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.

Design, Synthesis, and Pharmacological Evaluation of Novel Tacrine Derivatives as Multi-target ANTI-Alzheimer's Agents In Rat Models.

BACKGROUND: Alzheimer's disease is a progressive neurodegenerative disorder for which no curative drugs are available and treatment available is just palliative. OBJECTIVES: Current research focused on design of Tacrine-Flavone hybrids as multitargeted cholinesterase and monoamine oxidase B inhibitors. METHODS: A total of 10 Tacrine- Flavone hybrids were designed, synthesized and characterized. The in vitro neurotoxicity and hepatotoxicity of the synthesized compounds determined using SHSY5Y cell line and HEPG2 cell line. One most active compound (AF1) with least toxicity in in vitro studies was chosen for in vivo studies. Acute and subacute toxicity of the novel compound AF1 conducted on Wistar rats according to OECD guideline 423 and 407. The LD50 value of the novel compound calculated according to Finney's method using Probit analysis. Anti-Alzheimer's activity studies conducted on male Wistar rats. Behavioral studies conducted and AChE and MAO-B activity determined in rat brain. RESULTS AND DISCUSSION: All the compounds exhibited good inhibitory effect on MAO B and AChE. The neurotoxicity studies of the active compound AF1 did not show toxicity up to 100µg. The hepatotoxicity study of the most active compound AF1, showed the compound to be safe up to 200µg. The LD 50 value of the novel compound after a single oral administration was found to be 64 mg/kg bodyweight in rats. Subacute toxicity studies did not show any remarkable toxicity in the vital organs up to 40 mg/kg. Activity studies showed comparable results with standard at 20 mg/kg. CONCLUSION: The results showed that the novel Tacrine-Flavone hybrids are multitarget-directed ligands, which are safe and active compared to tacrine and can be a promising lead molecule for further study.

Mutation in the FUS nuclear localisation signal domain causes neurodevelopmental and systemic metabolic alterations.

Variants in the ubiquitously expressed DNA/RNA-binding protein FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS). Most FUS mutation studies have focused on motor neuron degeneration; little is known about wider systemic or developmental effects. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the pathogenic FUSDelta14 mutation in homozygosity. RNA sequencing of multiple organs aimed to identify pathways altered by the mutant protein in the systemic transcriptome, including metabolic tissues, given the link between ALS-frontotemporal dementia and altered metabolism. Few genes were commonly altered across all tissues, and most genes and pathways affected were generally tissue specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. Magnetic resonance imaging brain scans and histological characterisation revealed that homozygous FUSDelta14 brains were smaller than heterozygous and wild-type brains and displayed significant morphological alterations, including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. These findings show that the disease aetiology of FUS variants can include both neurodevelopmental and systemic alterations.

Optically active organic and inorganic nanomaterials for biological imaging applications: A review.

Recent advancements in the field of nanotechnology have enabled targeted delivery of drug agents in vivo with minimal side effects. The use of nanoparticles for bio-imaging has revolutionized the field of nanomedicine by enabling non-invasive targeting and selective delivery of active drug moieties in vivo. Various inorganic nanomaterials like mesoporous silica nanoparticles, gold nanoparticles, magnetite nanoparticles graphene-based nanomaterials etc., have been created for multimodal therapies with varied multi-imaging modalities. These nanomaterials enable us to overcome the disadvantages of conventional imaging contrast agents (organic dyes) such as lack of stability in vitro and in vivo, high reactivity, low-quantum yield and poor photo stability. Inorganic nanomaterials can be easily fabricated, functionalised and modified as per requirements. Recently, advancements in synthesis techniques, such as the ability to generate molecules and construct supramolecular structures for specific functionalities, have boosted the usage of engineered nanomaterials. Their intrinsic physicochemical properties are unique and they possess excellent biocompatibility. Inorganic nanomaterial research has developed as the most actively booming research fields in biotechnology and biomedicine. Inorganic nanomaterials like gold nanoparticles, magnetic nanoparticles, mesoporous silica nanoparticles, graphene-based nanomaterials and quantum dots have shown excellent use in bioimaging, targeted drug delivery and cancer therapies. Biocompatibility of nanomaterials is an important aspect for the evolution of nanomaterials in the bench to bedside transition. The conduction of thorough and meticulous study for safety and efficacy in well-designed clinical trials is absolutely necessary to determine the functional and structural relationship between the engineered nanomaterial and its toxicity. In this article an attempt is made to throw some light on the current scenario and developments made in the field of nanomaterials in bioimaging.

