A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis

Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms1-3. The steroid biosynthetic pathway is highly conserved and is key in eukaryote evolution4-7. The flavoprotein squalene epoxidase (SQE) catalyses the first oxygenation reaction in this pathway and is rate limiting. However, despite its conservation in animals, plants and fungi, several phylogenetically widely distributed eukaryote genomes lack an SQE-encoding gene7,8. Here, we discovered and characterized an alternative SQE (AltSQE) belonging to the fatty acid hydroxylase superfamily. AltSQE was identified through screening of a gene library of the diatom Phaeodactylum tricornutum in a SQE-deficient yeast. In accordance with its divergent protein structure and need for cofactors, we found that AltSQE is insensitive to the conventional SQE inhibitor terbinafine. AltSQE is present in many eukaryotic lineages but is mutually exclusive with SQE and shows a patchy distribution within monophyletic clades. Our discovery provides an alternative element for the conserved steroid biosynthesis pathway, raises questions about eukaryote metabolic evolution and opens routes to develop selective SQE inhibitors to control hazardous organisms

Chemical and biological studies of Siddha medicinal plants

Theoretical thesis."A thesis submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy from Macquarie University, Sydney"."October 2010".Includes bibliographical references.Chapter 1. Introduction -- Chapter 2. Literature studies on Siddha medicinal plants -- Chapter 3. Biological studies on Siddha medicinal plants -- Chapter 4. Bioassay guided studies on Cardiospermum halicacabum -- Chapter 5. Bioassay guided isolation of bioactive compounds from Caralluma fimbriata -- Chapter 6. Conclusions and future directions -- Appendices.Siddha is a traditional system of medicine being practiced in the southern part of India. Siddha medicines are predominantly plant based, usually containing polyherbal combinations. Following the establishment of a collaborative research partnership with a Siddha practitioner, Dr R. Velmurugan, information on some medicinal plants that he identified to be effective in treating cancer, pain and swelling were provided. The overall aim of this study was to isolate and identify biologically active molecules from some of these plants. This study consisted of reviewing the literature on the Siddha medicinal plants identified by Dr Velmurugan, biological studies and isolation and characterisation of bioactive constituents from a selection of these plants.A broad literature search was undertaken on these plants to identify those plants that have not been well studied phytochemically and/or biologically, thus having the potential for further investigations. This led to the selection of nine plants for biological screening studies.Antiproliferative and antiinflammatory studies were employed for the evaluation of the biological activities. The antiproliferative screenings were performed against SKNMC (neuroepithelioma), MCF 7 (Breast Cancer) and MRC 5 (normal) cell lines using the MTT assay. Antiinflammatory studies were carried out by using cyclooxygenase inhibitory assays. Significant antiproliferative activity of Cardiospermum halicacabum and Caralluma fimbriata was identified in the MTT assay. Both these plants also exhibited potent COX 1 and COX 2 inhibitory activity.Inhibition studies with the enzyme indoleamine 2,3-dioxygenase (IDO), which is involved in cancers and inflammatory diseases, were also conducted on all plants. IDO inhibitory studies revealed the IDO inhibitory activity of Marsdenia tinctoria, Evolvulus alsinoides and C. halicacabum.Based on the initial antiproliferative screening results, C. halicacabum and C. fimbriata were selected for further biological and chemical investigations. The bioassay guided isolation of the water partition of the ethanolic extract of the leaves of C. halicacabum led to the isolation of five compounds: quercetin 3-O-β-D-rutinoside, apigenin 7-O-β-Dvii glucoside, chrysoeriol, luteolin and scutellarein. The antiproliferative activities of the isolated compounds were evaluated against the SKNMC, MCF 7 and MRC 5 cell lines. Chrysoeriol and scutellarein exhibited the highest antiproliferative activity. The other three molecules also demonstrated antiproliferative activity. A comparison of the activity exhibited by the compounds against the MCF 7 and SKNMC cell lines indicated some selectivity of these molecules for the breast cancer cell line. This is the first report of apigenin 7-O-β-D-glucoside and scutellarein from C. halicacabum.Bioassay guided studies on the ethyl acetate fraction of C. fimbriata led to the isolation of cleomiscosin A, N-(p-trans-coumaroyl)tyramine, aristolochic acid 1 and aristolactam1a-N-β-D-glucoside . This is the first report of all these molecules from this plant and the first report of aristolochic acid 1 and aristolactam1a-N-β-D-glucoside from the Ascelpedeaceae family. The compounds were tested for antiproliferative activities. Aristolochic acid 1 demonstrated the most potent activity. Cleomiscosin A and N-(ptrans-coumaroyl)tyramine also demonstrated antiproliferative activity and aristolactam1a-N-β-D-glucoside was found to be less active. No remarkable selectivity in activity was observed with these compounds.This study also identified the IDO inhibitory activity of N-(p-trans-coumaroyl)tyramine for the first time.The biological activities of the plant extracts and the isolated molecules were consistent with the use of these plants by Dr Velmurugan, thus providing strong support for their use in the Siddha system of medicine for treatment of cancers.Mode of access: World Wide Web.1 online resource (xiv, 183 pages) illustrations (some colour

