Search results for: RNA binding

Authors: Mark-Adam Kellerman

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Proteins are capable of exploring vast mutational spaces. This makes it difficult for protein engineers to devise rational methods to improve stability and function via mutagenesis. Deciding which residues to mutate requires knowledge of the characteristics they elicit. We probed the characteristics of residues in granulocyte-colony stimulating factor (G-CSF) using a thermal melt (from 295K to 323K) to denature it in a 700 MHz Bruker spectrometer. These characteristics included dynamics, micro-environmental changes experienced/ induced during denaturing and structure-function relationships. 15N-1H HSQC experiments were performed at 2K increments along with this thermal melt. We observed that dynamic residues that also undergo a lot of change in their microenvironment were predominantly in unstructured regions. Moreover, we were able to identify four residues (G4, A6, T133 and Q134) that we class as high priority targets for mutagenesis, given that they all appear in both the top 10% of measures for environmental changes and dynamics (∑Δ and ∆PI). We were also able to probe these NMR observables and combine them with molecular dynamics (MD) to elucidate what appears to be an opening motion of G-CSFs binding site III. V48 appears to be pivotal to this opening motion, which also seemingly distorts the loop region between helices A and B. This observation is in agreement with previous findings that the conformation of this loop region becomes altered in an aggregation-prone state of G-CSF. Hence, we present here an approach to profile the characteristics of residues in order to highlight their potential as rational mutagenesis targets and their roles in important conformational changes. These findings present not only an opportunity to effectively make biobetters, but also open up the possibility to further understand epistasis and machine learn residue behaviours.

Keywords: protein engineering, rational mutagenesis, NMR, molecular dynamics

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Authors: Redouane Krini, Lutz Nuhn, Hicham El Mard Cheol Woo Ha, Yoondeok Han, Kwang-Sup Lee, Dong-Yol Yang, Jinsoo Joo, Rudolf Zentel

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To use Quantum Dots (QDs) in the two photon initiated polymerization technique (TPIP) for 3D patternings, QDs were modified on the surface with photosensitive end groups which are able to undergo a photopolymerization. We were able to fabricate fluorescent 3D lattice structures using photopatternable QDs by TPIP for photonic devices such as photonic crystals and metamaterials. The QDs in different diameter have different emission colors and through mixing of RGB QDs white light fluorescent from the polymeric structures has been created. Metamaterials are capable for unique interaction with the electrical and magnetic components of the electromagnetic radiation and for manipulating light it is crucial to have a negative refractive index. In combination with QDs via TPIP technique polymeric structures can be designed with properties which cannot be found in nature. This makes these artificial materials gaining a huge importance for real-life applications in photonic and optoelectronic. Understanding of interactions between nanoparticles and biological systems is of a huge interest in the biomedical research field. We developed a synthetic strategy of polymer functionalized nanoparticles for biomedical studies to obtain hybrid systems of QDs and copolymers with a strong binding network in an inner shell and which can be modified in the end through their poly(ethylene glycol) functionalized outer shell. These hybrid systems can be used as models for investigation of cell penetration and drug delivery by using measurements combination between CryoTEM and fluorescence studies.

Keywords: biomedical study models, lithography, photo induced polymerization, quantum dots

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Authors: Supannika Klangphukhiew, Roongnapa Srichana, Rina Patramanon

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Cortisol has been used as a well-known commercial stress biomarker. A homeostasis response to psychological stress is indicated by an increased level of cortisol produced in hypothalamus-pituitary-adrenal (HPA) axis. Chronic psychological stress contributing to the high level of cortisol relates to several health problems. In this study, the cortisol biosensor was fabricated that mimicked the natural receptors. The artificial antibodies were prepared using molecular imprinted polymer technique that can imitate the performance of natural anti-cortisol antibody with high stability. Cortisol-molecular imprinted polymer (cortisol-MIP) was obtained using the multi-step swelling and polymerization protocol with cortisol as a target molecule combining methacrylic acid:acrylamide (2:1) with bisacryloyl-1,2-dihydroxy-1,2-ethylenediamine and ethylenedioxy-N-methylamphetamine as cross-linkers. Cortisol-MIP was integrated to the sensor. It was coated on the disposable screen-printed carbon electrode (SPCE) for portable electrochemical analysis. The physical properties of Cortisol-MIP were characterized by means of electron microscope techniques. The binding characteristics were evaluated via covalent patterns changing in FTIR spectra which were related to voltammetry response. The performance of cortisol-MIP modified SPCE was investigated in terms of detection range, high selectivity with a detection limit of 1.28 ng/ml. The disposable cortisol biosensor represented an application of MIP technique to recognize steroids according to their structures with feasibility and cost-effectiveness that can be developed to use in point-of-care.

Keywords: stress biomarker, cortisol, molecular imprinted polymer, screen-printed carbon electrode

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Authors: Muhammad Shahzad Iqbal, Zobia Sarwar, Salah-ud-Din

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Tomato spotted wilt virus (TSWV) belongs to the genus Tospoviruses (family Bunyaviridae). It is one of the most devastating pathogens of tomato (Solanum Lycopersicum) and heavily damages the crop yield each year around the globe. In this study, we retrieved 329 mature miRNA sequences from two microRNA databases (miRBase and miRSoldb) and checked the putative target sites in the downloaded-genome sequence of TSWV. A consensus of three miRNA target prediction tools (RNA22, miRanda and psRNATarget) was used to screen the false-positive microRNAs targeting sites in the TSWV genome. These tools calculated different target sites by calculating minimum free energy (mfe), site-complementarity, minimum folding energy and other microRNA-mRNA binding factors. R language was used to plot the predicted target-site data. All the genes having possible target sites for different miRNAs were screened by building a consensus table. Out of these 329 mature miRNAs predicted by three algorithms, only eight miRNAs met all the criteria/threshold specifications. MC-Fold and MC-Sym were used to predict three-dimensional structures of miRNAs and further analyzed in USCF chimera to visualize the structural and conformational changes before and after microRNA-mRNA interactions. The results of the current study show that the predicted eight miRNAs could further be evaluated by in vitro experiments to develop TSWV-resistant transgenic tomato plants in the future.

