Long-term outcomes of ADEM-like and tumefactive presentations of CNS demyelination: a case-comparison analysis.

A minority of initial multiple sclerosis (MS) presentations clinically or radiologically resemble other central nervous system (CNS) pathologies, acute disseminated encephalomyelitis (ADEM) or tumefactive demyelination (atypical demyelination presentations). With the aim of better defining the long-term outcomes of this group we have performed a retrospective cohort comparison of atypical demyelination versus 'typical' MS presentations. Twenty-seven cases with atypical presentations (both first and subsequent demyelinating events) were identified and compared with typical MS cases. Disease features analysed included relapse rates, disability severity, whole brain and lesion volumes, lesion number and distribution. Atypical cases represented 3.9% of all MS cases. There was considerable overlap in the magnetic resonance imaging (MRI) features of ADEM-like and tumefactive demyelination cases. ADEM-like cases tended to be younger but not significantly so. Atypical cases showed a trend towards higher peak expanded disability severity score (EDSS) score at the time of their atypical presentation. Motor, cranial nerve, cerebellar, cerebral and multifocal presentations were all more common in atypical cases, and less likely to present with optic neuritis. Cerebrospinal fluid (CSF) white cell counts were higher in atypical cases (p = 0.002). One atypical case was associated with peripheral blood myelin oligodendrocyte glycoprotein (MOG) antibodies, but subsequent clinical and radiological course was in keeping with MS. There was no difference in long-term clinical outcomes including annualised relapse rates (ARR), brain volume, lesion numbers or lesion distributions. Atypical demyelination cases were more likely to receive high potency disease modifying therapy early in the course of their illness. Despite the severity of initial illness, our cohort analysis suggests that atypical demyelination presentations do not confer a higher risk of long-term adverse outcomes.

gRNAde: Geometric Deep Learning for 3D RNA inverse design.

Computational RNA design tasks are often posed as inverse problems, where sequences are designed based on adopting a single desired secondary structure without considering 3D geometry and conformational diversity. We introduce gRNAde, a geometric RNA design pipeline operating on 3D RNA backbones to design sequences that explicitly account for structure and dynamics. Under the hood, gRNAde is a multi-state Graph Neural Network that generates candidate RNA sequences conditioned on one or more 3D backbone structures where the identities of the bases are unknown. On a single-state fixed backbone re-design benchmark of 14 RNA structures from the PDB identified by Das et al. [2010], gRNAde obtains higher native sequence recovery rates (56% on average) compared to Rosetta (45% on average), taking under a second to produce designs compared to the reported hours for Rosetta. We further demonstrate the utility of gRNAde on a new benchmark of multi-state design for structurally flexible RNAs, as well as zero-shot ranking of mutational fitness landscapes in a retrospective analysis of a recent RNA polymerase ribozyme structure.

gRNAde: Geometric Deep Learning for 3D RNA inverse design.

Computational RNA design tasks are often posed as inverse problems, where sequences are designed based on adopting a single desired secondary structure without considering 3D geometry and conformational diversity. We introduce gRNAde, a geometric RNA design pipeline operating on 3D RNA backbones to design sequences that explicitly account for structure and dynamics. Under the hood, gRNAde is a multi-state Graph Neural Network that generates candidate RNA sequences conditioned on one or more 3D backbone structures where the identities of the bases are unknown. On a single-state fixed backbone re-design benchmark of 14 RNA structures from the PDB identified by Das et al. [2010], gRNAde obtains higher native sequence recovery rates (56% on average) compared to Rosetta (45% on average), taking under a second to produce designs compared to the reported hours for Rosetta. We further demonstrate the utility of gRNAde on a new benchmark of multi-state design for structurally flexible RNAs, as well as zero-shot ranking of mutational fitness landscapes in a retrospective analysis of a recent RNA polymerase ribozyme structure. Open source code: https://github.com/chaitjo/geometric-rna-design.

A facile one step route that introduces functionality to polymer powders for laser sintering.

Laser Sintering (LS) is a type of Additive Manufacturing (AM) exploiting laser processing of polymeric particles to produce 3D objects. Because of its ease of processability and thermo-physical properties, polyamide-12 (PA-12) represents ~95% of the polymeric materials used in LS. This constrains the functionality of the items produced, including limited available colours. Moreover, PA-12 objects tend to biofoul in wet environments. Therefore, a key challenge is to develop an inexpensive route to introduce desirable functionality to PA-12. We report a facile, clean, and scalable approach to modification of PA-12, exploiting supercritical carbon dioxide (scCO2) and free radical polymerizations to yield functionalised PA-12 materials. These can be easily printed using commercial apparatus. We demonstrate the potential by creating coloured PA-12 materials and show that the same approach can be utilized to create anti-biofouling objects. Our approach to functionalise materials could open significant new applications for AM.

