Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Inhibition Conversion of Aspirin into Salicylic Acid in Presence of Glycine.

Aspirin (AS) is a common drug having anti-pyretic and anti-inflammatory properties which is widely used in diverse medical conditions. The intake of AS may cause adverse effects such as gastrointestinal ulcer, tinnitus and Reye's syndrome. The adverse effects of AS arise due to conversion of AS into salicylic acid (SAL). Glycine (Gly) is a simplest non essential amino acid having anti-oxidative and anti-inflammatory effects. It also reduces the risk of obesity, hypertension, and diabetes mellitus. AS with Gly is well accepted form of the drug for the treatment of rheumatic conditions in comparisons to the bare AS. In the present work using UV-Visible absorption, fluorescence and DFT/ TD-DFT techniques confirmed that in presence of Gly inhibited the conversion of AS into SAL effectively.

Unveiling the Influence of Glutathione in Suppressing the Conversion of Aspirin to Salicylic Acid: A Fluorescence and DFT Study.

Aspirin is a commonly used nonsteroidal anti-inflammatory drug, associated with many adverse effects. The adverse effects of aspirin such as tinnitus, Reye's syndrome and gastrointestinal bleeding are caused due to conversion of aspirin into its active metabolite salicylic acid after oral intake. Glutathione is a naturally occurring antioxidant produced by the liver and nerve cells in the central nervous system. It helps to metabolize toxins, break down free radicles, and support immune function. This study aims to investigate and explore the possibility of inhibiting aspirin to salicylic acid conversion in presence of glutathione at a molecular level using spectroscopic techniques such as UV-Visible absorption, time-Resolved and time-dependent fluorescence and theoretical DFT/ TD-DFT calculations. The results of steady state fluorescence spectroscopy and time-dependent fluorescence indicated that the aspirin to salicylic acid conversion is considerably inhibited in presence of glutathione. Further, the results presented here might have significant clinical implications for individuals with variations in glutathione level.

Detection of Malachite Green in Water Using Edge Excited Label Free Fluorescent Probe NCQDs.

The fluorescent properties of nitrogen doped carbon quantum dots (NCQDs) prepared through microwave assisted green method has been used as label free fluorescent probe for selective and sensitive detection of malachite green (MG) in water. The optical responses revealed that the NCQDs are highly stable and have good fluorescent quantum yield. The NCQDs were used to detect the Malchite Green in Mili Q water. Reduction in the fluorescence response was monitored in the range 17.12-128.43 µM of MG dissolved in Mili Q water. Linear response was observed in the range, 10-80 µM. The calculated value of limit of detection is 5.16 µM and the sensitivity is (0.03536 ± 0.00001) µM-1. The future application of this work is that it can be employed to detect MG in the tap water and other natural sources of water.

Spectroscopic Studies of CDPy Molecule in Different Protic and Aprotic Solvents and Investigation of Antioxidant Property.

The properties of 3-Cyano-4, 6-Dimethyl-2-Pyridone (CDPy) were analyzed to study the antioxidant behavior. The UV-Visible absorption and fluorescence properties of CDPy have been studied in two protic (water and methanol) and two aprotic (acetonitrile and dimethyl sulfoxide) solvents. Its antioxidant properties were compared with well known antioxidant ascorbic acid. This compound, CDPy was found to exhibits moderate antioxidant properties. The experimental results were reproduced by theoretical density functional methods, which helped to understand the experimental result better.

Nitrogen Doped Carbon Quantum Dots Modified by Lens culinaris ß-Galactosidase as a Fluorescent Probe for Detection of Lactose.

Nitrogen doped carbon quantum dots (NCQDs) were synthesized via hydrothermal route. The NCQDs are thermally and optically stable with high flouresence yield. For the synthesis of NCQDs, citric acid and urea was taken as carbon and nitrogen sources, respectively. The Transmission Electron Microscopy (TEM) of these quantum dots revealed nearly spherical shape and average size of 1.5 nm, which was calculated using Image J software. The quantum dots were also well-characterized using spectroscopic techniques such as FTIR, UV-Visible absorption and fluorescence. These synthesized and characterized dots were utilized for selective detection of lactose in Milli Q water. The bioprobe provide a wide linear range varying from (10.00-77.41) µM with limit of detection 11.36 µM and sensitivity equal to (0.0065 ± 0.0002) µM-1. Graphical Abstract.