Keywords: tomato spotted wild virus (TSWV), Solanum lycopersicum, plant virus, miRNAs, microRNA target prediction, mRNA

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Authors: Yves Mann Elate Lea Mbassi, Marie Solange Evehe Bebandoue, Wilfred Fon Mbacham

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Improving the catalytic performance of enzymes has been a long-standing theme of analytical biochemistry research. Induction of peroxidase activity by metals is a common reaction in higher plants. We thought that this increase in peroxidase activity may be due, on the one hand, to the stimulation of the gene expression of these enzymes but also to a modification of their chemical reactivity following the binding of some metal ions on their active site. We tested the effect of some metal salts (MgCl₂, MnCl₂, ZnCl₂, CaCl₂ and CuSO₄) on the activity and thermostability of peroxidase A6, a thermostable peroxidase that we discovered and purified in a previous study. The chromogenic substrate used was 3,3′,5,5′-tetramethylbenzidine. Of all the metals tested for their effect on A6, only magnesium and copper had a significant effect on the activity of the enzyme at room temperature. The Mann-Whitney test shows a slight inhibitory effect of activity by the magnesium salt (P = 0.043), while the activity of the enzyme is 5 times higher in the presence of the copper salt (P = 0.002). Moreover, the thermostability of peroxidase A6 is increased when calcium and copper salts are present. The activity in the presence of CaCl₂ is 8 times higher than the residual activity of the enzyme alone after incubation at 80°C for 10 min and 35 times higher in the presence of CuSO4 under the same conditions. In addition, manganese and zinc salts slightly reduce the thermostability of the enzyme. The activity and structural stability of peroxidase A6 can clearly be activated by Cu₂+, which therefore enhance the oxidation of 3,3′,5,5′-tetramethylbenzidine, which was used in this study as a chromogenic substrate. Ca₂+ likely has a more stabilizing function for the catalytic site.

Keywords: peroxidase activity, copper ions, calcium ions, thermostability

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Authors: Harish Rajak, Kamlesh Raghuwanshi

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Histone deacetylase (HDAC) are promising target for cancer treatment. The panobinostat (Farydak; Novartis; approved by USFDA in 2015) and chidamide (Epidaza; Chipscreen Biosciences; approved by China FDA in 2014) are the novel HDAC inhibitors ratified for the treatment of patients with multiple myeloma and peripheral T cell lymphoma, respectively. On the other hand, two other HDAC inhibitors, Vorinostat (SAHA; approved by USFDA in 2006) and Romidepsin (FK228; approved by USFDA in 2009) are already in market for the treatment of cutaneous T-cell lymphoma. Several hydroxamic acid based HDAC inhibitors i.e., belinostat, givinostat, PCI24781 and JNJ26481585 are in clinical trials. HDAC inhibitors consist of three pharmacophoric features - an aromatic cap group, zinc binding group (ZBG) and a linker chain connecting cap group to ZBG. Herein, we report synthesis, characterization and biological evaluation of HDAC inhibitors possessing substituted isatin moiety as cap group which recognize the surface of active enzyme pocket and thiosemicarbazide moiety incorporated as linker group responsible for connecting cap group to ZBG (hydroxamic acid). Several analogues were found to inhibit HDAC and cellular proliferation of Hela cervical cancer cells with GI50 values in the micro molar range. Some of the compounds exhibited promising results in vitro antiproliferative studies. Attempts were also made to establish the structure activity relationship among synthesized HDAC inhibitors.

Keywords: HDAC inhibitors, hydroxamic acid derivatives, isatin derivatives, antiproliferative activity, docking

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Authors: Joel Ballesteros, Harriet Jane Caleja, Florian Del Mundo, Cherrie Pascual

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Diffraction-based sensing was utilized in the quantification of human ferritin in blood serum to provide an alternative to label-based immunoassays currently used in clinical diagnostics and researches. The diffraction intensity was measured by the diffractive optics technology or dotLab™ system. Two methods were evaluated in this study: direct immunoassay and direct sandwich immunoassay. In the direct immunoassay, human ferritin was captured by human ferritin antibodies immobilized on an avidin-coated sensor while the direct sandwich immunoassay had an additional step for the binding of a detector human ferritin antibody on the analyte complex. Both methods were repeatable with coefficient of variation values below 15%. The direct sandwich immunoassay had a linear response from 10 to 500 ng/mL which is wider than the 100-500 ng/mL of the direct immunoassay. The direct sandwich immunoassay also has a higher calibration sensitivity with value 0.002 Diffractive Intensity (ng mL-1)-1) compared to the 0.004 Diffractive Intensity (ng mL-1)-1 of the direct immunoassay. The limit of detection and limit of quantification values of the direct immunoassay were found to be 29 ng/mL and 98 ng/mL, respectively, while the direct sandwich immunoassay has a limit of detection (LOD) of 2.5 ng/mL and a limit of quantification (LOQ) of 8.2 ng/mL. In terms of accuracy, the direct immunoassay had a percent recovery of 88.8-93.0% in PBS while the direct sandwich immunoassay had 94.1 to 97.2%. Based on the results, the direct sandwich immunoassay is a better diffraction-based immunoassay in terms of accuracy, LOD, LOQ, linear range, and sensitivity. The direct sandwich immunoassay was utilized in the determination of human ferritin in blood serum and the results are validated by Chemiluminescent Magnetic Immunoassay (CMIA). The calculated Pearson correlation coefficient was 0.995 and the p-values of the paired-sample t-test were less than 0.5 which show that the results of the direct sandwich immunoassay was comparable to that of CMIA and could be utilized as an alternative analytical method.