Non-invasive Analysis of Fiber Type Composition in Lower Limb Skeletal Muscles Using Reduced Interference Rihaczek Distribution.

Fiber composition is an important factor influencing force generation and endurance of different skeletal muscles. The analysis of the heterogeneous composition of muscles has gained importance in the field of sports biomechanics and biomedicine. In this work, an attempt is made to analyze the fiber composition of Rectus femoris (type II dominant) and Soleus (type I dominant) muscles using surface electromyography. Isometric signals are acquired from the muscles of 15 participants using a well-defined protocol and are further processed using reduced interference Rihaczek distribution. Instantaneous median frequency (IMDF) is extracted from the non-fatigue (NF) and fatigue (F) segments of the signals and is analyzed. From the distributions, it is found that the spectral power increases in the lower frequencies of the signal recorded from the rectus femoris and in the higher frequencies of signals recorded from the soleus during fatigue. The soleus is showing higher IMDF values than the rectus femoris in both segments. A reduction of 14% and an increase of 10% is observed in the IMDF during fatigue for rectus femoris and soleus, respectively. Thus, the extracted feature is found to be sensitive and statistically significant (p<0.05) to differentiate fiber types as well as the NF and F states of the two muscles.Clinical Relevance- This study may be extended to non-invasively analyze the fiber type shifts in muscles due to athletic training and pathological conditions.

Design Synthesis and in vitro Evaluation of Tacrine-flavone Hybrids as Multifunctional Cholinesterase Inhibitors for Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder. The multifactorial etiology of AD has led to the design of multitarget directed ligands (MTDL) for AD. Tacrine an acetylcholinesterase (AChE) inhibitor was the first FDA approved drug for AD but is discontinued due to hepatotoxicity. OBJECTIVE: Present research focused on incorporating a flavone to the tacrine nucleus to enhance the anti-Alzheimer's property of the tacrine with the synergistic effect of flavone which is a very good antioxidant. It is expected that the antioxidant property and hepatoprotective nature of flavones will reduce the hepatotoxic side effect of tacrine. METHODS: We designed and synthesized ten flavone substituted tacrine derivatives and evaluated for in vitro AChE and BuChE inhibitoy activity by modified Ellman's method using eeAChE and eqBuChE. In vitro antioxidant activity was studied by DPPH radical scavenging assay. Molecular modeling studies were conducted in Schrodinger and AutoDock Vina with TcAChE(PDB ID:1H23),hAChE(PDB ID:4EY7) and hBuChE(PDB ID:4TPK). RESULTS: All the compounds exhibited potent inhibitory effect on AChE and BuChE with IC50 values in µM concentration. The compounds exhibited very good antioxidant activity in DPPH radical scavenging assay. Among the compounds the compound AF1 showed highest activity with IC50 value of 0.93 µM for AChE and 1.48 µM for BuChE and also showed significant antioxidant activity (2.6 nM). A correlation graph was plotted for IC 50 values vs Dock score and the results are promising with r2 values of 0.62 and 0.73 for AChE and BuChE inhibition respectively which proved the reliability of docking approaches. CONCLUSION: The results highlighted the multifunctional nature of the novel Tacrine-Flavone hybrids and they may be promising MTDL for AD.

Erratum: Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

[This corrects the article DOI: 10.1016/j.isci.2021.103463.].

Use of biomass-derived biochar in wastewater treatment and power production: A promising solution for a sustainable environment.

