Search results for: NOD2 (nucleotide binding oligomerization receptor 2)

Authors: Chakrapani Pullagummi, Arun Jyothi Bheemagani, B. Chandra Sekhar Singh, Prem Kumar, A. Roja Rani

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Diabetes mellitus describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and its action. The objective of present study was alloxan induced diabetes in S.D (Sprague Dawley) rats, treated with leaf extract of Andrographis paniculata and bark extract of Erythrina indica. Plant extract treated rats were analyzed biochemically and molecularly. on normal and diabetic rats. The changes in MDA (lipid peroxidation) and glucose (by GOD method) levels in blood of both normal and diabetic rat were analyzed. Diabetes induced rats were treated with methanolic extracts of Andrographis paniculata leaf and Erythrina indica bark which are of medicinal importance. Later after inducing diabetes the rats were treated with medicinal plant extracts, Andrographis paniculata leaf and Erythrina indica bark which are well known for their anti diabetic and antioxidative property in order to control the glucose and MDA levels. The blood plasma of diabetic and normal rats was analyzed for the levels of MDA (lipid peroxidation) and glucose levels. Results of this study suggested that the Andrographis paniculata leaf and Erythrina indica can be used as a potential natural antidiabetic agent for treating and postponing the appearance of complications that arise due to Diabetes. Molecular study deals with the analysis of binding mechanism of 2 selected natural compounds from Andrographis and Erythrina extracts against the novel target for type T2D namely PPAR-γ compared with Rosiglitazone (standard compound). The results revealed that most of the selected herbal lead compounds were effective targets against the receptors. These compounds showed favorable interactions with the amino acid residues thereby substantiating their proven efficacy as anti-diabetic compounds.

Keywords: andrographis paniculata, erythrina indica, alloxan, lipid peroxidation, blood glucose level, PPAR-γ

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: Maki Mizogami, Hironori Tsuchiya, Yoshiroh Hayabuchi, Kenji Shigemi

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Since drugs exhibit significant structure-dependent differences in activity and toxicity, their differentiation based on the mechanism of action should have implications for comparative drug efficacy and safety. We aimed to differentiate drug stereoisomers by their stereostructure-selective membrane interactions underlying pharmacological and toxicological effects. Biomimetic lipid bilayer membranes were prepared with phospholipids and sterols (either cholesterol or epicholesterol) to mimic the lipid compositions of neuronal and cardiomyocyte membranes and to provide these membranes with the chirality. The membrane preparations were treated with different classes of stereoisomers at clinically- and pharmacologically-relevant concentrations (25-200 μM), followed by measuring fluorescence polarization to determine the membrane interactivity of drugs to change the physicochemical property of membranes. All the tested drugs acted on lipid bilayers to increase or decrease the membrane fluidity. Drug stereoisomers could not be differentiated when interacting with the membranes consisting of phospholipids alone. However, they stereostructure-selectively interacted with neuro-mimetic and cardio-mimetic membranes containing 40 mol% cholesterol ((3β)-cholest-5-en-3-ol) to show the relative potencies being local anesthetic R(+)-bupivacaine > rac-bupivacaine > S(‒)-bupivacaine, α2-adrenergic agonistic D-medetomidine > rac-medetomidine > L-medetomidine, β-adrenergic antagonistic R(+)-propranolol > rac-propranolol > S(–)-propranolol, NMDA receptor antagonistic S(+)-ketamine > rac-ketamine, analgesic monoterpenoid (+)-menthol > (‒)-menthol, non-steroidal anti-inflammatory S(+)-ibuprofen > rac-ibuprofen > R(‒)-ibuprofen, and bioactive flavonoid (+)-epicatechin > (‒)-epicatechin. All of the order of membrane interactivity were correlated to those of beneficial and adverse effects of the tested stereoisomers. In contrast, the membranes prepared with epicholesterol ((3α)-chotest-5-en-3-ol), an epimeric form of cholesterol, reversed the rank order of membrane interactivity to be S(‒)-enantiomeric > racemic > R(+)-enantiomeric bupivacaine, L-enantiomeric > racemic > D-enantiomeric medetomidine, S(–)-enantiomeric > racemic > R(+)-enantiomeric propranolol, racemic > S(+)-enantiomeric ketamine, (‒)-enantiomeric > (+)-enantiomeric menthol, R(‒)-enantiomeric > racemic > S(+)-enantiomeric ibuprofen, and (‒)-enantiomeric > (+)-enantiomeric epicatechin. The opposite configuration allows drug molecules to interact with chiral sterol membranes enantiomer-selectively. From the comparative results, it is speculated that a 3β-hydroxyl group in cholesterol is responsible for the enantioselective interactions of drugs. In conclusion, the differentiation of drug stereoisomers by their stereostructure-selective membrane interactions would be useful for designing and predicting drugs with higher activity and/or lower toxicity.