Keywords: biosensor, diffraction, ferritin, immunoassay

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Authors: Bhuvanesh Baniya

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Alzheimer disease is illnesses that are responsible for neuronal cell death and resulting in lifelong cognitive problems. Due to their unclear mechanism, there are no effective drugs available for the treatment. For a long time, herbal drugs have been used as a role model in the field of the drug discovery process. Holy basil in the Indian medicinal system (Ayurveda) is used for several neuronal disorders like insomnia and memory loss for decades. This study aims to identify active components of holy basil as potential inhibitors for the treatment of Alzheimer disease. To fulfill this objective, the Network pharmacology approach, gene ontology, pharmacokinetics analysis, molecular docking, and molecular dynamics simulation (MDS) studies were performed. A total of 7 active components in holy basil, 12 predicted neurodegenerative targets of holy basil, and 8063 Alzheimer-related targets were identified from different databases. The network analysis showed that the top ten targets APP, EGFR, MAPK1, ESR1, HSPA4, PRKCD, MAPK3, ABL1, JUN, and GSK3B were found as significant target related to Alzheimer disease. On the basis of gene ontology and topology analysis results, APP was found as a significant target related to Alzheimer’s disease pathways. Further, the molecular docking results to found that various compounds showed the best binding affinities. Further, MDS top results suggested could be used as potential inhibitors against APP protein and could be useful for the treatment of Alzheimer’s disease.

Keywords: holy basil, network pharmacology, neurodegeneration, active phytochemicals, molecular docking and simulation

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Authors: Larissa M. Vargas, Julia M. Sacchi, Renata O. Pereira, Lucas S. Asano, Iara C. Araújo, Patricia Fiorino, Vera Farah

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The aim of this study was to evaluate the occurrence of subclinical organ injuries induced by high-fat diet. Male wistar rats (n=5/group) were divided in control diet group (CD), commercial rat chow, and hyperlipidic diet (30% lipids) group (HD) administrated during 8 weeks, starting after weaning. All the procedures followed the rules of the Committee of Research and Ethics of the Mackenzie University (CEUA Nº 077/03/2011). At the end of protocol the animals were euthanized by anesthesia overload and the left kidney was removed. Intrarenal lipid deposition was evaluated by histological analyses with oilred. Kidney slices were stained with picrosirius red to evaluate the area of the Bowman's capsule (AB) and space (SB), and glomerular tuft area (GT). The renal expression of sterol regulatory element–binding protein (SREBP-2) was performed by Western Blotting. Creatinine concentration (serum and urine) and lipid profile were determined by colorimetric kit (Labtest). At the end of the protocol there was no differences in body weight between the groups, however the HD showed a marked increase in lipid deposits, glomeruli and tubules, and biochemical analysis for cholesterol and triglycerides. Moreover, in the kidney, the high-fat diet induced a reduction in the AB (13%), GT (18%) and SB (17%) associated with a reduction in glomerular filtration rate (creatinine clearance). The renal SRBP2 expression was increased in HD group. These data suggests that consumption of high-fat diet starting in childhood is associated with subclinical renal damage and function.

Keywords: high-fat diet, kidney, intrarenal lipid deposition, SRBP2

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Authors: Giorgio Visentin, Alexei A. Buchachenko

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Recent proposals to use Yb dimer as an optical clock and as a sensor for non-Newtonian gravity imply the knowledge of its interaction potential. Here, the ground-state Born-Oppenheimer Yb₂ potential energy curve is represented by a semi-analytical function, consisting of short- and long-range contributions. For the former, the systematic ab initio all-electron exact 2-component scalar-relativistic CCSD(T) calculations are carried out. Special care is taken to saturate diffuse basis set component with the atom- and bond-centered primitives and reach the complete basis set limit through n = D, T, Q sequence of the correlation-consistent polarized n-zeta basis sets. Similar approaches are used to the long-range dipole and quadrupole dispersion terms by implementing the CCSD(3) polarization propagator method for dynamic polarizabilities. Dispersion coefficients are then computed through Casimir-Polder integration. The semiclassical constraint on the number of the bound vibrational levels known for the ¹⁷⁴Yb isotope is used to scale the potential function. The scaling, based on the most accurate ab initio results, bounds the interaction energy of two Yb atoms within the narrow 734 ± 4 cm⁻¹ range, in reasonable agreement with the previous ab initio-based estimations. The resulting potentials can be used as the reference for more sophisticated models that go beyond the Born-Oppenheimer approximation and provide the means of their uncertainty estimations. The work is supported by Russian Science Foundation grant # 17-13-01466.

Keywords: ab initio coupled cluster methods, interaction potential, semi-analytical function, ytterbium dimer

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Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

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PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provides better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: Abigail Tan, Heng Liang Tan, Vanessa Ding, James Hui, Eng Hin Lee, Andre Choo

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Introduction: Comparative oncology investigates the similarities in spontaneous carcinogenesis between humans and animals, in order to identify treatments that can benefit these patients. Companion animals (CA), like canines and felines, are of special interest when it comes to studying human cancers due to their exposure to the same environmental factors and develop tumours with similar features. The purpose of this study is to explore the cross-reactivity of monoclonal antibodies (mAbs) across cancers in humans and CA. Material and Methods: A panel of CA mAbs generated in the lab was screened on multiple human cancer cell lines through flow cytometry to identify for positive binders. Shortlisted candidates were then characterised by biochemical and functional assays e.g., antibody-drug conjugate (ADC) and western blot assays, including glycan studies. Results: Candidate mAb KP52 was generated from whole-cell immunisation using feline mammary carcinoma. KP52 showed strong positive binding to human cancer cells, such as breast cancer and ovarian cancer. Furthermore, KP52 demonstrated strong killing ( > 50%) as an ADC with Saporin as the payload. Western blot results revealed the molecular weight of the antigen targets to be approximately 45kD and 50kD under reduced conditions. Glycan studies suggest that the epitope is glycan in nature, specifically an O-linked glycan. Conclusion: Candidate mAb KP52 has a therapeutic potential as an ADC against feline mammary cancer, human ovarian cancer, human mammary cancer, human pancreatic cancer, and human gastric cancer.