Distinct forms of structural plasticity of adult-born interneuron spines in the mouse olfactory bulb induced by different odor learning paradigms.

The morpho-functional properties of neural networks constantly adapt in response to environmental stimuli. The olfactory bulb is particularly prone to constant reshaping of neural networks because of ongoing neurogenesis. It remains unclear whether the complexity of distinct odor-induced learning paradigms and sensory stimulation induces different forms of structural plasticity. In the present study, we automatically reconstructed spines in 3D from confocal images and performed unsupervised clustering based on morphometric features. We show that while sensory deprivation decreased the spine density of adult-born neurons without affecting the morphometric properties of these spines, simple and complex odor learning paradigms triggered distinct forms of structural plasticity. A simple odor learning task affected the morphometric properties of the spines, whereas a complex odor learning task induced changes in spine density. Our work reveals distinct forms of structural plasticity in the olfactory bulb tailored to the complexity of odor-learning paradigms and sensory inputs.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Manipulating stereoselectivity of parahydrogen addition to acetylene to unravel interconversion of ethylene nuclear spin isomers.

Symmetric molecules exist as distinct nuclear spin isomers (NSIMs). A deeper understanding of their properties, including interconversion of different NSIMs, requires efficient techniques for NSIM enrichment. In this work, selective hydrogenation of acetylene with parahydrogen (p-H2) was used to achieve the enrichment of ethylene NSIMs and to study their equilibration processes. The effect of the stereoselectivity of H2 addition to acetylene on the imbalance of ethylene NSIMs was experimentally demonstrated by using three different heterogeneous catalysts (an immobilized Ir complex and two supported Pd catalysts). The interconversion of NSIMs with time during ethylene storage was studied using NMR spectroscopy by reacting ethylene with bromine water, which rendered the p-H2-derived protons in the produced 2-bromoethan(2H)ol (BrEtOD) magnetically inequivalent, thereby revealing the non-equilibrium nuclear spin order of ethylene. A thorough analysis of the shape and transformation of the 1H NMR spectra of hyperpolarized BrEtOD allowed us to reveal the initial distribution of produced ethylene NSIMs and their equilibration processes. Comparison of the results obtained with three different catalysts was key to properly attributing the derived characteristic time constants to different ethylene NSIM interconversion processes: ∼3-6 s for interconversion between NSIMs with the same inversion symmetry (i.e., within g or u manifolds) and ∼1700-2200 s between NSIMs with different inversion symmetries (i.e., between g and u manifolds).

Hyperpolarized long-lived spin state of methylene protons of 2-bromoethanol obtained from ethylene with non-equilibrium nuclear spin order.

In this work we achieve a significant overpopulation (PLLS≈1%) of the long-lived spin state (LLS) of methylene protons in 2-bromoethan(2H)ol (BrEtOD) obtained in a reaction between ethylene with non-equilibrium nuclear spin order and bromine water. Given all protons in ethylene are magnetically equivalent, its nuclear states are classified into nuclear spin isomers (NSIM) with total spin I = 2,1,0. Addition of parahydrogen to acetylene produces ethylene with a population of only those NSIMs with I = 1,0. As a result of the reaction with bromine water the non-equilibrium spin order of ethylene is partly transferred to the singlet LLS involving the two methylene groups of BrEtOD. The 1H NMR signal enhancement (SE≈200) obtained as a result of the LLS readout is approximately equal to the SE of the hyperpolarized BrEtOD obtained with a single π/4 pulse. The LLS relaxation time (TLLS) was shown to be approximately 40 s (≈8T1) in the argon-bubbled sample.

A potential alternative to fungicides using actives-free (meth)acrylate polymers for protection of wheat crops from fungal attachment and infection.