The Potential of autofluorescence spectroscopy to detect human urinary tract infection

Urinary tract infections (UTIs) are known to alter the normal urine composition which, in principle, can lead to changes in urine autofluorescence. This paper describes the study of human urine (normal and UTI) by using UV fluorescence excitation/emission matrices and synchronous spectra and proposes a method of diagnosing UTI without any sample preparation. The method is based on excitation in the shorter UV region (250–350 nm) which shows good discrimination between the normal urine and UTI samples. The synchronous scans with an offset of Δλ = 90 nm were also able to differentiate between normal urines and UTI samples. These differences were observed even though the two known major urine fluorophores, tryptophan and indoxyl sulfate were present in the normal urine and UTI samples in similar concentration as established by HPLC analysis. Although the identity of substances responsible for the altered autofluorescence in UTI is not established, our study shows that autofluorescence has the potential to differentiate between normal human urine samples and those with UTI.6 page(s

The Influence of indoxyl sulfate and ammonium on the autofluorescence of human urine

Despite biological variability the spectral characteristics of undiluted human urine show relatively low autofluorescence at short UV (250–300 nm) excitation. However with dilution the fluorescence intensity remarkably increases. This paper examines the mechanisms behind this effect, by using excitation–emission matrices. Corrections for the inner filter effect were made for improved understanding of the spectral patterns. We focused on three major fluorophores (tryptophan, indoxyl sulfate and 5-hydroxyindole-3-acetate) that are excited at these wavelengths, and whose content in urine is strongly linked with various health conditions. Their fluorescence was studied both individually and in combinations. We also examined the effect of ammonium on the fluorescence of these major fluorophores individually and in combinations. Through these studies we have identified the leading effects that reduce the UV fluorescence, namely higher concentration of indoxyl sulfate producing the inner filter effect and concentration quenching and quenching of fluorophores by ammonium. This result will assist in broader utilisation of UV fluorescence in medical diagnostics.8 page(s

Bioanalysis of urine samples after manipulation by oxidizing chemicals: technical considerations

Drug testing programs are established to help achieve a drug-free work environment, promote fair competition in sport, facilitate harm minimization and rehabilitation programs, better manage patient care by clinicians and service law enforcement authorities. Urine remains the most popular and appropriate testing matrix for such purposes. However, urine is prone to adulteration, where chemicals, especially oxidizing chemicals, are purposely added to the collected urine specimens to produce a false-negative test result. This article will describe the effect of various popular oxidizing adulterants on urine drug test results, the countermeasures taken by laboratories in dealing with adulterated urine samples and the prospect of developing more robust and economical methods to combat urine adulteration in the future

