Antiviral properties of Berembang Bukit and Kandis Hutan against psedorabies virus in animal cell culture

Tropical rainforest in Malaysia represents and untapped potential source of antiviral compounds. Bioactive compounds in plant species from the same genus as Kandis Hutan such as xanthones, benzophenones, biflavonoids and lupeol had been studied. Eugeniin is an anti-herpesvirus compound which had also been found n Berembang Bukit. This preliminary study was carried out to discover the presence of antiviral properties in Berembang Bukit and Kandis Hutan using different antiviral assays. In this study, MTT cell viability assay was used in addition to microscopic evaluation of pseudorabies virus (PrV)- induced cytopathic effects (CPE) on Vero cells. The cellular toxicity of DMSO was also evaluated. DMSO was less than 10% cytotoxic at concentration of 0.1% to Vero cells and its effect can be negligible. Both plants had demonstrated antiviral properties in thyl acetate and ethanol extracts. From our findings from all three antiviral assays, the ethanol-extracted Kandis Hutan possessed the most promising antiviral properties. Nevertheless, antiviral potential of ethyl acetate and ethanol-extracted Berembang Bukit and ethyl acetate-extracted Kandis Hutan also merit further investigatio

Association between water and carbon dioxide transport in leaf plasma membranes: Assessing the role of aquaporins

The role of some aquaporins as CO2 permeable channels has been controversial. Low CO2 permeability of plant membranes has been criticized because of unstirred layers and other limitations. Here we measured both water and CO2 permeability (Pos, PCO2) using stopped flow on plasma membrane vesicles (pmv) isolated from Pisum sativum (pea) and Arabidopsis thaliana leaves. We excluded the chemical limitation of carbonic anhydrase (CA) in the vesicle acidification technique for PCO2 using different temperatures and CA concentrations. Unstirred layers were excluded based on small vesicle size and the positive correlation between vesicle diameter and PCO2. We observed high aquaporin activity (Pos 0.06 to 0.22 cm s−1) for pea pmv based on all the criteria for their function using inhibitors and temperature dependence. Inhibitors of Pos did not alter PCO2. PCO2 ranged from 0.001 to 0.012 cm s−1 (mean 0.0079 + 0.0007 cm s−1) with activation energy of 30.2 kJ mol−1. Intrinsic variation between pmv batches from normally grown or stressed plants revealed a weak (R2 = 0.27) positive linear correlation between Pos and PCO2. Despite the low PCO2, aquaporins may facilitate CO2 transport across plasma membranes, but probably via a different pathway than for water.This research was supported by The Australian Research Council (ARC) Centre of Excellence in Plant Energy Biology (CE140100008), DP0771413 and ARC COE for Translational Photosynthesis (CE140100015)

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens. Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis

COVID-19 Antibody Surveillance Among Healthcare Workers in A Non-COVID designated Cardiology Centre

BACKGROUND: Reports on healthcare worker antibody response to COVID-19 infection are scarce. We aim to determine theCOVID-19 antibody prevalence among healthcare workers in a cardiology centre and the relationship between case definitioncriteria with the COVID-19 antibody result. METHODS: Convenience sampling was applied. Healthcare workers in SarawakHeart Centre (SHC) cardiology, radiology, and emergency unit were recruited. A survey form on clinical symptoms and closecontact history was distributed. HEALGEN COVID-19 IgG/IgM rapid test was performed using serum/ whole blood specimen.Staff with positive COVID-19 antibody results were referred to the infectious disease specialist for assessment. RESULTS: Atotal of 310 staff were screened. 220(71%) were female, and the mean age was 36±7.7 years old. 46(14.8%) staff reported havingclinical symptoms at some stage from the end of January 2020 to the time of this surveillance. Number of staff who had a historyof overseas travel, close contact with confirmed COVID-19 patients, or had visited places with identified COVID-19 clusterswere 4(1.3%), 24(7.7%) and 24(7.7%) respectively. There were 14 staff (4.5%) with positive tests positive, 2 for IgM, and 12for IgG. All those with positive antibody were subsequently tested negative with RT-PCR test. The history of having clinicalsymptoms and exposure to COVID-19 cluster area were independently associated with a positive IgG result. CONCLUSION:The application of COVID-19 antibody serology rapid tests could determine true exposure of staff to the infection and allowus to reassess existing measures of infection control within the hospital

Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.

Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

Characterizing and Prognosticating Heart Failure with Improved Ejection Fraction Using NT-proBNP, Growth Differentiation Factor 15 and Global Longitudinal Strain

Background: Heart failure with improved ejection fraction (HFiEF) is a novel heart failure (HF) subgroup. There are sparse data on using NT-proBNP, growth differentiation factor 15 (GDF15) and global longitudinal strain (GLS) to characterize and prognosticate HFiEF patients. Objectives: (1) To determine the level and correlation between NT-proBNP, GDF-15 and GLS in HFiEF patients. (2) To examine the correlation of each marker with NYHA, MAGGIC prognostic score, HF etiologies, comorbidities status, degree of LVEF/ LV end-diastolic diameter change from baseline and diastolic dysfunction. (3) To look for association of each marker with follow-up LVEF change and 1-year composite mortality or HF events outcome. Materials & Methods: This was a cross-sectional observational study in Sarawak Heart Centre HF clinic. 53 HfiEF patients who had NT-proBNP and GDF15 tests performed were selected. This cohort had no HF events in the past 6 months during the blood tests. Clinical characteristics, echocardiography parameters, and 1-year composite clinical outcome were analyzed retrospectively. Results: The mean age of the cohort was 52 years old and 81% were male. The cohort was highly comorbid (hypertension 71%; diabetes 45.3%; AF 17.3%). Most of the patients (87%) were asymptomatic by NYHA (I) and low rate of composite outcome was observed, 5.7%. The mean NT-proBNP, GDF-15, GLS were 357 pg/ml, 1572 pg/ml, and -12.1% respectively. There were significant moderate correlation between GDF15 with NT-proBNP (r=0.414) and NT-proBNP with GLS (r=-0.351). Higher NT-proBNP and GDF15 levels were associated with poorer MAGGIC prognostic scores (r=0.549, 0.41 respectively). NT-proBNP was the only marker associated with a higher degree of LVEF improvement compare to baseline echocardiography. NT-proBNP was also related to severe diastolic echo parameters. Hypertension and diabetes were strongly associated with higher elevated GDF15 levels. The lower mean GLS level was significantly associated with the presence of composite outcome (-6.45% vs -12.47%, p=0.0). Patients with NT-proBNP levels below the median cutoff had favourable follow-up LVEF improvement (+9.73%, p=0.035). Conclusion: In our HFiEF study cohort, NT-proBNP best correlate and prognosticate future LV remodelling. GDF15 was closely related to systemic illnesses such as diabetes. The role of GLS in our HFiEF cohort remains uncertain

Analysis of cellulose synthase genes and regulatory elements in barley and arabidopsis.

Genetic manipulation of cellulose synthase genes (CesA) might be expected to increase cellulose content and improve the stem strength of cereals. Such a possibility would have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Thus, an attempt to manipulate cellulose content was made via three approaches: (1) Driving barley HvCesA genes using the CaMV 35S constitutive promoter. (2) Driving barley secondary cell wall (SCW) HvCesA genes with specific promoters from barley and maize, and (3) Identifying novel transcription factors interacting with CesA promoters and over-expressing those candidate transcription factors in transgenic Arabidopsis lines. In Chapter 2, attempts to drive HvCesA genes involved in both primary and secondary cell wall biosynthesis via the CaMV 35S promoter are described. Five sets of barley plants transformed with primary cell wall (PCW) and SCW HvCesA genes were analysed for transcript levels, phenotype, tissue morphology, cellulose content and crystalline cellulose distribution in cell walls. Only the lines transgenic for the SCW HvCesA genes exhibited aberrant phenotypes. One of the two constructs showed a dwarfed phenotype, where xylem cells in the vascular tissue were irregular in shape and partially collapsed. Transcript silencing, together with cellulose content reduction, were also observed in the dwarf plants and TEM images showed a significant decrease in cell wall thickness. Overall, there was no increase in either crystalline cellulose content or in stem strength. In Chapter 3, experiments are described that further investigated if specific barley or maize promoters could ameliorate the aberrant phenotypes reported in Chapter 2. Promoter/gene combination constructs made up of either a barley HvCesA4 or maize ZmCesA10 (orthologue) promoters fused with either the HvCesA4 or HvCesA8 gene were used for transformation. Phenotyping, transcript profiling, stem strength measurements and cellulose content determination were conducted. There was no reduction in stature for any To transgenic plants, although other aberrant phenotypes were observed. One out of four promoter/gene combinations, namely HvCesA4pro:HvCesA4, produced an increase in crystalline cellulose content and stem strength. In situ PCR results showed that the HvCesA4 transgene transcripts in this line were concentrated in the vascular bundles of the stem. Finally, an attempt to manipulate cellulose content was conducted by over-expressing candidate transcription factors shown to interact with the CesA4 promoter, as described in Chapter 4. Candidate transcription factors in apple (Malus x domestica) were identified using the dual luciferase transient expression system in tobacco, with Arabidopsis and barley CesA promoters as bait. Transgenic Arabidopsis lines transformed with two candidate MYB transcription factors were analysed for phenotype, transcript abundance and cellulose content. Over-expression of these transcription factors did not increase crystalline cellulose content in Arabidopsis, despite an induction of SCW AtCesA transcript levels. In conclusion, the results presented demonstrate the importance of controlled expression of HvCesA genes to increase cellulose content and stem strength as well as the existence of a finely balanced regulatory system that controls the cellulose biosynthetic process.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Analysis of key glycosyltransferase (GT) families in barley.