Fungicidal compounds are actives widely used for crop protection from fungal infection, but they can also kill beneficial organisms, enter the food chain and promote resistant pathogen strains from overuse. Here we report the first field crop trial of homopolymer materials that prevent fungal attachment, showing successful crop protection via an actives-free approach. In the trial, formulations containing two candidate polymers were applied to young wheat plants that were subject to natural infection with the wheat pathogen Zymoseptoria tritici. A formulation containing one of the candidate polymers, poly(di(ethylene glycol) ethyl ether acrylate) (abbreviated DEGEEA), produced a significant reduction (26%) in infection of the crop by Z. tritici, delivering protection against fungal infection that compared favourably with three different commercially established fungicide programmes tested in parallel. Furthermore, the sprayed polymers did not negatively affect wheat growth. The two lead polymer candidates were initially identified by bio-performance testing using in vitro microplate- and leaf-based assays and were taken forward successfully into a programme to optimize and scale-up their synthesis and compound them into a spray formulation. Therefore, the positive field trial outcome has also established the validity of the smaller-scale, laboratory-based bioassay data and scale-up methodologies used. Because fungal attachment to plant surfaces is a first step in many crop infections, this non-eluting polymer: (i) now offers significant potential to deliver protection against fungal attack, while (ii) addressing the fourth and aligning with the eleventh principles of green chemistry by using chemical products designed to preserve efficacy of function while reducing toxicity. A future focus should be to develop the material properties for this and other applications including other fungal pathogens.

Adaptation to sorbic acid in low sugar promotes resistance of yeast to the preservative.

The weak acid sorbic acid is a common preservative used in soft drink beverages to control microbial spoilage. Consumers and industry are increasingly transitioning to low-sugar food formulations, but potential impacts of reduced sugar on sorbic acid efficacy are barely characterised. In this study, we report enhanced sorbic acid resistance of yeast in low-glucose conditions. We had anticipated that low glucose would induce respiratory metabolism, which was shown previously to be targeted by sorbic acid. However, a shift from respiratory to fermentative metabolism upon sorbic acid exposure of Saccharomyces cerevisiae was correlated with relative resistance to sorbic acid in low glucose. Fermentation-negative yeast species did not show the low-glucose resistance phenotype. Phenotypes observed for certain yeast deletion strains suggested roles for glucose signalling and repression pathways in the sorbic acid resistance at low glucose. This low-glucose induced sorbic acid resistance was reversed by supplementing yeast cultures with succinic acid, a metabolic intermediate of respiratory metabolism (and a food-safe additive) that promoted respiration. The results indicate that metabolic adaptation of yeast can promote sorbic acid resistance at low glucose, a consideration for the preservation of foodstuffs as both food producers and consumers move towards a reduced sugar landscape.

Impact of intraspecific variation in insect microbiomes on host phenotype and evolution.

Microbes can be an important source of phenotypic plasticity in insects. Insect physiology, behaviour, and ecology are influenced by individual variation in the microbial communities held within the insect gut, reproductive organs, bacteriome, and other tissues. It is becoming increasingly clear how important the insect microbiome is for insect fitness, expansion into novel ecological niches, and novel environments. These investigations have garnered heightened interest recently, yet a comprehensive understanding of how intraspecific variation in the assembly and function of these insect-associated microbial communities can shape the plasticity of insects is still lacking. Most research focuses on the core microbiome associated with a species of interest and ignores intraspecific variation. We argue that microbiome variation among insects can be an important driver of evolution, and we provide examples showing how such variation can influence fitness and health of insects, insect invasions, their persistence in new environments, and their responses to global environmental changes. A and B are two stages of an individual or a population of the same species. The drivers lead to a shift in the insect associated microbial community, which has consequences for the host. The complex interplay of those consequences affects insect adaptation and evolution and influences insect population resilience or invasion.

Increased Free Radical Generation during the Interaction of a Quinone-Quinoline Chelator with Metal Ions and the Enhancing Effect of Light.

Schiff bases and similar molecules forming metal complexes may cause redox effects, which may also be influenced by light. Anthraquinones such as doxorubicin and idarubicin are widely used antitumor agents, which can generate reactive oxygen species (ROS), stimulated by both the presence of iron and copper ions and also by light. The generated ROS can cause DNA scission, cell membrane oxidation, and many other toxic effects. The redox activity of the quinone-quinoline chelator 2-phenyl-4-(butylamino)naphtho [2,3-h]quinoline-7,12-dione (Q1) was investigated in the presence of iron, copper, and zinc. The influence of light in these interactions was also examined. The chemically induced dynamic nuclear polarization (CIDNP), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) methods were used to elucidate the molecular changes and ROS generation effects of the Q1 metal interactions. A model electron transfer reaction system between 1,4-dihydropyridine and Q1 was utilized to demonstrate that the chelate complexes of Q1 with both Fe(III) and Cu(II) ions were more redox active than Q1 itself. Similarly, CIDNP and NMR data showed that the concentration dependence of the free radicals yield is much higher in the presence of Fe(III) and Cu(II) ions, in comparison to Zn(II), and also that it increased in the presence of light. These findings underline the role of transition metal ions and Q1 in cyclic redox chain reactions and increase the prospect of the development of copper- and iron-based chelating agents, including Q1 and its derivatives, for anticancer therapy. Furthermore, these findings also signify the effect of light on enhancing ROS formation by Q1 and the prospect of utilizing such information for designing target specific anticancer drugs for photodynamic therapy.