Development and validation of a simple, rapid and sensitive LC-MS/MS method for the measurement of urinary neurotransmitters and their metabolites

Neurotransmitters play crucial roles in physiological functions and their imbalances have demonstrated association in the pathology of several diseases. The measurement of neurotransmitters possesses a great potential as a significant clinical tool. This study presents the development and validation of an LC-MS/MS method for simultaneous quantification of multi-class neurotransmitters associated with dopamine, tryptophan and glutamate-γ-aminobutyric acid pathways. A total of ten neurotransmitters and their metabolites (dopamine, epinephrine, metanephrine, tryptophan, serotonin, kynurenic acid, kynurenine, anthranilic acid, GABA, glutamic acid) were determined based on a simple and rapid \u27dilute and shoot\u27 method using minimal urine volume. The chromatographic separation was achieved using a Poroshell 120 Bonus-RP LC Column in combination with a gradient elution within an 8.5-min time frame. The method exhibited good sensitivity as the limits of quantification ranged between 0.025 and 0.075 μg/mL with acceptable matrix effects ( \u3c ± 14.5%), no carryover and good linearity (r2\u3e 0.98). The accuracy and precision for all analytes were within tolerances, at \u3c ± 9.9% mean relative error (MRE) and \u3c 8.6% relative standard deviation (RSD), respectively. The method was successfully applied in measuring the neurotransmitter concentrations in urine of healthy donors. Furthermore, the undertaken stability experiments indicated that acidified urine specimens allowed the analytes to be stable for prolonged durations in comparison to those untreated. The study also reveals the performance of the method is unaffected by the absence of expensive deuterated reference standards under the experimental conditions employed which further simplifies the analytical procedures and provides a significant cost saving for running the assay

Biomasonry products from macroalgae: A design driven approach to developing biomaterials for carbon storage

Lowering the embodied carbon of building materials requires a transition away from fossil derived products towards bio-based alternatives, alongside the design and development of new clean tech biomaterials that can function as carbon sinks. This paper presents an overview of historical and existing uses of seaweeds in construction to identify gaps and opportunities for the development of seaweed-based construction materials that can support atmospheric carbon removal through algal photosynthesis. This study highlights the value of interdisciplinary research collaborations that can be situated within the expanding field of biodesign where design research and methods are used to influence the development materials science. It presents as a case study the design of seaweed bricks utilising a biorefinery framework that aims to valorise residual seaweed biomass being grown for waste-water management, identifying value-adding opportunities for this seaweed by-product and new possibilities for carbon storage in the built environment. It details the development of a 1:1 scale prototype for the purposes of an exhibition at the Art Gallery of South Australia in order to demonstrate what biomasonry products from macroalgae can look like, to build social acceptance and to encourage future uptake of sustainable seaweed construction products

Biomasonry products from macroalgae: A design driven approach to developing biomaterials for carbon storage

Lowering the embodied carbon of building materials requires a transition away from fossil derived products towards bio-based alternatives, alongside the design and development of new clean tech biomaterials that can function as carbon sinks. This paper presents an overview of historical and existing uses of seaweeds in construction to identify gaps and opportunities for the development of seaweed-based construction materials that can support atmospheric carbon removal through algal photosynthesis. This study highlights the value of interdisciplinary research collaborations that can be situated within the expanding field of biodesign where design research and methods are used to influence the development materials science. It presents as a case study the design of seaweed bricks utilising a biorefinery framework that aims to valorise residual seaweed biomass being grown for waste-water management, identifying value-adding opportunities for this seaweed by-product and new possibilities for carbon storage in the built environment. It details the development of a 1:1 scale prototype for the purposes of an exhibition at the Art Gallery of South Australia in order to demonstrate what biomasonry products from macroalgae can look like, to build social acceptance and to encourage future uptake of sustainable seaweed construction products.</p