Over the past few years, we are witnessing the advent of a revolutionary bioengineering technology in biochar production and its application in waste treatment and an important component in power generation devices. Biochar is a solid product, highly rich in carbon, whose adsorption properties are ideal for wastewater decontamination. Due to its high specific surface area to volume ratio, it can be utilized for many environmental applications. It has diverse applications in various fields. This review focuses on its various applications in wastewater treatment to remove various pollutants such as heavy metals, dyes, organic compounds, and pesticides. This review also highlights several energy-based applications in batteries, supercapacitors, and microbial fuel cells. It described information about the different feedstock materials to produce LB-derived biochar, the various conditions for the production process, i.e., pyrolysis and the modification methods of biochar for improving properties required for wastewater treatment. The present review helps the readers understand the importance of biochar in wastewater treatment and its application in power generation in terms of batteries, supercapacitors, microbial fuel cells, applications in fuel production, pollutant and dye removal, particularly the latest development on using LB-derived biochar. This review also highlights the economic and environmental sustainability along with the commercialization of biochar plants. It also describes various pyrolytic reactors utilized for biochar production.

ALS-related FUS mutations alter axon growth in motoneurons and affect HuD/ELAVL4 and FMRP activity.

Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3'UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

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.

Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

Submarine groundwater discharge and associated nutrient flux from southwest coast of India.

The nutrient discharge associated with submarine groundwater discharge (SGD) into the Arabian Sea has been investigated for the first time using the Radon isotope (222Rn) mass balance model at three sites along the southwest coast of India. The SGD flux varied in the range of 49.48×104 - 335.84×104 m3/day, with high (low) discharge during the low (high) tide. SGD delivers a considerable amount of the nutrient into the Arabian Sea with 2.10×104 to 11.66×104 mol/day dissolved inorganic nitrogen (DIN), 1.23×102 to 56.31×102 mol/day dissolved inorganic phosphate (DIP), and 7.28×104 to 24.44×104 mol/day dissolved silicate (DSi). This significant nutrient input to the coastal waters through SGD is mainly attributed to the land-use practices like agricultural activities, improper waste disposal, and thickly populated coastal settlement zones. The increase in the nutrient discharge may lead to phytoplankton bloom in the nearshore environment and can accelerate seasonal coastal hypoxia over the western Indian shelf. This evidence of considerably high nutrient flux through SGD advocates the importance of understanding SGD associated flux along the southwest coast of India to maintain a sustainable ecological balance.

Correction to: Binding of Brucella protein, Bp26, to select extracellular matrix molecules.

Following publication of the original article [1], an error was reported in the tagging of Eugene H. Johnson and Remya R. Nair in the author group.

Monilethrix: A Report of Three Cases in Children Confirmed with Dermoscopy.

Monilethrix is a rare hereditary disorder affecting hair resulting in hair fragility and alopecia. We report three patients of monilethrix who presented with complaints of sparse and brittle hair from early childhood. All three patients had multiple discrete hyperkeratotic papules over the scalp. Dermoscopy revealed beaded appearance of hair with the presence of elliptical nodes and intermittent constrictions on the hair shafts as well as broken hairs, which were confirmed with routine microscopic examination of hair. Dermoscopy helps in easier and faster diagnosis of monilethrix.

Binding of Brucella protein, Bp26, to select extracellular matrix molecules.

BACKGROUND: Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). RESULTS: ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein. CONCLUSIONS: Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

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.

Humanising the mouse genome piece by piece.

To better understand human health and disease, researchers create a wide variety of mouse models that carry human DNA. With recent advances in genome engineering, the targeted replacement of mouse genomic regions with orthologous human sequences has become increasingly viable, ranging from finely tuned humanisation of individual nucleotides and amino acids to the incorporation of many megabases of human DNA. Here, we examine emerging technologies for targeted genomic humanisation, we review the spectrum of existing genomically humanised mouse models and the insights such models have provided, and consider the lessons learned for designing such models in the future.