Keywords: ADC, comparative oncology, mAb, therapeutic

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Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

Abstract:

PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: M. Cabral, A. Verdin, G. Garçon, A. Touré, C. Diop, M. Fall, S. Bouhsina, D. Dewaele, F.Cazier, A. Tall Dia, P. Shirali, A. Diouf

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This case-control study dealt with the health adverse effects within the population neighboring the Mbeubeuss waste dump, which is located near the district of Malika (Diamalaye II) in Dakar (Senegal). All the household and industrial waste arising from Dakar are stored in this open landfill without being covered and are therefore possible sources of Pb and Cd contaminated air emissions and lixiviates. The objective of this study is part of improving the health of the population neighboring Mbeubeuss by determining Pb and Cd concentrations both in environment and humans, and studying possible renal function alterations within the adults. Soil and air samples were collected in the control site (Darou Salam) and the waste dump neighboring site (Diamalaye II). Control and exposed adults were recruited as living in Darou Salam (n = 52) and in Diamalaye II (n = 77). Pb and Cd concentrations in soil, air and biological samples were determined. Moreover, we were interested in analyzing some impregnation (zinc protoporphyrin, d-aminolevulinic acid dehydratase) and oxidative stress biomarkers (malonedialdehyde, gluthatione status), in addition to several nephrotoxicity parameters (creatinuria, proteinuria, lactate dehydrogenase, CC16 protein, glutathione S-transferase-alpha and retinol binding protein) in blood and/or urine. The results showed the significant Pb and Cd contamination of the soil and air samples derived from the landfill, and therefore of the neighboring population of adults. This critical exposure to environmental Pb and Cd had some harmful consequences for their health, as shown by the reported oxidative stress and nephrotoxicity signs.

Keywords: Pb and Cd environmental exposure, impregnation markers, landfill, nephrotoxicity markers

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Authors: A. Katsman, L. Bloch, Y. Etinger, Y. Kauffmann, B. Pokroy

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Various nanosized amorphous alumina thin films in the range of (2.4 - 63.1) nm were deposited onto amorphous carbon and amorphous Si3N4 membrane grids. Transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), X-ray photoelectron spectroscopy (XPS) and differential scanning calorimetry (DSC) techniques were used to probe the size effect on the short range order and the amorphous to crystalline phase transition temperature. It was found that the short-range order changes as a function of size: the fraction of tetrahedral Al sites is greater in thinner amorphous films. This result correlates with the change of amorphous alumina density with the film thickness demonstrated by the reflectivity experiments: the thinner amorphous films have the less density. These effects are discussed in terms of surface reconstruction of the amorphous alumina films. The average atomic binding energy in the thin film layer decreases with decease of the thickness, while the average O-Al interatomic distance increases. The reconstruction of amorphous alumina is induced by the surface reconstruction, and the short range order changes being dependent on the density. Decrease of the surface energy during reconstruction is the driving force of the alumina reconstruction (density change) followed by relaxation process (short range order change). The amorphous to crystalline phase transition temperature measured by DSC rises with the decrease in thickness from 997.6°C for 13.9 nm to 1020.4 °C for 2.7 nm thick. This effect was attributed to the different film densities: formation of nanovoids preceding and accompanying crystallization process influences the crystallization rate, and by these means, the temperature of crystallization peak.

Keywords: amorphous alumina, density, short range order, size effect

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Authors: Mukunda Upreti, Jill Storry, Rafael Björk, Emilie Louvet, Johan Normark, Sven Bergström

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Borrelia duttonii and most other relapsing fever species are Gram-negative bacteria which cause a blood borne infection characterized by the binding of bacterium to erythrocytes. The bacteria associate with two or more erythrocytes to form clusters of cells into rosettes. Rosetting is a major virulence factor and the mechanism is believed to facilitate persistence of bacteria in the circulatory system and the avoidance of host immune cells through masking or steric hindrance effects. However, the molecular mechanisms of rosette formation are still poorly understood. This study aims at determining the molecules involved in the rosette formation phenomenon. Fractionated serum, using different affinity purification methods, was investigated as a rosetting agent and IgG and at least one other serum components were needed for rosettes to form. An IgG titration curve demonstrated that IgG alone is not enough to restore rosette formation level to the level whole serum gives. IgG hydrolysis by IdeS ( Immunoglobulin G-degrading enzyme of Streptococcus pyogenes) and deglycosylation using N-Glycanase proved that the whole IgG molecule regardless of saccharide moieties is critical for Borrelia induced rosetting. Complement components C3 and C4 were also important serum molecules necessary to maintain optimum rosetting rates. The deactivation of complement network and serum depletion with C3 and C4 significantly reduced the rosette formation rate. The dependency of IgG and complement components also implied involvement of the complement receptor (CR1). Rosette formation test with Knops null RBC and sCR1 confirmed that CR1 is also part of Borrelia induced rosette formation.

Keywords: complement components C3 and C4, complement receptor 1, Immunoglobulin G, Knops null, Rosetting

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Authors: Indu Barwal, Shloka Thakur, Subhash C. Yadav

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The plant virus capsid protein based nanoparticles are extensively studied for their application in biomedical research for development of nanomedicines and drug delivery systems. We have developed a chimeric drug delivery system by controlled in vitro assembly of separately bioconjugated fluorescent dye (as reporting molecule), folic acid (as receptor binding biomolecule for targeted delivery) and doxorubicin (as anticancer drug) using modified EDC NHS chemistry on heterologously overexpressed (E. coli) capsid proteins of cowpea chlorotic mottle virus (CCMV). This chimeric vehicle was further encapsidated on gold nanoparticles (20nm) coated with 5≠ thiolated DNA probe to neutralize the positive charge of capsid proteins. This facilitates the in vitro assembly of modified capsid subunits on the gold nanoparticles to develop chimeric GNPs encapsidated targeted drug delivery system. The bioconjugation of functionalities, number of functionality on capsid subunits as well as virus like nanoparticles, structural stability and in vitro assembly were confirmed by SDS PAGE, relative absorbance, MALDI TOF, ESI-MS, Circular dichroism, intrinsic tryptophan fluorescence, zeta particle size analyzer and TEM imaging. This vehicle was stable at pH 4.0 to 8.0 suitable for many organelles targeting. This in vitro assembled chimeric plant virus like particles could be suitable for ideal drug delivery vehicles for subcutaneous cancer treatment and could be further modified for other type of cancer treatment by conjugating other functionalities (targeting, drug) on capsids.

Keywords: chimeric drug delivery vehicles, bioconjugated plant, virus, capsid

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Authors: Stefan Gruden, Charlott Brunmark, Bo Holmqvist, Erwin D. Brenndorfer, Martin Johansson, Jian Liu, Ying Zhao, Niklas Axen, Moustapha Hassan

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Aim: The study was designed to evaluate the ability of the calcium sulfate-based NanoZolid® drug delivery technology to locally release the epidermal growth factor (EGF) protein while maintaining its biological activity. Methods: NanoZolid-formulated EGF protein labelled with a near-infrared dye (EGF-NIR) depots or EGF-NIR dissolved in PBS were injected subcutaneously into mice bearing EGF receptor (EGFR) positive human A549 lung cancer tumors inoculated subcutaneously. The release and biodistribution of the EGF-NIR were investigated in vivo longitudinally up to 96 hours post-administration, utilizing whole-body fluorescence imaging. In order to confirm the in vivo findings, histological analysis of tumor cryosections was performed to investigate EGF-NIR fluorescent signal and EGFR expression level by immunofluorescence labelling. Results: The in vivo fluorescence imaging showed a controlled release profile of the EGF-NIR loaded in the NanoZolid depots compared to free EGF-NIR. Histological analysis of the tumors further demonstrated a prevailing distribution of EGF-NIR in regions with high levels of EGFR expression. Conclusion: Calcium sulfate based depots can be used to formulate EGF while maintaining its biological activity, e.g., receptor binding capability. This may have good clinical potential for local delivery of biomolecules to enhance treatment efficacy and minimize systemic adverse effects.

Keywords: bioresorbable, calcium sulfate, controlled release, NanoZolid

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Authors: Khin Thanda Win, Chunying Zhang, Sanghyeob Lee

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Mildew resistance locus o (Mlo), a plant-specific gene family with seven-transmembrane (TM), plays an important role in plant resistance to powdery mildew (PM). PM caused by Podosphaera xanthii is a widespread plant disease and probably represents the major fungal threat for many Cucurbits. The recent Cucurbita maxima genome sequence data provides an opportunity to identify and characterize the MLO gene family in this species. Total twenty genes (designated CmaMLO1 through CmaMLO20) have been identified by using an in silico cloning method with the MLO gene sequences of Cucumis sativus, Cucumis melo, Citrullus lanatus and Cucurbita pepo as probes. These CmaMLOs were evenly distributed on 15 chromosomes of 20 C. maxima chromosomes without any obvious clustering. Multiple sequence alignment showed that the common structural features of MLO gene family, such as TM domains, a calmodulin-binding domain and 30 important amino acid residues for MLO function, were well conserved. Phylogenetic analysis of the CmaMLO genes and other plant species reveals seven different clades (I through VII) and only clade IV is specific to monocots (rice, barley, and wheat). Phylogenetic and structural analyses provided preliminary evidence that five genes belonged to clade V could be the susceptibility genes which may play the importance role in PM resistance. This study is the first comprehensive report on MLO genes in C. maxima to our knowledge. These findings will facilitate the functional analysis of the MLOs related to PM susceptibility and are valuable resources for the development of disease resistance in pumpkin.

Keywords: Mildew resistance locus o (Mlo), powdery mildew, phylogenetic relationship, susceptibility genes

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Authors: L. Mentar, O. Baka, M. R. Khelladi, A. Azizi

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Zinc oxide (ZnO), as a transparent semiconductor with a wide band gap of 3.4 eV and a large exciton binding energy of 60 meV at room temperature, is one of the most promising materials for a wide range of modern applications. With the development of film growth technologies and intense recent interest in nanotechnology, several varieties of ZnO nanostructured materials have been synthesized almost exclusively by thermal evaporation methods, particularly chemical vapor deposition (CVD), which generally require a high growth temperature above 550 °C. In contrast, wet chemistry techniques such as hydrothermal synthesis and electro-deposition are promising alternatives to synthesize ZnO nanostructures, especially at a significantly lower temperature (below 200°C). In this study, the electro-deposition method was used to produce zinc oxide (ZnO) nanostructures on fluorine-doped tin oxide (FTO)-coated conducting glass substrate from chloride bath. We present the influence of KCl concentrations on the electro-deposition process, morphological, structural and optical properties of ZnO nanostructures. The potentials of electro-deposition of ZnO were determined using the cyclic voltammetry. From the Mott-Schottky measurements, the flat-band potential and the donor density for the ZnO nanostructure are determined. Field emission scanning electron microscopy (FESEM) images showed different sizes and morphologies of the nanostructures which depends on the concentrations of Cl-. Very netted hexagonal grains are observed for the nanostructures deposited at 0.1M of KCl. X-ray diffraction (XRD) study confirms the Wurtzite phase of the ZnO nanostructures with a preferred oriented along (002) plane normal to the substrate surface. UV-Visible spectra showed a significant optical transmission (~80%), which decreased with low Cl-1 concentrations. The energy band gap values have been estimated to be between 3.52 and 3.80 eV.

