Methods Development for the Fluorescent Determination of Inorganic Phosphate and Phospho-Proteins

The micronutrient orthophosphate loss into the aquatic environment is one of the primary drivers of harmful algal blooms (HABs) which result in the contamination of drinking water and negatively affects recreation and tourism industries. The aim of this study was to develop fluorescence-based Pi sensors with sensitivity in the low micromolar range Various solid supports were surveyed as potential sensor optimizing components. Quinacrine mustard dihydrochloride bound to Sephadex® LH-20 yielded fluorescence detection of inorganic phosphate concentrations between 0.5-1.25 mM. Quercetin-Al (III), shared a direct negative fluorescence relationship with inorganic phosphate concentrations between 0-1 mM, in aqueous solutions of pH 5. Binding studies of quercetin-Zr (IV) led to an extended investigation for the detection of phospho-biomolecules. Phosphorylation of proteins are prevalent post-translational modifications responsible for signal transduction pathways and gene expression. As such, our quercetin-Zr (IV) probe was studied as a simple and cost-effective stain for phosphorylated proteins immobilized on nitrocellulose membrane. Our studies also explored the potential of an aqueous phosphatase assay with the o-aminobenzoyl-phosphoserine as a potential probe. These studies yielded significant fluorescence differences between phosphorylated and nonphosphorylated L-serine amino acid conjugates in the presence of lanthanide metal, europium (III). These findings can be further utilized for the optimization of inorganic phosphate and phospho-protein/peptide assays

Building a Toolbox for Drug Delivery: Lipid-based Conjugated Polymer Nanoparticles

Conjugated polymer nanoparticles, or CPNs, are highly versatile nano-structured materials that have amassed great interest due to their straightforward synthesis, biocompatibility, and tunable properties.1 The properties of CPNs can be tuned by varying the composition of the surfactant conjugated to the polymer core of these nanoparticles, rendering them suitable for a variety of applications including many in the realm nanomedicine.1 This tunability is key for the design of new drug-delivery systems and therapeutics as the CPN size and structure directly impact important properties, such as the blood brain barrier (BBB) permeability and drug target selectivity.2 Similarly, lipids and lipid-based nanomaterials have been widely studied as nanocarriers transporting various therapeutics in drug delivery systems because of their non-toxic and biocompatible nature.3-5 In this work, the synthesis of an isoindigo - based CPN system is demonstrated with four different lipid surfactants; – DMPC (14:0), DMPS (14:0), DPPC (16:0) and DPPS (16:0). The size, morphology and fluorescence properties of the resulting nanoparticles have been characterized using dynamic light scattering (DLS), small angle neutron scattering (SANS), transmission electron microscopy (TEM), and fluorescence spectroscopy. The development of this lipid-containing CPN system places an emphasis on elucidating lipid – CPN structural relationships by harnessing the differences in the properties of lipids to control the shape and size of the CPNs produced. The resulting lipid-CPN systems and the new structure-relationships unraveled in this work contribute to the refinement of nanomedicine by unveiling novel design criteria in nanomaterials. This new knowledge will open new avenues for improved efficiency in treatments and consequently establish a novel family of nanomaterials as an alternative drug delivery system for cancer treatment. References: 1. Tuncel, D.; Demir, H. V., Conjugated polymer nanoparticles. Nanoscale 2010, 2 (4), 484-494. 2. Rizvi, S. A. A.; Saleh, A. M., Applications of nanoparticle systems in drug delivery technology. Saudi Pharm J 2018, 26 (1), 64-70. 3. Yingchoncharoen, P.; Kalinowski, D. S.; Richardson, D. R., Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and What Is Yet to Come. Pharmacol Rev 2016, 68 (3), 701-787. 4. Scioli Montoto, S.; Muraca, G.; Ruiz, M. E., Solid Lipid Nanoparticles for Drug Delivery: Pharmacological and Biopharmaceutical Aspects. Frontiers in Molecular Biosciences 2020, 7. 5. Bhalekar, M. R.; Madgulkar, A. R.; Desale, P. S.; Marium, G., Formulation of piperine solid lipid nanoparticles (SLN) for treatment of rheumatoid arthritis. Drug Development and Industrial Pharmacy 2017, 43 (6), 1003-1010

Covalently Functionalized Sawdust for the Remediation of Phosphate from Agricultural Wastewater

Phosphate remediation from wastewater is rapidly becoming an ever more attractive process due to a combination of both the economic pressure of increasing phosphate scarcity and the environmental damage caused by untreated agricultural runoff. Ideally, remediated phosphate will be recoverable and would be able to be reused as fertilizer. Many different resins have been investigated, but due to the scale of the challenge, any feasible solution will involve the use of very inexpensive waste products as the solid support. Sawdust, functionalized with iron-binding ligands, is such a potential resin. Sawdust alone binds 0.3 g/kg of phosphate which is insufficient. Iron has a strong affinity for phosphate, making the formation of iron-phosphate bonds a promising avenue for the development of recyclable resins. Previously prepared iron-chitosan complexes bound 8.2 g/kg. However, as the price of chitosan has rapidly increased, alternatives are required. In this current study, the covalent modification of the sawdust using either carboxymethylcellulose-supported ligands, or direct functionalization of the sawdust can increase this to 40 g/kg using ethylene diamine as the iron-binding ligand. Binding decreases over repeated cycles of phosphate exposure and elution, but can be fully restored through regeneration using iron salts. The simple green synthesis of this material, and the iron-binding capability of the investigated ligands is discussed. These sawdust-based resins show promise as potential candidates for industrial-scale phosphate recovery efforts in the future