Exploring the Effects of Residence Time on the Utility of Stable Isotopes and S/C Ratios as Proxies for Ocean Connectivity.

Various geochemical proxies have been developed to determine if ancient sedimentary strata were deposited in marine or nonmarine environments. A critical parameter for proxy reliability is the residence time of aqueous species in seawater, which is rarely considered for proxies relying on stable isotopes and elemental abundance ratios. Differences in residence time may affect our ability to track geologically short-lived alternations between marine and nonmarine conditions. To test this effect for sulfur and nitrogen isotopes and sulfur/carbon ratios, we investigated a stratigraphic section in the Miocene Oberpullendorf Basin in Austria. Here, previous work revealed typical seawater-like rare earth element and yttrium (REY) systematics transitioning to nonmarine-like systematics. This shift was interpreted as a brief transition from an open marine depositional setting to a restricted embayment with a reduced level of exchange with the open ocean and possibly freshwater influence. Our isotopic results show no discernible response in carbonate-associated sulfate sulfur isotopes and carbon/sulfur abundance ratios during the interval of marine restriction inferred from the REY data, but nitrogen isotopes show a decrease by several permil. This observation is consistent with the much longer residence time of sulfate in seawater compared with REY and nitrate. Hence, this case study illustrates that the residence time is a key factor for the utility of seawater proxies. In some cases, it may make geochemical parameters more sensitive to marine water influx than paleontological observations, as in the Oberpullendorf Basin. Particular care is warranted in deep time, when marine residence times likely differ markedly from the modern.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Evaluating the potential of natural product combinations with sorbic acid for improving preservative action against food-spoilage yeasts.

Fungal control methods commonly involve the use of antifungals or preservatives, which can raise concerns about broader effects of these stressors on non-target organisms, spread of resistance and regulatory hurdles. Consequently, control methods enabling lower usage of such stressors are highly sought, for example chemical combinations that synergistically inhibit target-organisms. Here, we investigated how well such a principle extends to improving efficacy of an existing but tightly controlled food preservative, sorbic acid. A screen of ∼200 natural products for synergistic fungal inhibition in combinations with sorbic acid, in either 2% or 0.1% (w/v) glucose to simulate high or reduced-sugar foods, did not reveal reproducible synergies in either of the spoilage yeast species Saccharomyces cerevisiae or Zygosaccharomyces bailii. Potentially promising screen candidates (e.g. lactone parthenolide, ethyl maltol) or a small additional panel of rationally-selected compounds (e.g. benzoic acid) all gave Fractional Inhibitory Concentration Indices (FICI) ≥ 0.5 in combinations with sorbic acid, corroborating absence of synergy in either glucose condition (although FICI values did differ between the glucose conditions). Synergies were not achieved either in a tripartite combination with screen candidates or in a soft-drink formulation as matrix. In previous work with other stressors synergy 'hits' have been comparatively frequent, suggesting that sorbic acid could be unusually resistant to forming synergies with other potential inhibitors and this may relate to the weak acid's known multifactorial inhibitory-actions on cells. The study highlights a challenge in developing appropriate natural product or other chemical combinations applicable to food and beverage preservation.

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids.

Prenylated isoflavonoids are phytochemicals with promising antifungal properties. Recently, it was shown that glabridin and wighteone disrupted the plasma membrane (PM) of the food spoilage yeast Zygosaccharomyces parabailii in distinct ways, which led us to investigate further their modes of action (MoA). Transcriptomic profiling with Z. parabailii showed that genes encoding transmembrane ATPase transporters, including Yor1, and genes homologous to the pleiotropic drug resistance (PDR) subfamily in Saccharomyces cerevisiae were upregulated in response to both compounds. Gene functions involved in fatty acid and lipid metabolism, proteostasis, and DNA replication processes were overrepresented among genes upregulated by glabridin and/or wighteone. Chemogenomic analysis using the genome-wide deletant collection for S. cerevisiae further suggested an important role for PM lipids and PM proteins. Deletants of gene functions involved in biosynthesis of very-long-chain fatty acids (constituents of PM sphingolipids) and ergosterol were hypersensitive to both compounds. Using lipid biosynthesis inhibitors, we corroborated roles for sphingolipids and ergosterol in prenylated isoflavonoid action. The PM ABC transporter Yor1 and Lem3-dependent flippases conferred sensitivity and resistance, respectively, to the compounds, suggesting an important role for PM phospholipid asymmetry in their MoAs. Impaired tryptophan availability, likely linked to perturbation of the PM tryptophan permease Tat2, was evident in response to glabridin. Finally, substantial evidence highlighted a role of the endoplasmic reticulum (ER) in cellular responses to wighteone, including gene functions associated with ER membrane stress or with phospholipid biosynthesis, the primary lipid of the ER membrane. IMPORTANCE Preservatives, such as sorbic acid and benzoic acid, inhibit the growth of undesirable yeast and molds in foods. Unfortunately, preservative tolerance and resistance in food spoilage yeast, such as Zygosaccharomyces parabailii, is a growing challenge in the food industry, which can compromise food safety and increase food waste. Prenylated isoflavonoids are the main defense phytochemicals in the Fabaceae family. Glabridin and wighteone belong to this group of compounds and have shown potent antifungal activity against food spoilage yeasts. The present study demonstrated the mode of action of these compounds against food spoilage yeasts by using advanced molecular tools. Overall, the cellular actions of these two prenylated isoflavonoids share similarities (at the level of the plasma membrane) but also differences. Tryptophan import was specifically affected by glabridin, whereas endoplasmic reticulum membrane stress was specifically induced by wighteone. Understanding the mode of action of these novel antifungal agents is essential for their application in food preservation.

Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis.

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure-function relationships to bring more nanomedicines to the clinic.

The Influence of Heteroresistance on Minimum Inhibitory Concentration, Investigated Using Weak-Acid Stress in Food Spoilage Yeasts.

Populations of microbial cells may resist environmental stress by maintaining a high population-median resistance (IC50) or, potentially, a high variability in resistance between individual cells (heteroresistance); where heteroresistance would allow certain cells to resist high stress, provided the population was sufficiently large to include resistant cells. This study sets out to test the hypothesis that both IC50 and heteroresistance may contribute to conventional minimal inhibitory concentration (MIC) determinations, using the example of spoilage-yeast resistance to the preservative sorbic acid. Across a panel of 26 diverse yeast species, both heteroresistance and particularly IC50 were positively correlated with predicted MIC. A focused panel of 29 different isolates of a particular spoilage yeast was also examined (isolates previously recorded as Zygosaccharomyces bailii, but genome resequencing revealing that several were in fact hybrid species, Z. parabailii and Z. pseudobailii). Applying a novel high-throughput assay for heteroresistance, it was found that IC50 but not heteroresistance was positively correlated with predicted MIC when considered across all isolates of this panel, but the heteroresistance-MIC interaction differed for the individual Zygosaccharomyces subspecies. Z. pseudobailii exhibited higher heteroresistance than Z. parabailii whereas the reverse was true for IC50, suggesting possible alternative strategies for achieving high MIC between subspecies. This work highlights the limitations of conventional MIC measurements due to the effect of heteroresistance in certain organisms, as the measured resistance can vary markedly with population (inoculum) size. IMPORTANCE Food spoilage by fungi is a leading cause of food waste, with specialized food spoilage yeasts capable of growth at preservative concentrations above the legal limit, in part due to heteroresistance allowing small subpopulations of cells to exhibit extreme preservative resistance. Whereas heteroresistance has been characterized in numerous ecological contexts, measuring this phenotype systematically and assessing its importance are not encompassed by conventional assay methods. The development here of a high-throughput method for measuring heteroresistance, amenable to automation, addresses this issue and has enabled characterization of the contribution that heteroresistance may make to conventional MIC measurements. We used the example of sorbic acid heteroresistance in spoilage yeasts like Zygosaccharomyces spp., but the approach is relevant to other fungi and other inhibitors, including antifungals. The work shows how median resistance, heteroresistance, and inoculum size should all be considered when selecting appropriate inhibitor doses in real-world antimicrobial applications such as food preservation.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

Cell Membrane State, Permeability, and Elasticity Assessment for Single Cells and Cell Ensembles.

The phase state and especially phase transitions of synthetic lipid membranes are known to drastically modulate mechanical membrane properties like permeability and bending modulus. Although the main transition of lipid membranes is typically detected employing differential scanning calorimetry (DSC), this technique is not suitable for many biological membranes. Moreover, often single cell data on the membrane state or order is of interest. We here first describe how to use a membrane polarity-sensitive dye, Laurdan, to optically determine the order of cell ensembles over a wide temperature range from T = -40 °C to +95 °C. This allows to quantify the position and width of biological membrane order-disorder transitions. Second, we show that the distribution of membrane order within a cell ensemble allows for correlation analysis of membrane order and permeability. Third, combining the technique with conventional atomic force spectroscopy allows for the quantitative correlation of an overall effective Young's modulus of living cells with the membrane order.