Keywords: chiral membrane, differentiation, drug stereoisomer, enantioselective membrane interaction

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Authors: Zohreh Bayat, Dariush Minai-Tehrani

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Pseudomonas aeruginosa is a Gram-negative, rode shape and aerobic bacterium that has shown to be resistance to many antibiotics. This resistance makes the bacterium very harmful in some diseases. It can also generate diseases in any part of the gastrointestinal tract from oropharynx to rectum. P. aeruginosa has become an important cause of infection, especially in patients with compromised host defense mechanisms. One of the most important reasons that make P. aeruginosa an emerging opportunistic pathogen in patients is its ability to use various compounds as carbon sources. Lipase is an enzyme that catalyzes the hydrolysis of lipids. Most lipases act at a specific position on the glycerol backbone of lipid substrate. Some lipases are expressed and secreted by pathogenic organisms during the infection. Muscarinic antagonist used as an antispasmodic and in urinary incontinence. The drug has little effect on glandular secretion or the cardiovascular system. It does have some local anesthetic properties and is used in gastrointestinal, biliary, and urinary tract spasms. Aim: In this study the inhibitory effect of a muscarinic antagonist on lipase of P. aeruginosa was investigated. Methods: P. aeruginosa was cultured in minimal salt medium with 1% olive oil as carbon source. The cells were harvested and the supernatant, which contained lipase, was used for enzyme assay. Results: Our results showed that the drug can inhibit P. aeruginosa lipase by competitive manner. In the presence of different concentrations of the drug, the Vmax (2 mmol/min/mg protein) of enzyme did not change, while the Km raised by increasing the drug concentration. The Ki (inhibition constant) and IC50 (the half maximal inhibitory concentration) value of drug was estimated to be about 30 uM and 60 uM which determined that the drug binds to enzyme with high affinity. Maximum activity of the enzyme was observed at pH 8 in the absence and presence of muscarinic antagonist, respectively. The maximum activity of lipase was observed at 600C and the enzyme became inactive at 900C. Conclusion: The muscarinic antagonist drug could inhibit lipase of P. aeruginosa and changed the kinetic parameters of the enzyme. The drug binded to enzyme with high affinity and did not chang the optimum pH of the enzyme. Temperature did not affect the binding of drug to musmuscarinic antagonist.

Keywords: Pseudomonas aeruginosa, drug, enzyme, inhibition

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Authors: Avrokomi Zavitsanou, Spiros Papadopoulos, Theofanis Alexandridis

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This paper presents the research project ‘Colour & Travel’, which is co-funded by the European Union and national resources through the Operational Programme “Competitiveness, Entrepreneurship and Innovation” 2014-2020, under the Single RTDI State Aid Action "RESEARCH - CREATE - INNOVATE". The research project proposes the design of an innovative, playful framework for exploring a variety of travel destinations and creating personalised travel narratives, aiming to entertain, educate, and promote culture and tourism. Gamification of the cultural and touristic environment can enhance its experiential, multi-sensory aspects and broaden the perception of the traveler. The latter's involvement in creating and shaping his personal travel narrations and the possibility of sharing it with others can offer him an alternative, more binding way of getting acquainted with a place. In particular, the paper presents the design of an infrastructure: (a) for the development of interactive travel guides for mobile devices, where sites with specific points of interest will be recommended, with which the user can interact in playful ways and then create his personal travel narratives, (b) for the development of innovative games within virtual reality environment, where the interaction will be offered while the user is moving within the virtual environment; and (c) for an online application where the content will be offered through the browser and the modern 3D imaging technologies (WebGL). The technological products that will be developed within the proposed project can strengthen important sectors of economic and social life, such as trade, tourism, exploitation and promotion of the cultural environment, creative industries, etc. The final applications delivered at the end of the project will guarantee an improved level of service for visitors and will be a useful tool for content creators with increased adaptability, expansibility, and applicability in many regions of Greece and abroad. This paper aims to present the research project by referencing the state of the art and the methodological scheme, ending with a brief reflection on the expected outcome in terms of results.