Keywords: Cl-, electro-deposition, FESEM, Mott-Schottky, XRD, ZnO

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Authors: Larisa Gheber

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Mitosis is an essential process by which duplicated genetic information is transmitted from mother to daughter cells. Incorrect chromosome segregation during mitosis can lead to genetic diseases, chromosome instability and cancer. This process is mediated by a dynamic microtubule-based intracellular structure, the mitotic spindle. One of the major factors that govern the mitotic spindle dynamics are the kinesin-5 biological nano motors that were believed to move unidirectionally on the microtubule filaments, using ATP hydrolysis, thus performing essential functions in mitotic spindle dynamics. Surprisingly, several reports from our and other laboratories have demonstrated that some kinesin-5 motors are bi-directional: they move in minus-end direction on the microtubules as single-molecules and can switch directionality under a number of conditions. These findings broke a twenty-five-years old dogma regarding kinesin directionality (1, 2). The mechanism of this bi-directional motility and its physiological significance remain unclear. To address this unresolved problem, we apply an interdisciplinary approach combining live cell imaging, biophysical single molecule, and structural experiments to examine the activity of these motors and their mutated variants in vivo and in vitro. Our data shows that factors such as protein phosphorylation (3, 4), motor clustering on the microtubules (5, 6) and structural elements (7, 8) regulate the bi-directional motility of kinesin motors. We also show, using Cryo-EM, that bi-directional kinesin motors obtain non-canonical microtubule binding, which is essential to their special motile properties and intracellular functions. We will discuss the implication of these findings to mechanism bi-directional motility and physiological roles in mitosis.

Keywords: mitosis, cancer, kinesin, microtubules, biochemistry, biophysics

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Authors: Yi-Hui Lai, Chih-Hsiang Yang, Li-Chung Hsu

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The inflammasome is a protein complex that modulates caspase-1 activity, resulting in proteolytic cleavage of proinflammatory cytokines such as IL-1β and IL-18, into their bioactive forms. It has been shown that the inflammasomes play a crucial role in the clearance of pathogenic infection and tissue repair. However, dysregulated inflammasome activation contributes to a wide range of human diseases such as cancers and auto-inflammatory diseases. Yet, regulation of NLRP3 inflammasome activation remains largely unknown. We discovered a novel DEAD box protein, whose biological function has not been reported, not only negatively regulates NLRP3 inflammasome activation by interfering NLRP3 inflammasome assembly and cellular localization but also mitigate pyroptosis upon pathogen evasion. The DEAD-box protein is the first DEAD-box protein gets involved in modulation of the inflammasome activation. In our study, we found that caspase-1 activation and mature IL-1β production were largely enhanced upon LPS challenge in the DEAD box-containing protein- deleted THP-1 macrophages and bone marrow-derived macrophages (BMDMs). In addition, this DEAD box-containing protein migrates from the nucleus to the cytoplasm upon LPS stimulation, which is required for its inhibitory role in NLRP3 inflammasome activation. The DEAD box-containing protein specifically interacted with the LRR motif of NLRP3 via its DEAD domain. Furthermore, due to the crucial role of the NLRP3 LRR domain in the recruitment of NLRP3 to mitochondria and binding to its adaptor ASC, we found that the interaction of NLRP3 and ASC was downregulated in the presence of the DEAD box-containing protein. In addition to the mechanical study, we also found that this DEAD box protein protects host cells from inflammasome-triggered cell death in response to broad-ranging pathogens such as Candida albicans, Streptococcus pneumoniae, etc., involved in nosocomial infections and severe fever shock. Collectively, our results suggest that this novel DEAD box molecule might be a key therapeutic strategy for various infectious diseases.

Keywords: inflammasome, inflammation, innate immunity, pyroptosis

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Authors: Amira M. Othman

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Despite the massive array of literature that engages with the Egyptian legislative system on a theoretical level, very little attention has been dedicated to the comparison between the legislative clauses on the one hand, and the (absence of their) real-world implementation on the other. This paper starts with this discrepancy, focusing on the legal proceedings in some recent cases dubbed ‘political,’ in which defendants received death sentences. Then, it sheds light on the trend of practical disregard of the law on behalf of the criminal justice apparatuses (whether security forces, public prosecution offices, lawyers, judges, prison wardens, and executioners) through the examination of case files and the conduction of interviews with some defense lawyers in the cases in question. It also identifies the resultant state of confusion among prison staff, as manifest in their treatment of defendants even before the death sentences against them is pronounced; in other words, the application of some aspects of the law in certain cases, and their simultaneous disregard of others. Then, the paper explores the effects of such execution of the law on the death row inmates, as it identifies the different strategies through which defendants who are sentenced to death appropriate a number of legal clauses to their benefit, thereby embarrassing - or highly irritating - the judges that pronounce their death sentences. In addition to appropriation, other strategies include the contestation of the law and their presence before the courts in general, as well as the complete disregard and dismissal of the legal system altogether. Finally, the paper investigates the consequent conceptual effect on the first degree families of death row inmates, namely how their daily encounters with the Egyptian legislative system - particularly its emphasis on the absence of the otherwise binding local legislation - continue to shape their conceptions of the ‘law,’ of ‘justice,’ and their trust in the ‘state.’

Keywords: death penalty, Egyptian law absence, justice, political cases

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Authors: Gajanan M. Sonwane

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The computational studies on 2-phenazinamines with their protein targets have been carried out to design compounds with potential anticancer activity. This strategy of designing compounds possessing selectivity over specific tyrosine kinase has been achieved through G-QSAR and molecular docking studies. The objective of this research has been to design newer 2-phenazinamine derivatives as Bcr-Abl tyrosine kinase inhibitors by G-QSAR, molecular docking studies followed by wet-lab studies along with evaluation of their anticancer potential. Computational chemistry was done by using VLife MDS 4.3 and Autodock 4.2 followed by wet-lab experiments for synthesizing 2-phenazinamine derivatives. The chemical structures of ligands in 2D were drawn by employing Chemdraw 2D Ultra 8.0 and were converted into 3D. These were optimized by using a semi-empirical method called MOPAC. The protein structure was retrieved from RCSC protein data bank as a PDB file. The binding interactions of protein and ligands were done by using PYMOL. The molecular properties of the designed compounds were predicted in silico by using Osiris property explorer. The parent compound 2-phenazinamine was synthesized by reduction of 2, 4-dinitro-N-phenyl-benzenamine in the presence of tin chloride followed by cyclization in the presence of nitrobenzene and magnesium sulfate. The derivatization at the amino function of 2-phenazinamine was performed by treating parent compound with various aldehydes in the presence of dicyclohexylcarbodiimide (DCC) and urea to afford 2-(2-chlorophenyl)-3-(phenazine-2-yl) thiazolidine-4-one. Synthesized 39 novel derivatives of 2-phenazinamine and performed antioxidant activity, anti antiproliferative on the bulb of onion and anticancer activity on cell line showing significant competition with marked blockbuster drug imatinib.

Keywords: computer-aided drug design, tyrosin kinases, anticancer, docking

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Authors: Yessenia Parrado, Ana Barbosa, Daniela Mahe, Sebastian Toro, Jhon Garcia

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The purpose of this work is to present the identification and characterization of the barriers and enablers to public innovation in the Central Region of Colombia from a mixed methodology in a research carried out in 2020 by the Laboratory of Innovation, Creativity and New Technologies of the National University of Colombia in alliance with the National Planning Department. Based on the research, the index of barriers to regional and departmental public innovation was built, which reflects the level of difficulty of the territorial entities to overcome the barriers present around three dimensions: organizational structure of the entity, generation of public value, and governance processes. The index was built from the item response methodology and the multiple correspondence analysis from the application of an institutional information form for public entities and a perception form for public servants. This investigation had the participation of 36 entities and 1038 servers and servants from the departments of Huila, Meta, Boyacá, Cundinamarca, Tolima, and the Capital District. In this exercise, it was identified that the departmental indices range between 13 and 44 and that the regional index was 30 out of 100. From the analysis of the information, it was possible to establish that the main barriers are the lack of specialized agencies for public innovation exercises, lack of qualified personnel and work methodologies for public innovation, inadequate information management, lack of feedback between the learning from governmental and non-governmental entities, the inability of the initiatives to generate binding participation mechanisms and the lack of qualification of citizens to participate in these processes.

Keywords: item response, public innovation, quantitative analysis, compared analysis

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Authors: Przemyslaw Nogly, Tobias Weinert, Daniel James, Sergio Carbajo, Dmitry Ozerov, Antonia Furrer, Dardan Gashi, Veniamin Borin, Petr Skopintsev, Kathrin Jaeger, Karol Nass, Petra Bath, Robert Bosman, Jason Koglin, Matthew Seaberg, Thomas Lane, Demet Kekilli, Steffen Brünle, Tomoyuki Tanaka, Wenting Wu, Christopher Milne, Thomas A. White, Anton Barty, Uwe Weierstall, Valerie Panneels, Eriko Nango, So Iwata, Mark Hunter, Igor Schapiro, Gebhard Schertler, Richard Neutze, Jörg Standfuss

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Ultrafast isomerization of retinal is the primary step in a range of photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin using an X-ray laser. Twenty snapshots with near-atomic spatial and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket prior to passing through a highly-twisted geometry and emerging in the 13-cis conformation. The aspartic acid residues and functional water molecules in proximity of the retinal Schiff base respond collectively to formation and decay of the initial excited state and retinal isomerization. These observations reveal how the protein scaffold guides this remarkably efficient photochemical reaction.

Keywords: bacteriorhodopsin, free-electron laser, retinal isomerization mechanism, time-resolved crystallography

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Authors: Sara Przetocka, Krzysztof Żak, Grzegorz Dubin, Tadeusz Holak

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Toll-like receptors (TLRs) play central role in the innate immune response and inflammation by recognizing pathogen-associated molecular patterns (PAMPs). A fundamental basis of TLR signalling is dependent upon the recruitment and association of adaptor molecules that contain the structurally conserved Toll/interleukin-1 receptor (TIR) domain. MyD88 (myeloid differentiation primary response gene 88) is the universal adaptor for TLRs and cooperates with Mal (MyD88 adapter-like protein, also known as TIRAP) in TLR4 response which is predominantly used in inflammation, host defence and carcinogenesis. Up to date two possible models of MyD88, Mal and TLR4 interactions have been proposed. The aim of our studies is to confirm or abolish presented models and accomplish the full structural characterisation of TIR domains interaction. Using molecular cloning methods we obtained several construct of MyD88 and Mal TIR domain with GST or 6xHis tag. Gel filtration method as well as pull-down analysis confirmed that recombinant TIR domains from MyD88 and Mal are binding in complexes. To examine whether obtained complexes are homo- or heterodimers we carried out cross-linking reaction of TIR domains with BS3 compound combined with mass spectrometry. To investigate which amino acid residues are involved in this interaction the NMR titration experiments were performed. 15N MyD88-TIR solution was complemented with non-labelled Mal-TIR. The results undoubtedly indicate that MyD88-TIR interact with Mal-TIR. Moreover 2D spectra demonstrated that simultaneously Mal-TIR self-dimerization occurs which is necessary to create proper scaffold for Mal-TIR and MyD88-TIR interaction. Final step of this study will be crystallization of MyD88 and Mal TIR domains complex. This crystal structure and characterisation of its interface will have an impact in understanding the TLR signalling pathway and possibly will be used in development of new anti-cancer treatment.

Keywords: cancer, MyD88, TIR domains, Toll-like receptors

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Authors: Vagolu S. Krishna, Shan Zheng, Estharla M. Rekha, Luke W. Guddat, Dharmarajan Sriram

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Tuberculosis (TB) remains a major threat to human health. This due to the fact that current drug treatments are less than optimal as well as the rising occurrence of multi drug-resistant and extensively drug-resistant strains of the etiological agent, Mycobacterium tuberculosis (Mt). Given the wide-spread significance of this disease, we have undertaken a design and evaluation program to discover new anti-TB drug leads. Here, our attention is focused on ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway. Importantly, this enzyme is present in bacteria but not in humans, making it an attractive proposition for drug discovery. In the present work, we used high-throughput virtual screening to identify seventeen potential inhibitors of KARI using the Birla Institute of Technology and Science in-house database. Compounds were selected based on high docking scores, which were assigned as the result of favourable interactions between the compound and the active site of KARI. The Ki values for two leads, compounds 14 and 16 are 3.71 and 3.06 µM, respectively for Mt KARI. To assess the mode of binding, 100 ns molecular dynamics simulations for these two compounds in association with Mt KARI were performed and showed that the complex was stable with an average RMSD of less than 2.5 Å for all atoms. Compound 16 showed an MIC of 2.06 ± 0.91 µM and a 1.9 fold logarithmic reduction in the growth of Mt in an infected macrophage model. The two compounds exhibited low toxicity against murine macrophage RAW 264.7 cell lines. Thus, both compounds are promising candidates for development as an anti-TB drug leads.

Keywords: ketol-acid reductoisomerase, macrophage, molecular docking and dynamics, tuberculosis

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Authors: Beatriz Muñiz Cano, J. Ripoll Sau, D. Pacile, P. M. Sheverdyaeva, P. Moras, J. Camarero, R. Miranda, M. Garnica, M. A. Valbuena

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Graphene, in its pristine state, is a semiconductor with a zero band gap and massless Dirac fermions carriers, which conducts electrons like a metal. Nevertheless, the absence of a bandgap makes it impossible to control the material’s electrons, something that is essential to perform on-off switching operations in transistors. Therefore, it is necessary to generate a finite gap in the energy dispersion at the Dirac point. Intense research has been developed to engineer band gaps while preserving the exceptional properties of graphene, and different strategies have been proposed, among them, quantum confinement of 1D nanoribbons or the introduction of super periodic potential in graphene. Besides, in the context of developing new 2D materials and Van der Waals heterostructures, with new exciting emerging properties, as 2D transition metal chalcogenides monolayers, it is fundamental to know any possible interaction between chalcogenide atoms and graphene-supporting substrates. In this work, we report on a combined Scanning Tunneling Microscopy (STM), Low Energy Electron Diffraction (LEED), and Angle-Resolved Photoemission Spectroscopy (ARPES) study on a new superstructure when Te is evaporated (and intercalated) onto graphene over Ir(111). This new superstructure leads to the electronic doping of the Dirac cone while the linear dispersion of massless Dirac fermions is preserved. Very interestingly, our ARPES measurements evidence a large band gap (~400 meV) at the Dirac point of graphene Dirac cones below but close to the Fermi level. We have also observed signatures of the Dirac point binding energy being tuned (upwards or downwards) as a function of Te coverage.

Keywords: angle resolved photoemission spectroscopy, ARPES, graphene, spintronics, spin-orbitronics, 2D materials, transition metal dichalcogenides, TMDCs, TMDs, LEED, STM, quantum materials

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Authors: Kolawole Oyekan Jamiu

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In Nigerian criminal law, a victim of an offence plays little or no role in the prosecution of an offender. The state concentrates only on imposing punishment on the offender while the victims of crime and abuse of power by security agencies are abandoned without any compensation either from the State or the offender. It has been stated that the victim of crime is the forgotten man in our criminal justice system. He sets the criminal law in motion but then goes into oblivion. Our present criminal law does not recognise the right of the victim to take part in the prosecution of the case or his right to compensation. The victim is merely a witness in a state versus case. This paper examines the meaning of the phrase ‘the victims of crime and abuse of power’. It needs to be noted that there is no definition of these two categories of victims in any statute in Nigeria. The paper also considers the United Nations General Assembly Declaration of Basic Principle of Justice for Victims and abuse of power. This declaration was adopted by the United Nations General Assembly on the 25th of November 1985. The declaration contains copious provisions on compensation for the victims of crime and abuse of power. Unfortunately, the declaration is not, in itself a legally binding instrument and has been given little or no attention since the coming into effect in1985. This paper examines the role of the judiciary in ensuring that victims of crime and abuse of power in Nigeria are compensated. While some Judges found it difficult to award damages to victims of abuse of power others have given some landmark rulings and awarded substantial damages. The criminal justice ( victim’s remedies) Bill shall also be examined. The Bill comprises of 74 sections and it spelt out the procedures for compensating the victims of crime and abuse of power in Nigeria. Finally, the paper also examines the practicability of awarding damages to victims of crime whether the offender is convicted or not and in addition, the possibility of granting all equitable remedies available in civil cases to victims of crime and abuse of power so that the victims will be restored to the earlier position before the crime.

Keywords: compensation, damages, restitution, victims

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