This research was performed over 10 months as part of a Masters in Biotechnology (Plant Biotechnology). This thesis was previously assessed and updated in accordance with the corrections suggested by the examiners. The main focus of the research is essentially the same as proposed initially in the literature review, although slight modifications have been made in the methodology and the focus of the study has been further narrowed. Only the E. coli expression system was used to express the proteins instead of both Pichia pastoris and E.coli. Also, one glycosylsyltransferase (GT) family, GT43 and one clade of GT47 family were characterized instead of three. Thus, the title in the manuscript is narrower as opposed to the title of the thesis. Although the research manuscript contained herein will provide the first draft of a future publication to be submitted to Plant Physiology, due to time constraints, all the data required for the publication has not been finalised. Further experiments are needed to verify and obtain more comprehensive data for the study. However, data which was collected but is not included in the manuscript due to space constraints is provided within the appendices such as the transcript profiling of glycosyltransferase family GT61 genes. Protein expression of genes in this family could not be achieved as amplification of the GT61 cDNA was not successful. The research manuscript begins by outlining the significance of heteroxylans in plant cell wall biology and in the utilization of cereals and grasses, and pointed to glycosyltransferases that had been implicated in their synthesis. The results of the study consisted of phylogenetic and bioinformatic analysis of barley GT43 family and one clade of barley GT47 family, transcript profiles of the GT43 and GT47 genes in a series of barley tissues, heterologous expression and the purification of two proteins of interest, and finally an assay of the purified proteins. In addition, the appendices contain data collected for family GT61, nucleotide and protein sequences for all the GT genes studied, buffer list, Plant Physiology’s “instructions for authors” acknowledgements, and a dedication.Thesis (M.Bio (PB)) - University of Adelaide, School of Agriculture, Food and Wine, 200

Association between water and carbon dioxide transport in leaf plasma membranes: Assessing the role of aquaporins

The role of some aquaporins as CO₂ permeable channels has been controversial. Low CO₂ permeability of plant membranes has been criticized because of unstirred layers and other limitations. Here we measured both water and CO₂ permeability (<em>P_os</em>, <em>P_CO2</em>) using stopped flow on plasma membrane vesicles (pmv) isolated from <em>Pisum sativum</em> (pea) and <em>Arabidopsis thaliana</em> leaves. We excluded the chemical limitation of carbonic anhydrase (CA) in the vesicle acidification technique for <em>P_CO2</em> using different temperatures and CA concentrations. Unstirred layers were excluded based on small vesicle size and the positive correlation between vesicle diameter and <em>P_CO2</em>. We observed high aquaporin activity (<em>P_os</em> 0.06 to 0.22 cm s^-1) for pea pmv based on all the criteria for their function using inhibitors and temperature dependence. Inhibitors of <em>P_os</em> did not alter <em>P_CO2</em>. <em>P_CO2</em> ranged from 0.001 to 0.012cm s^-1 (mean 0.0079 <span class="underlined ">+</span>0.0007 cm s^-1) with activation energy of 30.2 kJ mol^-1. Intrinsic variation between pmv batches from normally grown or stressed plants revealed a weak (<em>R</em>²= 0.27) positive linear correlation between <em>P_os</em> and <em>P_CO2</em>. Despite the low <em>P_CO2</em>, aquaporins may facilitate CO₂ transport across plasma membranes, but probably via a different pathway than for water

Development of a prediction model to estimate the risk of walking limitations in patients with total knee arthroplasty

Objective: Early and accurate risk prediction of walking limitations after total knee arthroplasty (TKA) is important for clinical and economic reasons. However, to our knowledge, no studies have systematically integrated multiple predictors into a single, clinically practical model. Our study aimed to develop a prediction model to estimate the risk of post-TKA walking limitations. Methods: We performed a prospective cohort study of 1096 patients who underwent elective, primary TKA between July 2013 and September 2014. Candidate predictors included patient demographics, surgical factors, and pre- and early (1-mo) post-TKA functional measures. The outcome of interest was self-reported walking limitations at 6 months of post-TKA. We used multivariable proportional odds regression with bootstrap internal validation to develop the model. Results: In all, 12% of patients reported walking limitations (maximum walk time ≤ 15 min) at 6 months postsurgery. The main predictors of increasing levels of walking limitations were preoperative walking limitations (overall p < 0.001), higher levels of body mass index [interquartile range (IQR)-OR 1.3, 95% CI 1.2–1.5], lower values of 1-month post-TKA gait speed (IQR-OR 1.9, 95% CI 1.3–2.6), the presence of contralateral knee pain (OR 1.9, 95% CI 1.2–3.0), and the use of a quadstick preoperatively (OR 3.5, 95% CI 1.7–7.3). The prediction model had an optimism-corrected concordance index of 0.71. Conclusion: A small but sizable proportion of patients with TKA had persistent mobility limitations. Our prediction model may help to risk-stratify patients, and external validation is required before the model can be used in clinical practice