Keywords: gamification, culture, tourism, AR, VR, applications

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Authors: Fausto Morais

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Artificial intelligence has been widely used in law. Programs are able to classify suits, to identify decision-making patterns, to predict outcomes, and to formalize legal arguments as well. In Brazil, the artificial intelligence victor has been classifying cases to supreme court’s standards. When those programs act doing those tasks, they simulate some kind of legal decision and legal arguments, raising doubts about how artificial intelligence can be integrated into legal reasoning. Taking this into account, the following three issues are identified; the problem of hypernormatization, the argument of legal anthropocentrism, and the artificial legal principles. Hypernormatization can be seen in the Brazilian legal context in the Supreme Court’s usage of the Victor program. This program generated efficiency and consistency. On the other hand, there is a feasible risk of over standardizing factual and normative legal features. Then legal clerks and programmers should work together to develop an adequate way to model legal language into computational code. If this is possible, intelligent programs may enact legal decisions in easy cases automatically cases, and, in this picture, the legal anthropocentrism argument takes place. Such an argument argues that just humans beings should enact legal decisions. This is so because human beings have a conscience, free will, and self unity. In spite of that, it is possible to argue against the anthropocentrism argument and to show how intelligent programs may work overcoming human beings' problems like misleading cognition, emotions, and lack of memory. In this way, intelligent machines could be able to pass legal decisions automatically by classification, as Victor in Brazil does, because they are binding by legal patterns and should not deviate from them. Notwithstanding, artificial intelligent programs can be helpful beyond easy cases. In hard cases, they are able to identify legal standards and legal arguments by using machine learning. For that, a dataset of legal decisions regarding a particular matter must be available, which is a reality in Brazilian Judiciary. Doing such procedure, artificial intelligent programs can support a human decision in hard cases, providing legal standards and arguments based on empirical evidence. Those legal features claim an argumentative weight in legal reasoning and should serve as references for judges when they must decide to maintain or overcome a legal standard.

Keywords: artificial intelligence, artificial legal principles, hypernormatization, legal anthropocentrism argument, legal reasoning

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Authors: Wei-Chou Hsu, Ching-Sung Lee, Han-Yin Liu

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This work uses H2O2 oxidation technique to improve the pH sensitivity of the AlGaN/GaN-based ion-sensitive field-effect transistors (ISFETs). 10-nm-thick Al2O3 was grown on the surface of the AlGaN. It was found that the pH sensitivity was improved from 41.6 mV/pH to 55.2 mV/pH. Since the H2O2-grown Al2O3 was served as a passivation layer and the problem of Fermi-level pinning was suppressed for the ISFET with the H2O2 oxidation process. Hysteresis effect in the ISFET with the H2O2 treatment also became insignificant. The hysteresis effect was observed by dipping the ISFETs into different pH value solutions and comparing the voltage difference between the initial and final conditions. The hysteresis voltage (Vhys) of the ISFET with the H2O2 oxidation process was improved from 8.7 mV to 4.8 mV. The hysteresis effect is related to the buried binding sites which are related to the material defects like threading dislocations in the AlGaN/GaN heterostructure which was grown by the hetero-epitaxy technique. The H2O2-grown Al2O3 passivate these material defects and the Al2O3 has less material defects. The long-term stability of the ISFET is estimated by the drift effect measurement. The drift measurement was conducted by dipping the ISFETs into a specific pH value solution for 12 hours and the ISFETs were operating at a specific quiescent point. The drift rate is estimated by the drift voltage divided by the total measuring time. It was found that the drift rate of the ISFET was improved from 10.1 mV/hour to 1.91 mV/hour in the pH 7 solution, from 14.06 mV/hour to 6.38 mV/pH in the pH 2 solution, and from 12.8 mV/hour to 5.48 mV/hour in the pH 12 solution. The drift effect results from the capacitance variation in the electric double layer. The H2O2-grown Al2O3 provides an additional capacitance connection in series with the electric double layer. Therefore, the capacitance variation of the electric double layer became insignificant. Generally, the H2O2 oxidation process is a simple, fast, and cost-effective method for the AlGaN/GaN-based ISFET. Furthermore, the performance of the AlGaN/GaN ISFET was improved effectively and the non-ideal effects were suppressed.

