pH of the cultures was also measured, where it shows that strains grown in cultures at 37°C produced a higher phytase activity and resulting a lower reading of pH compared when grown at 30°C. All around, L3 strains has the lowest value of pH when cultured at 30°C and 37°C, with the pH value of 3.62 and 2.37, respectively.  From the result obtained, the lower pH indicates the process of phytic acid degradation take place by the phytase in producing inorganic phosphate (Pi) due to the accumulation of organic acid. Since these bacterial strains were originally taken from Hot Springs, further analysis of temperature optimization using 55°C and even 60°C should be carried out. In the future, biochemical research and molecular identification may also be carried out to identify molecular identity in the strains

The Impact of Processing Methods on the Quality of Honey: A Review

The quality of honey are naturally differs depending on its geographical origin and the botanical source of honeydew collected by the bees. These characteristics influence the physical appearance of honey such as its colour lightness and liquid thickness; which then may influence the honey attractiveness to the consumers. The thickness of honey is correlate with its moisture content. The higher the moisture content of honey, the higher the risk of fermentation, thus honey is commonly process before marketed. However, the processing methods may change the nutritional content, original taste and physical characteristics of the honey. This paper reviews the effect of five types of processing methods on the quality of honey; which are the spray-drying process, thermal treatment technique, thermosonication technique, high-pressure processing technique and microwave processing method. Overall, it can be concluded that all processing methods reviewed in this article altered the nutritional quality and physicochemical characteristics of honey. However, the different processing methods may alter the quality of honey differently. This information is very important for consumer to understand the reasons on the variability of honey quality in the market. It also will help the honey industry players to choose the best method in processing their honey and ensuring the best quality of honey can be produce to fulfil the consumers need and satisfaction

Proses identifikasi jenis bakteria dengan menggunakan aplikasi BactFinder© (bacterial identification process using BactFinder© mobile application) / Nadiawati Alias … [et al.]

BactFinder© merupakan aplikasi interaktif dan mudah alih yang dibina untuk memudahkan identifikasi dan pengenalpastian jenis bakteria yang ingin dikaji. Seperti yang diketahui umum, bakteria terdiri daripada domain mikroorganisma prokariotik yang besar dan pelbagai jenis, justeru itu pengenalpastian kumpulan bakteria ini memerlukan satu proses identifikasi yang sistematik dan teratur. Melalui aplikasi mudah alih ini, maklumat hasil daripada uji kaji biokimia dan ujian morfologi yang diperoleh daripada uji kaji di makmal perlu dimasukkan ke dalam aplikasi ini sebelum pengenalpastian bakteria boleh dilakukan. Sebelum ini, kaedah untuk pengenalpastian jenis bakteria memerlukan ahli akademik serta para pelajar untuk merujuk kepada buku-buku rujukan, artikel-artikel serta jurnal saintifik yang berkaitan. Proses ini sudah tentu memakan masa yang agak panjang dan kurang efektif. Aplikasi mudah alih ini dilengkapkan dengan pangkalan data bagi 19 jenis ujian biokimia termasuk bakteria morfologi. Pangkalan data ini dibina merujuk kepada Bergey’s Manual of Systematic Bacteriology, Bergey’s Manual of Determinative Bacteriology dan juga jurnal-jurnal saintifik yang berkaitan. Aplikasi mudah alih BactFinder© ini terbukti dapat memberikan respon yang cepat (±2 saat) dan tepat dalam membantu para pelajar dan ahli akademik dalam proses pengenalpastian bakteria yang sedang dikaji di makmal. Aplikasi ini juga amat sesuai digunakan oleh pelajar prasiswazah, pascasiswazah dan ahli akademik dalam bidang mikrobiologi, bioteknologi dan sains secara umumnya

Seaweed effects on plant growth and environmental remediation: a review

Seaweeds are plants found in sea that have tremendous applications in the fields of agriculture and environment. It comprises of three giant classes with a large number of different species. their ability to adopt to various conditions qualifies them more applicable to various environmental and agricultural arena. Agriculturally, both three classes Phaeophyta, Rhodophyta and Chlorophyta, have significant roles in promoting plant growth and productivity and soil protection as well as reclamation with class Phaeophyta has highest contribution due to its alginic acid content and other multifaceted components that are higher followed by Rhodophyta and Chlorophyta. Seaweed (living or dead biomass) has ability to phycoremediate environment against heavy toxic metals and lessen the excessiveness of non-metal inorganic elements via physisorption, chemisorption with the aid of binding sites provided by proteins and carbohydrates functional groups existing in their cell walls and secretion of organic acids and intracellular transformation and accumulation. Seaweed is an important factor in environmental remediation and soil restoration processes

Genetic diversity and differentiation of Aquilaria malaccensis Lam. using RAPD markers

Aquilaria malaccensis Lam. (family Thymelaeaceae) commonly known as agarwood or gaharu producing tree in Malaysia. The tree is being heavily exploited due to its highly valuable agar oil used in the production of high grade perfumes and traditional medicines. Consequently, their population in nature is threatened greatly. Conservation of this tree species is of the main concern, however, identification of A. malaccensis from other Aquilaria sp. based on morphology is very difficult and time consuming. This study aimed to determine the genetic diversity among three selected Aquilaria sp. namely A. malaccensis, A. sinensis (Lour.) Spreng. And A. subintegra Ding Hou using random amplified polymorphic DNA (RAPD) markers and to differentiate A. malaccensis from A. sinensis and A. subintegra. Out of ten RAPD primers, four primers (G12, R15, U13 and OPA 05) produced the most clear and reproducible bands. A total of 24 bands were scored from the four primers. Construction of dendrogram resulted in two major clusters; cluster I consisted of only A. malaccensis accessions, and cluster II consisted of A. subintegra and A. sinensis accessions. This indicates that A. subintegra is more closely related to A. sinensis while A. malaccensis is genetically distant from both. Species-specific bands for A. malaccensis were produced at 875, 1000 and 2500 bp by G12 primer, and at 2500 bp by OPA 05 primer. This study laid the foundation for a creation of rapid and cost effective molecular identification of A. malaccensis

Isolation and Molecular Cloning of Carbohydrate Binding Module (CBM40) From Vibrio cholerae Non-O1 Neuraminidase.

Carbohydrate binding modules (CBMs) are discrete contiguous amino acid sequence within a carbohydrate-active enzyme which are, non-catalytic modules that primarily exist to target parent enzyme to its substrate for efficient hydrolysis. Although many sialidase proteins have been identified from various pathogenic bacteria, only a few enzymes are commercially available which have been used for chemoenzymatic syntheses and therapeutics application. In order to study family 40 CBM domain, a number of bacteria have been screened including Pseudomonas aeruginosa ATCC 27853,Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 33862, Salmonella thypii ATCC 14028  andVibrio cholerae Non-O1. A gene encoding CBM40 domain was screened from all the bacteria strains and subjected to PCR amplification. From all the samples tested, only Vibrio choleraeNon-O1 amplified a PCR product with approximate size of 530 bp. From BLAST sequence analysis, result has shown 99% similarity with the target Vibrio choleraeneuraminidase, NanH (M83562). Next, the confirmed CBM40 gene was further ligated into pGEMT Easy Vector system and transformed into E. coliJM109 host to secure the clone before re-ligated into suitable expression vector.