Probing self-associated intermolecular H-bonding using low-frequency SERS coupled with mid-IR SERS and DFT study: a case study of 2-MBA adsorbed on ZnO nanoparticles.

In the present study, low-frequency SERS is reported for the first time to investigate intermolecular interactions. Low-frequency SERS in the THz region (>50 cm-1) and the mid-IR region is used to probe the H-bonding interaction in 2-mercaptobenzoic acid (2-MBA) molecules adsorbed on the surface of ZnO nanoparticles. The self-association due to H-bonding leads to dimer formation of 2-MBA through carboxylic acid groups. The characteristic Raman bands of the 2-MBA dimer, H-bonded O-H stretching and out-of-plane O-H bending modes, are observed. Subsequently, this dimer formation causes the evolution of two new low-frequency modes at 90 cm-1 (shear dimer in-plane bending) and 110 cm-1 (shear dimer stretching) of intermolecular H-bonding and a blue-shift of the torsional mode of (-COOH) + (-SH). In the THz region (50-200 cm-1) the vibrational modes are blue-shifted, while in the mid-IR region the symmetric out-of-plane O-H bending is red-shifted. The present work shows that SERS can be used to study intermolecular H-bonding of molecules at very low concentrations.

Evidence of self-aggregation of cationic surfactants in a choline chloride+glycerol deep eutectic solvent.

Based on fluorescence probe, electrical conductivity, surface tension, small-angle X-ray/dynamic light scattering, and transmission electron microscopy experiments, we present the first clear lines of evidence for self-aggregation of cationic surfactants of the n-alkyltrimethylammonium family within an archetypical deep eutectic solvent comprised of a 1:2 molar mixture of choline chloride and glycerol. Estimated thermodynamic parameters suggest this self-aggregation process to be less entropically driven than that in water. These novel water-free self-assemblies might serve as dynamic soft templates to direct the growth of size- or shape-tailored nanoparticles within water-restricted media.

Involvement of the ß3-α3 loop of the proline dehydrogenase domain in allosteric regulation of membrane association of proline utilization A.

Proline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induce high affinity membrane association of PutA and relieve repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the ß3-α3 loop of the proline dehydrogenase domain (ßα)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the ß3-α3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analogue l-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the ß3-α3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain.

Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli.

The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain deletion analysis were used to obtain solution structural information for the 1320-residue PutA from Escherichia coli. Shape reconstructions show that PutA is a symmetric V-shaped dimer having dimensions of 205 × 85 × 55 Å. The particle consists of two large lobes connected by a 30-Å diameter cylinder. Domain deletion analysis shows that the N-terminal DNA-binding domain mediates dimerization. Rigid body modeling was performed using the crystal structure of the DNA-binding domain and a hybrid x-ray/homology model of residues 87-1113. The calculations suggest that the DNA-binding domain is located in the connecting cylinder, whereas residues 87-1113, which contain the two catalytic active sites, reside in the large lobes. The SAXS data and amino acid sequence analysis suggest that the Δ(1)-pyrroline-5-carboxylate dehydrogenase domains lack the conventional oligomerization flap, which is unprecedented for the aldehyde dehydrogenase superfamily. The data also provide insight into the function of the 200-residue C-terminal domain. It is proposed that this domain serves as a lid that covers the internal substrate channeling cavity, thus preventing escape of the catalytic intermediate into the bulk medium. Finally, the SAXS model is consistent with a cloaking mechanism of gene regulation whereby interaction of PutA with the membrane hides the DNA-binding surface from the put regulon thereby activating transcription.

A Case of Anaemia With High-Grade Splenomegaly.

In tropical areas, there are a variety of parasitic and nonparasitic causes of high-grade splenomegaly. An adolescent male patient with haemoglobin E/ß-thalassaemia came with high-grade splenomegaly and severe anaemia, requiring blood transfusions on a regular basis. Treatment with folic acid and antioxidant vitamins reduced the requirement for blood transfusions, brought haemoglobin levels back to near normal, and reduced splenic enlargement.  Haemoglobin E/ß-thalassaemia is a haematological condition that causes anaemia and high-grade splenomegaly in the tropics. Initially, the disease was only seen in Southeast Asia, but it has since spread around the world due to migration from that region.

Lymphocutaneous Nocardiosis in a Patient With Human Immunodeficiency/Tuberculosis Coinfection.

Nocardia spp. are Gram-positive bacteria, which are acid-fast as well. Nocardiosis is characterized by abscess formation anywhere in the body, especially in the lungs, brain, and skin. The disease manifests as pulmonary disease, brain abscess, or disseminated lesions in immunocompromised individuals. However, skin involvement in the form of lymphocutaneous abscess is found in immunocompetent individuals. Nocardia spp. appear as thin, branched filaments in fine needle aspirate under the microscope. Diagnosis of the nocardiosis is done by fine needle aspiration cytology (FNAC) and identification through matrix-assisted laser desorption/ionization-time of flight mass spectrometry from aspirated materials. Our case is lymphocutaneous nocardiosis in a patient having human immunodeficiency (HIV)/tuberculosis coinfection.

Interactions of a biological macromolecule with thermotropic liquid crystals: Applications of liquid crystals in biosensing platform.

Liquid crystal biosensor was developed based on a 4'-octyl-4-biphenylcarbonitrile (8CB) by adsorption of biological macromolecule bovine serum albumin (BSA) at the 8CB interface. BSA was detected by examining the changes in the director configurations of 8CB molecules under a polarizing optical microscope. The transitions in the director configuration were due to the non-covalent bonds. This technique demonstrated high sensitivity at a concentration of 100 µM of BSA. The binding events between the 8CB and BSA were investigated through molecular docking studies that confirmed the protein-ligand interaction. The most probable binding location of 8CB to dock with BSA were determined at a subdomain IB of Sudlow's site I. The active residues on analyzing were found to stabilize the 8CB molecules through different interactions. These active residues that were involved in the protein-ligand interaction were further confirmed with Raman spectroscopy. This study provided the vibrational properties and structural changes that occurred due to the various interactions between the 8CB and BSA. The results presented in this work lead to a potential biosensing tool for detecting and sensing proteins using LCs.

Thermotropic liquid crystals in the detection of albumins through a microscopic, spectroscopic and computational approach.

Liquid crystals (LCs) are a promising system of molecules for biosensing as a transducing agent for detecting protein human serum albumin (HSA). Herein, we investigate the detection of HSA by a liquid crystal 4'-octyl-4-biphenylcarbonitrile (8CB) intending to develop an LC-based biosensor. The change in the alignment of liquid crystal molecules in the presence of protein results in the transfigurations of the director through interactions. The limit of 8CB to detect HSA is found to be at a reliable concentration in the development of biosensors. The transition in the director configurations from radial to bipolar during the crystalline to the isotropic phase of the liquid crystals are studied under polarizing optical microscopy. These transitions confirm the detection of HSA by 8CB. The docking analysis depicts the interactions by which 8CB liquid crystal molecules bind with protein HSA. The binding energy, binding active residues and their distances between the docked residues of HSA and molecules of ligand 8CB are calculated by molecular docking. Temperature-dependent Raman spectroscopy is used to analyse the spectral behaviour of the interactions. The residues validated by molecular docking studies correlate well with the findings of Raman spectra for the interaction between 8CB and HSA.

Electrical Injury and Wandering Atrial Pacemaker.