Emerging targeted therapies for melanoma treatment (Review)

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Evolution of Rabies in South America and Inter-Species Dynamics (2009–2018)

Rabies is listed as one of the World Health Organisation’s (WHO) Neglected Tropical Diseases Worldwide, with a significant impact in South America. This paper explores the dynamics of rabies cases in humans, pets (dogs and cats), livestock and wildlife (bats in particular) in South America during the period 2009–2018. The data used in this study were derived from the two main databases for rabies in South America: the OIE-WAHIS from the World Organisation for Animal Health (OIE) and PANAFTOSA’s Regional Information System for the Epidemiological Surveillance of Rabies (SIRVERA). Being a neglected disease with possible underreporting in some areas, the reported rabies cases may not always represent the real disease burden. The analysis focuses on the evolution of the number of cases in time and their spatial distribution, as well as on the main source of infections in humans, determined by laboratory assays of the antigenic variant or through epidemiological investigations. Additionally, Generalised Linear Mixed Models (GLMM) were used to evaluate the risk factors associated with the occurrence of human cases. Our results show that the highest impact of the disease in terms of number of cases was reported on livestock, while the overall number of cases (in animals and humans) progressively decreased along the study period. The spatial distribution of rabies in livestock showed two main clusters in the north-western (mainly Colombia) and in the south-eastern part of the affected area (Brazil), and a third smaller cluster in Peru. A cluster in dogs was observed in Bolivia. Out of the 192 human cases reported during the study period, 70% of them were transmitted by bats. The number of human cases reported during the study period were significantly associated with the number of rabies cases reported in livestock, pets and wildlife. Despite the overall decreasing case report rate, the disease still represents a major animal and public health concern in South America, and new strategies for compiling systematic information, networking and education are needed, as well as the education and training of veterinary staff

Asymmetric Side-Chain Engineering In Semiconducting Polymers: A Platform For Greener Processing and Post-Functionalization of Organic Electronics

Organic semiconducting polymers are a powerful platform for the design of next-generation technologies due to their excellent optoelectronic properties and solution processability, allowing access to low-cost and scalable manufacturing techniques such as spin-coating, slot-die coating and roll-to-roll printing. However, their extended π-conjugation results in low solubility, requiring the use of toxic halogenated solvents to generate thin films and devices. Furthermore, accessible post-functionalization of semiconductors toward the development of multifunctional devices and sensors remains a challenge due to limited solid-state chemistry for alkyl side chains. In this work, an asymmetric side-chain engineering approach was used to introduce terminal hydroxyl moieties alongside traditional solubilizing branched alkyl chains into an isoindigo-based polymer. The hydroxyl moieties led to significantly improved processability in alcohol-based solvents without sacrificing electronic performance in thin film organic field-effect transistors. Solid state morphologies of the thin films processed from both alcohol-based and traditional halogenated solvents were further characterized using atomic force microscopy and grazing incidence wide angle X-ray scattering. Additionally, Cryo-EM was utilized in order to characterize the role of asymmetric side-chain functionality in solution state aggregation. The versatility of this design was further probed using fluorescein isothiocyanate to directly functionalize the asymmetric polymer in thin film. This facile solid-state post-functionalization further demonstrates asymmetric side-chain engineering to be a viable approach toward the development of sustainably manufactured multifunctional electronics

Impairing Proliferation of Glioblastoma Multiforme with CD44+ Selective Conjugated Polymer Nanoparticles

Glioblastoma is one of the most aggressive types of cancer with median survival of only 15 months. Successful therapy is hampered by the existence of treatment resistant populations of stem-like tumour initiating cells (TICs) and poor blood-brain barrier drug penetration. Therapies capable of effectively targeting the TIC population are in high demand. Here, we synthesize spherical diketopyrrolopyrrole (DPP)-based conjugated polymer nanoparticles (CPNs) with an average diameter of 109 nm. The CPN were designed to include fluorescein-conjugated hyaluronic acid (HA), a ligand for the CD44 receptor present on one population of TICs. We demonstrate blood-brain barrier permeability of this system and concentration and cell cycle phase-dependent selective uptake of HA-CPNs in CD44 positive GBM-patient derived cultures. Interestingly, we found that uptake alone decreases stemness, invasive properties and proliferation of the CD44-TIC population in zebrafish PDX models in vivo. This study is the first to show surface moiety-driven selectivity of conjugated polymer nanoparticles in targeting TIC populations in brain cancer