Advances in molecular genetic systems in malaria

Robust tools for analysing gene function in Plasmodium parasites, which are the causative agents of malaria, are being developed at an accelerating rate. Two decades after genetic technologies for use in Plasmodium spp. were first described, a range of genetic tools are now available. These include conditional systems that can regulate gene expression at the genome, transcriptional or protein level, as well as more sophisticated tools for gene editing that use piggyBac transposases, integrases, zinc-finger nucleases or the CRISPR-Cas9 system. In this Review, we discuss the molecular genetic systems that are currently available for use in Plasmodium falciparum and Plasmodium berghei, and evaluate the advantages and limitations of these tools. We examine the insights that have been gained into the function of genes that are important during the blood stages of the parasites, which may help to guide the development and improvement of drug therapies and vaccines.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.

Fluorescent photoinduced electron transfer (PET) sensors for anions; from design to potential application

This mini review highlights the synthesis and photophysical evaluation of anion sensors, for nonaqueous solutions, that have been developed in our laboratories over the last few years. We have focused our research mainly on developing fluorescent photoinduced electron transfer (PET) sensors based on the fluorophore-spacer-anion receptor principle using several anthracene (emitting in the blue) and 1,8-naphthalimide (emitting in the green) fluorophores, with the aim of targeting biologically and industrially relevant anions such as acetates, phosphate and amino acids, as well as halides such as fluoride. The receptors and the fluorophore are separated by a short methyl or ethyl spacer, where the charge neutral anion receptors are either aliphatic or aromatic urea (or thiourea) moieties. For these, the anion recognition is through hydrogen bonding, yielding anion:receptor complexes. Such bonding gives rise to enhanced reduction potential in the receptor moieties which causes enhancement in the rate of PET quenching of the fluorophore excited state from the anion:receptor moiety. This design can be further elaborated on by incorporating either two fluorophores, or urea/thiourea receptors into the sensor structures, using anthracene as a fluorophore. For the latter design, the sensors were designed to achieve sensing of bis-anions, such as di-carboxylates or pyrophosphate, where the anion bridged the anthracene moiety. In the case of the naphthalimide based mono-receptor based PET sensors, it was discovered that in DMSO the sensors were also susceptible to deprotonation by anions such as F− at high concentrations. This led to substantial changes in the absorption spectra of these sensors, where the solution changed colour from yellow/green to deep blue, which was clearly visible to the naked eye. Hence, some of the examples presented can act as dual fluorescent-colorimetric sensors for anions. Further investigations into this phenomenon led to the development of simple colorimetric sensors for fluorides, which upon exposure to air, were shown to fix carbon dioxide as bicarbonate.

Subcellular localisation of proteins in fluorescent microscope images using a random forest

This paper presents a system that employs random forests to formulate a method for subcellular localisation of proteins. A random forest is an ensemble learner that grows classification trees. Each tree produces a classification decision, and an integrated output is calculated. The system classifies the protein-localisation patterns within fluorescent microscope images. 2D images of HeLa cells that include all major classes of subcellular structures, and the associated feature set are used. The performance of the developed system is compared against that of the support vector machine and decision tree approaches. Three experiments are performed to study the influence of the training and test set size on the performance of the examined methods. The calculated classification errors and execution times are presented and discussed. The lowest classification error (2.9%) has been produced by the developed system.

Characterization of the drug binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Dual responsive chemosensors for anions : the combination of fluorescent PET (Photoinduced Electron Transfer) and colorimetric chemosensors in a single molecule

The design and synthesis of two novel fluorescent PET anion sensors is described, based on the principle of ‘fluorophore-spacer-(anion)receptor’. The sensors 1 and 2 employ simple diaromatic thioureas as anion receptors, and the fluorophore is a naphthalimide moiety that absorbs in the visible part of the spectrum and emits in the green. Upon recognition of anions such as F⁻ and AcO⁻ in DMSO, the fluorescence emission of 1 and 2 was ‘switched off’, with no significant changes in the UV–vis spectra. This recognition shows a 1:1 binding between the receptor and the anions. In the case of F⁻, further additions of the anion, gave rise to large changes in the UV–vis spectra, where the λ_max at 455 nm was shifted to 550 nm. These changes are thought to be due to the deprotonation of the 4-amino moiety of the naphthalimide fluorophore. This was in fact found to be the case, using simple naphthalimide derivatives such as 6. Sensors 1 and 2 can thus display dual sensing action; where at low concentrations, the fluorescence emission is quenched, and at higher concentrations the absorption spectra are modulated.

