TDP-43-M323K causes abnormal brain development and progressive cognitive and motor deficits associated with mislocalised and increased levels of TDP-43.

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

Exploring intermixed magnetic nanoparticles: insights from atomistic spin dynamics simulations.

Binary nanoparticles, composed of both rare-earth elements with substantial magnetic properties and transition metals known for their high magnetic ordering temperatures, hold great promise as innovative materials for novel magnetic applications. In this study, we employ an atomistic spin dynamics framework to investigate how the magnetic properties change at finite temperatures in mixed NiGd nanoparticles. We specifically examine parameters such as saturation magnetization and spin-reorientation in relation to the nanoparticle's size, which ranges from 4 nm to 16 nm, and composition. Our findings reveal that Ni75Gd25 nanoparticles demonstrate exceptional magnetic properties at finite temperatures, marked by significantly increased saturation magnetizations and magnetic ordering temperatures. In contrast, nanoparticles containing 50% and 75% Gd contents exhibit notably reduced saturation magnetizations and magnetic ordering temperatures. Theoretical findings of our study shed light on the pivotal role that the Gd content plays in determining the magnetic behaviour at finite temperatures.

Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs.

Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.

Erratum: Fundamental Physics Implications for Higher-Curvature Theories from Binary Black Hole Signals in the LIGO-Virgo Catalog GWTC-1 [Phys. Rev. Lett. 123, 191101 (2019)].

This corrects the article DOI: 10.1103/PhysRevLett.123.191101.

Acoustic Energy Harvesting and Sensing via Electrospun PVDF Nanofiber Membrane.

This paper introduces a new usage of piezoelectric poly (vinylidene fluoride) (PVDF) electrospun nanofiber (NF) membrane as a sensing unit for acoustic signals. In this work, an NF mat has been used as a transducer to convert acoustic signals into electric voltage outcomes. The detected voltage has been analyzed as a function of both frequency and amplitude of the excitation acoustic signal. Additionally, the detected AC signal can be retraced as a function of both frequency and amplitude with some wave distortion at relatively higher amplitudes and within a certain acoustic spectrum region. Meanwhile, the NFs have been characterized through piezoelectric responses, beta sheet calculations and surface morphology. This work is promising as a low-cost and innovative solution to harvest acoustic signals coming from wide resources of sound and noise.

Impaired Mitochondrial Fatty Acid Synthesis Leads to Neurodegeneration in Mice.

There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties ∼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.

Emerging Mechanisms of Drug Resistance in Candida albicans.

Drug resistance mechanisms in the commensal human pathogen Candida albicans are continually evolving. Over time, Candida species have implemented diverse strategies to vanquish the effects of various classes of drugs, thereby emanating as a serious life threat. Apart from the repertoire of well-established strategies, which predominantly comprise permeability constraints, increased drug efflux or compromised drug import, alteration, overexpression of drug targets, and chromosome duplication, C. albicans has evolved novel regulatory mechanisms of drug resistance. For instance, recent evidences point to newer circuitry involving different mediators of the stress-responsive machinery of oxidative, osmotic, thermal, nitrosative, and nutrient limitation, which contribute to the emergence of drug resistance. Contemporary advances in genome-wide studies of transcription factors, for instance, the Zn2Cys6 transcription factors, TAC1 (transcriptional activator of CDR) in Candida albicans, or YRR1 in yeast have made it feasible to dissect their involvement for the elucidation of unexplored regulatory network of drug resistance. The coordination of implementers of the conventional and nonconventional drug resistance strategies provides robustness to this commensal human pathogen. In this review, we shed light not only on the established strategies of antifungal resistance but also discuss emerging cellular circuitry governing drug resistance of this human pathogen.

Genetic modifications of Mecr reveal a role for mitochondrial 2-enoyl-CoA/ACP reductase in placental development in mice.

Mitochondrial fatty acid synthesis (mtFAS) is an underappreciated but highly conserved metabolic process, indispensable for mitochondrial respiration. It was recently reported that dysfunction of mtFAS causes childhood onset of dystonia and optic atrophy in humans (MEPAN). To study the role of mtFAS in mammals, we generated three different mouse lines with modifications of the Mecr gene, encoding mitochondrial enoyl-CoA/ACP reductase (Mecr). A knock-out-first type mutation, relying on insertion of a strong transcriptional terminator between the first two exons of Mecr, displayed embryonic lethality over a broad window of time and due to a variety of causes. Complete removal of exon 2 or replacing endogenous Mecr by its functional prokaryotic analogue fabI (Mecrtm(fabI)) led to more consistent lethality phenotypes and revealed a hypoplastic placenta. Analyses of several mitochondrial parameters indicate that mitochondrial capacity for aerobic metabolism is reduced upon disrupting mtFAS function. Further analysis of the synthetic Mecrtm(fabI) models disclosed defects in development of placental trophoblasts consistent with disturbed peroxisome proliferator-activated receptor signalling. We conclude that disrupted mtFAS leads to deficiency in mitochondrial respiration, which lies at the root of the observed pantropic effects on embryonic and placental development in these mouse models.

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast.

Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast Saccharomyces cerevisiae but also in the pathogenic yeast Candida albicans We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of S. cerevisiae, Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in C. albicans, Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.

Uses for humanised mouse models in precision medicine for neurodegenerative disease.

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer's Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.

Multidrug transporters of Candida species in clinical azole resistance.

Over-expression of the human P-glycoprotein (P-gp) in tumor cells is a classic example of an ABC protein serving as a hindrance to effective chemotherapy. The existence of proteins homologous to P-gp in organisms encompassing the entire living kingdom highlights extrusion of drugs as a general mechanism of multidrug resistance. Infections caused by opportunistic human fungal pathogens such as Candida species are very common and has intensified in recent years. The typical hosts, who possess suppressed immune systems due to conditions such as HIV and transplantation surgery etc., are prone to fungal infections. Prolonged chemotherapy induces fungal cells to eventually develop tolerance to most of the antifungals currently in clinical use. Amongst other prominent mechanisms of antifungal resistance such as manipulation of the drug target, rapid efflux achieved through overexpression of multidrug transporters has emerged as a major resistance mechanism for azoles. Herein, the azole-resistant clinical isolates of Candida species utilize a few select efflux pump proteins belonging to the ABC and MFS superfamilies, to deter the toxic accumulation of therapeutic azoles and thus, facilitating cell survival. In this article, we summarize and discuss the clinically relevant mechanisms of azole resistance in Candida albicans and non-albicans Candida (NAC) species, specifically highlighting the role of multidrug efflux proteins in the phenomenon.

Fundamental Physics Implications for Higher-Curvature Theories from Binary Black Hole Signals in the LIGO-Virgo Catalog GWTC-1.

Gravitational-wave astronomy offers not only new vistas into the realm of astrophysics, but it also opens an avenue for probing, for the first time, general relativity in its strong-field, nonlinear, and dynamical regime, where the theory's predictions manifest themselves in their full glory. We present a study of whether the gravitational-wave events detected so far by the LIGO-Virgo scientific collaborations can be used to probe higher-curvature corrections to general relativity. In particular, we focus on two examples: Einstein-dilaton-Gauss-Bonnet and dynamical Chern-Simons gravity. We find that the two events with a low-mass m≈7 M_{⊙} BH (GW151226 and GW170608) place stringent constraints on Einstein-dilaton-Gauss-Bonnet gravity, α_{EDGB}^{1/2}≲5.6 km, whereas dynamical Chern-Simons gravity remains unconstrained by the gravitational-wave observations analyzed.

Non-heat shock responsive roles of HSF1 in Candida albicans are essential under iron deprivation and drug defense.

Recently, we have reported that the conditional mutant of the heat shock factor-1 (HSF1) in Candida albicans displays enhanced susceptibility not only towards a plant alkaloid, berberine, but also to diverse antifungal drugs. The present study attempts to identify additional phenotypes highlighting the non-heat shock responsive roles of HSF1 that could be correlated with the enhanced drug susceptibility. We uncover an intricate relationship between cellular iron and HSF1 mediated drug susceptibility of C. albicans. Interestingly, at 30°C, the conditional deletion of HSF1 while presented no growth defect, exhibited low intracellular iron. Notably, exogenous supplementation of iron reversed growth defects of HSF1 mutant when grown at 37°C. We provide evidence that the HSF1 mutant presents interesting phenotypes at basal conditions and are implicated in enhanced drug susceptibilities, dysfunctional mitochondria, decreased resistance towards oxidative stress and compromised cell wall integrity, all of which could be fully reversed upon iron supplementation. The HSF1 mutant also displayed defective filamentation at basal conditions under various solid hypha inducing media. Further, chelation of iron of HSF1 mutant cells led to severe growth defects and apparently triggers an iron starvation signal in the cell thus, demonstrating that HSF1 is essential for C. albicans cells to tolerate the iron deprivation stress. Together, apart from the well-established roles of HSF1 in reciprocation of thermal stress, this study extends its role under basal conditions and provides molecular insights into the role of HSF1 in iron deprivation and drug defense of C. albicans.

Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.

Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both ß-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Phytol is lethal for Amacr-deficient mice.

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.

Implicit Social Biases in People With Autism.

Implicit social biases are ubiquitous and are known to influence social behavior. A core diagnostic criterion of autism spectrum disorders (ASD) is abnormal social behavior. We investigated the extent to which individuals with ASD might show a specific attenuation of implicit social biases, using Implicit Association Tests (IATs) involving social (gender, race) and nonsocial (nature, shoes) categories. High-functioning adults with ASD showed intact but reduced IAT effects relative to healthy control participants. We observed no selective attenuation of implicit social (vs. nonsocial) biases in our ASD population. To extend these results, we supplemented our healthy control data with data collected from a large online sample from the general population and explored correlations between autistic traits and IAT effects. We observed no systematic relationship between autistic traits and implicit social biases in our online and control samples. Taken together, these results suggest that implicit social biases, as measured by the IAT, are largely intact in ASD.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

Cholesterol is catabolized to bile acids by peroxisomal ß-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this ß-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae.

Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell.