Probing the activation sequence of NMDA receptors with lurcher mutations

N-methyl-d-aspartate (NMDA) receptor activation involves a dynamic series of structural rearrangements initiated by glutamate binding to glycine-loaded receptors and culminates with the clearing of the permeation pathway, which allows ionic flux. Along this sequence, three rate-limiting transitions can be quantified with kinetic analyses of single-channel currents, even though the structural determinants of these critical steps are unknown. In inactive receptors, the major permeation barrier resides at the intersection of four M3 transmembrane helices, two from each GluN1 and GluN2 subunits, at the level of the invariant SYTANLAAF sequence, known as the lurcher motif. Because the A7 but not A8 residues in this region display agonist-dependent accessibility to extracellular solutes, they were hypothesized to form the glutamate-sensitive gate. We tested this premise by examining the reaction mechanisms of receptors with substitutions in the lurcher motifs of GluN1 or GluN2A subunits. We found that, consistent with their locations relative to the proposed activation gate, A8Y decreased open-state stability, whereas A7Y dramatically stabilized open states, primarily by preventing gate closure; the equilibrium distribution of A7Y receptors was strongly shifted toward active states and resulted in slower microscopic association and dissociation rate constants for glutamate. In addition, for both A8- and A7-substituted receptors, we noticed patterns of kinetic changes that were specific to GluN1 or GluN2 locations. This may be a first indication that the sequence of discernible kinetic transitions during NMDA receptor activation may reflect subunit-dependent movements of M3 helices. Testing this hypothesis may afford insight into the activation mechanism of NMDA receptors.     

Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1-E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric 'core' provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an 'average' 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.     

Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens

The aim of this work was to optimize the cultural and production parameters through the statistical approach for the synthesis of alpha amylase by Bacillus amyloliquefaciens in submerged fermentation (SmF) using a combination of wheat bran and groundnut oil cake (1:1) as the substrate. The process parameters influencing the enzyme production were identified using Plackett-Burman design. Among the various variables screened, the substrate concentration, incubation period and CaCl2 concentration were most significant. The optimum levels of these significant parameters were determined employing the response surface Box-Behnken design, which revealed these as follows: substrate concentration (12.5%), incubation period (42 h) and CaCl2 (0.0275 M).     

Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma

BackgroundGlioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine if GBM organoids may offer a platform, complimentary to traditional sphere culture methods, to recapitulate patterns of clinical drug resistance arising from 3D growth.MethodsAdult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy.ResultsGBM organoids showed diverse and spatially distinct proliferative cell niches and include heterogeneous populations of CSCs/non-CSCs (marked by SOX2) and cycling/senescent cells. Organoid cultures display a comparatively blunted response to current standard-of-care therapy (combination temozolomide and radiotherapy) that reflects what is seen in practice. Treatment of organoids with clinically relevant drugs showed general therapeutic resistance with drug- and patient-specific antiproliferative, apoptotic, and senescent effects, differing from those of matched sphere cultures.ConclusionsTherapeutic resistance in organoids appears to be driven by altered biological mechanisms rather than physical limitations of therapeutic access. GBM organoids may therefore offer a key technological approach to discover and understand resistance mechanisms of human cancer cells.     

Effect of glutamate and riluzole on manganese-induced apoptotic cell signaling in neuronally differentiated mouse P19 Cells

Excess exposure to Mn causes a neurological disorder known as manganism which is similar to dystonic movements associated with Parkinson's disease. Manganism is largely restricted to occupations in which high atmospheric levels are prevalent which include Mn miners, welders and those employed in the ferroalloy processing or related industrial settings. T1 weighted MRI images reveal that Mn is deposited to the greatest extent in the globus pallidus, an area of the brain that is presumed to be responsible for the major CNS associated symptoms. Neurons within the globus pallidus receive glutamatergic input from the subthalamic nuclei which has been suggested to be involved in the toxic actions of Mn. The neurotoxic actions of Mn and glutamate are similar in that they both affect calcium accumulation in the mitochondria leading to apoptotic cell death. In this paper, we demonstrate that the combination of Mn and glutamate potentiates toxicity of neuronally differentiated P19 cells over that observed with either agent alone. Apoptotic signals ROS, caspase 3 and JNK were increased in an additive fashion when the two neurotoxins were combined. The anti-glutamatergic drug, riluzole, was shown to attenuate these apoptotic signals and prevent P19 cell death. Results of this study confirm, for the first time, that Mn toxicity is potentiated in the presence of glutamate and that riluzole is an effective antioxidant which protects against both Mn and glutamate toxicity.     

Reversal of reduced parvalbumin neurons in hippocampus and amygdala of Angelman syndrome model mice by chronic treatment of fluoxetine

Angelman syndrome (AS) is a neuropsychiatric disorder characterized by autism, intellectual disability and motor disturbances. The disease is primarily caused by the loss of function of maternally inherited UBE3A. Ube3a maternal‐deficient mice recapitulates many essential feature of AS. These AS mice have been shown to be under chronic stress and exhibits anxiety‐like behaviour because of defective glucocorticoid receptor signalling. Here, we demonstrate that chronic stress in these mice could lead to down‐regulation of parvalbumin‐positive interneurons in the hippocampus and basolateral amygdala from early post‐natal days. Down‐regulation of parvalbumin‐positive interneurons number could be because of decrease in the expression of parvalbumin in these neurons. We also find that treatment with fluoxetine, a selective serotonin reuptake inhibitor, results in restoration of impaired glucocorticoid signalling, elevated serum corticosterone level, parvalbumin‐positive interneurons and anxiety‐like behaviours. Our findings suggest that impaired glucocorticod signalling in hippocampus and amygdala of AS mice is critical for the decrease in parvalbumin interneurons number, emergence of anxiety and other behavioural deficits and highlights the importance of fluoxetine in the recovery of these abnormalities.

     

Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities.

Abbreviations

ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - – Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - – Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.     

Bare fiber Bragg grating immunosensor for real‐time detection of Escherichia coli bacteria

Escherichia coli (E. coli) bacteria have been identified to be the cause of variety of health outbreaks resulting from contamination of food and water. Timely and rapid detection of the bacteria is thus crucial to maintain desired quality of food products and water resources. A novel methodology proposed in this paper demonstrates for the first time, the feasibility of employing a bare fiber Bragg grating (bFBG) sensor for detection of E. coli bacteria. The sensor was fabricated in a photo‐sensitive optical fiber (4.2 µm/80 µm). Anti‐E. coli antibody was immobilized on the sensor surface to enable the capture of target cells/bacteria present in the sample solution. Strain induced on the sensor surface as a result of antibody immobilization and subsequent binding of E. coli bacteria resulted in unique wavelength shifts in the respective recording of the reflected Bragg wavelength, which can be exploited for the application of biosensing. Functionalization and antibody binding on to the fiber surface was cross validated by the color development resulting from the reaction of an appropriate substrate solution with the enzyme label conjugated to the anti‐E. coli antibody. Scanning electron microscope image of the fiber, further verified the E. coli cells bound to the antibody immobilized sensor surface.