The supply of household electricity remains a low-voltage (110-220 V) energy source, and its effects on the human body depend on several factors, including the type of contact and duration of contact, among other things. In a significant number of cases, direct contact with household electricity causes reversible cardiac arrhythmia-ventricular fibrillation, ventricular premature beats, atrial tachycardia, and atrial fibrillation. Wandering atrial pacemaker (WAP) is a benign atrial arrhythmia observed in elderly patients suffering from obstructive pulmonary diseases that result from an ischemic heart. This report discusses WAP as observed in a patient who suffered an electrical injury.

Late-Onset Dystrophinopathy.

Dystrophinopathy is a spectrum of muscular dystrophies resulting from absolute to relative deficiency of dystrophin - a protein essential for muscle fiber integrity. This includes a severe form called Duchenne muscular dystrophy, a mild form called Becker muscular dystrophy, and intermediate muscular dystrophy. Becker muscular dystrophy relates to late-onset and slow progression muscle dystrophy caused by deletions or duplications in the dystrophin gene. Individuals with this type of tardive slow progression have a life expectancy of 60 years. A patient in his late 40s presented this disease with duplication of exon 2 in the dystrophin gene.

Chronic Pulmonary Aspergillosis in a Patient With AIDS.

Chronic pulmonary aspergillosis (CPA) is caused by saprophytic fungi Aspergillus spp. Certain conditions predispose individuals to pulmonary aspergilloses, for example, neutropenia, prolonged steroid therapy, immunosuppressive drugs, and solid organ transplants. Individuals are infected with Aspergillus spores by inhalation. CPA is diagnosed through imaging features, such as cavities, fungal balls, peripheral air crescent signs, and the direct visualization of the Aspergillus spp. (microscopy or culture from biopsy) or immunological response to Aspergillus spp. (serum IgG confirms the diagnosis of CPA). All these should be present for at least three months. An Aspergillus infection is uncommon in those with the human immunodeficiency virus (HIV) due to intact phagocytic cell function. However, HIV-infected individuals with CD4+ T cell < 100 cells/mL are more likely to experience disease progression. Chronic tubercular cavities predispose one to the colonization of cavities with Aspergillus spp. When HIV advances to AIDS (acquired immunodeficiency syndrome), the aspergilloma transforms into an invasive form.

Potential liquid crystal-based biosensor depending on the interaction between liquid crystals and proteins.

Liquid crystal biosensors serve an essential role to detect biomolecules and chemical events as an effective, simple and early detection tool. The detection of Human serum albumin protein by a room temperature liquid crystal 4́-pentyl-4-biphenylcarbonitrile has been investigated using multiple techniques such as Polarizing optical microscope, Raman spectroscopy and molecular docking. The dynamics of director transfigurations of the liquid crystal molecule in the presence of protein through interactions are reported in the study. The change in the alignment of liquid crystal molecules during the nematic phase is observed under a polarizing optical microscope. The interactions through which the liquid crystal molecules bind with protein is depicted from the docking analysis. The residues in the active sites confirm their presence from docking studies. The spectral behaviour has been investigated by temperature-dependent Raman spectroscopy. The findings from Raman spectra for the interaction between these compounds correlates with the residues confirmed from molecular docking analysis.

Allosteric Inhibition of Parkinson's-Linked LRRK2 by Constrained Peptides.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson's Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

Extraordinary magnetic properties of double perovskite Eu2CoMnO6 wide band gap semiconductor.

Some novel magnetic behaviours in double perovskite Eu2CoMnO6 (ECMO) have been reported. The x-ray photoemission spectroscopy study shows the presence of mixed valence states of transition metal ions. The UV-visible absorption spectroscopic study suggests that the ECMO has a direct wide band gap. A second-order magnetic phase transition as a sudden jump in the magnetization curve has been observed around 124.5 K. The large bifurcation between the zero field cooling and field cooling, suggests existence of strong spin frustration in the system. The inverse DC susceptibility confirms the presence of the Griffiths like phase. Sharp steps in magnetization have been observed in the M-H curve at 2 K, which vanishes on increasing temperature. The AC susceptibility study demonstrates the Hopkinson like effect as well as the presence of volume spin-glass-like behaviour. The temperature dependent Raman spectrum shows the presence of spin-phonon coupling.