Polycarbonate (PC) membrane fouling by protein and sodium alginate

This data was obtained from an experiment, where polycarbonate (PC) membranes were used to filter two types of organic foulants, including protein and sodium alginate, from suspension in a dead-end filtration cell. These model foulants were stained with fluorescent dyes before filtration. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM). This data collection contains 105 2D images of polycarbonate (PC) membranes fouling layer. This data collection would be useful to investigate membrane fouling mechanism by membrane materials researchers and water researchers.

Production of green nanomaterials

While our awareness towards sustainable society and environment grows, the importance of ‘green’ materials and manufacturing is gaining significant recognition. We have demonstrated that naturally-occurring fibers as renewable raw materials can be converted into nanoparticles and nano fibers using simple top-down methods without introducing hazardous chemicals. This new class of green nanomaterials will have a wide range of environmental and biomedical applications owing to the inherent biocompatible, biodegradable and carbon-neutral nature.

Study on the fouling phenomena of poly(vinylidene fluoride) (PVDF) membrane by protein and yeast

This sub-collection is the result of an investigation into the mechanism of organic fouling in membrane filtration processes. In this experiment, poly(vinylidene fluoride) (PVDF) membranes were used to filter two types of organic foulants, protein and yeast with a concentration of 50mg/l and 20 mg/l, respectively, from suspension in a dead-end filtration cell. These model foulants were stained with fluorescent dyes before filtration. This dataset contains a stack of images of the fouling layer on the PVDF membrane surface captured by a confocal laser scanning microscope (CLSM) and its associated acquisition software. This dataset would be useful to researchers who are investigating the membrane organic fouling mechanism so that new membrane materials and new anti-fouling surface treatment technologies can be developed for water and wastewater industry in the future.

Evaluating backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by protein and yeast

This collection is the result of an investigation into the backwashing efficiency of poly(vinylidene fluoride) (PVDF) membrane fouled by two types of organic foulants, protein and yeast. In this experiement, poly(vinylidene fluoride) (PVDF) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PVDF membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of poly(vinylidene fluoride) (PVDF) membranes' fouling layer when two types of organic foulants (protein and yeast) present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers who are evaluating the backwashing efficiency of PVDF membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water.

Evaluating backwashing efficiency of polycarbonate (PC) membrane fouled by protein and sodium alginate

This collection is the result of an investigation into the backwashing efficiency of polycarbonate (PC) membrane fouled by two types of organic foulants, protein and sodium alginate. In this experiement, polycarbonate (PC) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of polycarbonate (PC) membranes' fouling layer when two types of organic foulants (protein and sodium alginate) are present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers evaluating the backwashing efficiency of PC membrane in order to optimize frequency and operational conditions of backwashing by membrane materials researchers and by water..

Evaluating backwashing efficiency of polycarbonate (PC) membrane fouled by protein, sodium alginate and yeast

This collection is the result of an investigation into the backwashing efficiency of polycarbonate (PC) membrane fouled by three types of organic foulants, protein, sodium alginate and yeast. In this experiement, polycarbonate (PC) membrane was used to filter those organic foulants from suspensions in a dead-end stirred cell. The organic foulants were stained with fluorescent dyes before filtration. After filtration, the PC membrane was backwashed. Consequently, a stack of images were captured from the fouling layers on the PC membrane surface using confocal laser scanning microscope (CLSM) and its associated image acquisition software. It contains image data of polycarbonate (PC) membranes' fouling layer when three types of organic foulants (protein, sodium alginate and yeast) are present. By comparing with the same membrane without backwashing, the efficiency of backwashing was computed. This data collection would be useful to researchers who are evaluating the backwashing efficiency of PC membrane in order to optimize frequency and operational conditions of backwashing by membrane materials and by water..

Study on the fouling phenomena of poly(vinylidene fluoride) (PVDF) membrane by protein and sodium alginate

This sub-collection is the result of an investigation into the mechanism of organic fouling in membrane filtration processes. In this experiment, poly(vinylidene fluoride) (PVDF) membranes were used to filter two types of organic foulants, protein and sodium alginate with a concentration of 50mg/l and 40 mg/l, respectively, from suspension in a dead-end filtration cell. These model foulants were stained with fluorescent dyes before filtration. This dataset contains a stack of images of the fouling layer on the PVDF membrane surface captured by a confocal laser scanning microscope (CLSM) and its associated acquisition software. This dataset would be useful to researchers who are investigating the membrane organic fouling mechanism so that new membrane materials and new anti-fouling surface treatment technologies can be developed for water and wastewater industry in the future.