Keywords: AlGaN/GaN, Al2O3, hysteresis effect, drift effect, reliability, passivation, pH sensors

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Authors: Tom Nakotte, Hongmei Luo

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Lead chalcogenide-based (PbS, PbSe, and PbTe) quantum dots (QDs) were synthesized for the purpose of implementing them in radiation detectors. Pb based materials have long been of interest for gamma and x-ray detection due to its high absorption cross section and Z number. The emphasis of the studies was on exploring how to control charge carrier transport within thin films containing the QDs. The properties of QDs itself can be altered by changing the size, shape, composition, and surface chemistry of the dots, while the properties of carrier transport within QD films are affected by post-deposition treatment of the films. The QDs were synthesized using colloidal synthesis methods and films were grown using multiple film coating techniques, such as spin coating and doctor blading. Current QD radiation detectors are based on the QD acting as fluorophores in a scintillation detector. Here the viability of using QDs in solid-state radiation detectors, for which the incident detectable radiation causes a direct electronic response within the QD film is explored. Achieving high sensitivity and accurate energy quantification in QD radiation detectors requires a large carrier mobility and diffusion lengths in the QD films. Pb chalcogenides-based QDs were synthesized with both traditional oleic acid ligands as well as more weakly binding oleylamine ligands, allowing for in-solution ligand exchange making the deposition of thick films in a single step possible. The PbS and PbSe QDs showed better air stability than PbTe. After precipitation the QDs passivated with the shorter ligand are dispersed in 2,6-difloupyridine resulting in colloidal solutions with concentrations anywhere from 10-100 mg/mL for film processing applications, More concentrated colloidal solutions produce thicker films during spin-coating, while an extremely concentrated solution (100 mg/mL) can be used to produce several micrometer thick films using doctor blading. Film thicknesses of micrometer or even millimeters are needed for radiation detector for high-energy gamma rays, which are of interest for astrophysics or nuclear security, in order to provide sufficient stopping power.

Keywords: colloidal synthesis, lead chalcogenide, radiation detectors, quantum dots

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Authors: Nusrat Praween, Krishna Thej Pammi Guru, Palash Kumar Basu

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Designing non-invasive biosensors for cancer diagnosis is essential for developing an affordable and specific tool to measure cancer-related exosome biomarkers. Exosomes, released by healthy as well as cancer cells, contain valuable information about the biomarkers of various diseases, including cancer. Despite the availability of various isolation techniques, ultracentrifugation is the standard technique that is being employed. Post isolation, exosomes are traditionally exposed to detergents for extracting their proteins, which can often lead to protein degradation. Further to this, it is very essential to develop a sensing platform for the quantification of clinically relevant proteins in a wider range to ensure practicality. In this study, exosomes were immobilized on the Au Screen Printed Electrode (SPE) using EDC/NHS chemistry to facilitate binding. After immobilizing the exosomes on the screen-printed electrode (SPE), we investigated the impact of the electric field by applying various voltages to induce exosome lysis and release their contents. The lysed solution was used for sensing TSG101, a crucial biomarker associated with various cancers, using both faradaic and non-faradaic electrochemical impedance spectroscopy (EIS) methods. The results of non-faradaic and faradaic EIS were comparable and showed good consistency, indicating that non-faradaic sensing can be a reliable alternative. Hence, the non-faradaic sensing technique was used for label-free quantification of the TSG101 biomarker. The results were validated using ELISA. Our electrochemical immunosensor demonstrated a consistent response of TSG101 from 125 pg/mL to 8000 pg/mL, with a detection limit of 0.125 pg/mL at room temperature. Additionally, since non-faradic sensing is label-free, the ease of usage and cost of the final sensor developed can be reduced. The proposed immunosensor is capable of detecting the TSG101 protein at low levels in healthy serum with good sensitivity and specificity, making it a promising platform for biomarker detection.

Keywords: biosensor, exosomes isolation on SPE, electric field lysis of exosome, EIS sensing of TSG101

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Authors: Ankita Singh, Chandan Gorain, Amirul I. Mallick

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Successful adherence of the mucosal epithelial cells is the key early step for Campylobacter jejuni pathogenesis (C. jejuni). A set of Surface Exposed Colonization Proteins (SECPs) are among the major factors involved in host cell adherence and invasion of C. jejuni. Among them, constitutively expressed surface-exposed lipoprotein adhesin of C. jejuni, JlpA, interacts with intestinal heat shock protein 90 (hsp90α) and contributes in disease progression by triggering pro-inflammatory response via activation of NF-κB and p38 MAP kinase pathway. Together with its ability to express in the bacterial surface, higher sequence conservation and predicted predominance of several B cells epitopes, JlpA protein reserves its potential to become an effective vaccine candidate against wide range of Campylobacter sps including C. jejuni. Given that chickens are the primary sources for C. jejuni and persistent gut colonization remain as major cause for foodborne pathogenesis to humans, present study explicitly used chickens as model to test the immune-protective efficacy of JlpA protein. Taking into account that gastrointestinal tract is the focal site for C. jejuni colonization, to extrapolate the benefit of mucosal (intragastric) delivery of JlpA protein, a food grade Nisin inducible Lactic acid producing bacteria, Lactococcus lactis (L. lactis) was engineered to express recombinant JlpA protein (rJlpA) in the surface of the bacteria. Following evaluation of optimal surface expression and functionality of recombinant JlpA protein expressed by recombinant L. lactis (rL. lactis), the immune-protective role of intragastric administration of live rL. lactis was assessed in commercial broiler chickens. In addition to the significant elevation of antigen specific mucosal immune responses in the intestine of chickens that received three doses of rL. lactis, marked upregulation of Toll-like receptor 2 (TLR2) gene expression in association with mixed pro-inflammatory responses (both Th1 and Th17 type) was observed. Furthermore, intragastric delivery of rJlpA expressed by rL. lactis, but not the injectable form, resulted in a significant reduction in C. jejuni colonization in chickens suggesting that mucosal delivery of live rL. lactis expressing JlpA serves as a promising vaccine platform to induce strong immune-protective responses against C. jejuni in chickens.

Keywords: chickens, lipoprotein adhesion of Campylobacter jejuni, immuno-protection, Lactococcus lactis, mucosal delivery

Procedia PDF Downloads 139

Authors: Amos Sunday Onikanni, Bashir Lawal, Babatunji Emmanuel Oyinloye, Gomaa Mostafa-Hedeab, Mohammed Alorabi, Simona Cavalu, Augustine O. Olusola, Chih-Hao Wang, Gaber El-Saber Batiha

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The increasing global burden of diabetes mellitus has called for the search for a therapeutic alternative that offers better activities and safety than conventional chemotherapy. Herein, we evaluated the neuroprotective and antioxidant properties of different fractions (ethyl acetate, N-butanol and residual aqueous) of Clompanus pubescens leaves in streptozotocin (STZ)-induced diabetic rats. Our results revealed a significant elevation in the levels of blood glucose, pro-inflammatory cytokines, lipid peroxidation, neuronal activities of acetylcholinesterase, butyrylcholinesterase, nitric oxide, epinephrine, norepinephrine, and Na+/K+-ATPase in diabetic non treated rats. In addition, decreased levels of enzymatic and non-enzymatic antioxidants were observed. Treatment with different fractions of C. pubescens leaves resulted in a significant reversal of the biochemical alteration and improved the neurocognitive deficit in STZ-induced diabetic rats. However, the ethyl-acetate fraction demonstrated higher activities than the other fractions and was characterized for its phytoconstituents, revealing the presence of Gallic acid (713.00 ppm), catechin (0.91 ppm), ferulic acid (0.98 ppm), rutin (59.82 ppm), quercetin (3.22 ppm) and kaempferol (4.07 ppm). Our molecular docking analysis revealed that these compounds exhibited different binding affinities and potentials for targeting BChE/AChE/ IL-1 β/Na+-K+-ATPase. However, only Kampferol and ferulic exhibited good drug-like, ADMET, and permeability properties suitable for use as a neuronal drug target agent. Hence, the ethyl-acetate fraction of C. pubescent leaves could be considered a source of promising bioactive metabolite for the treatment and management of cognitive impairments related to type II diabetes mellitus.

Keywords: diabetes mellitus, neuroprotective, antioxidant, pro-inflammatory cytokines

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Authors: Hanaa A. Al-Gaoudi, Hassan Ebeid

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The research investigates the materials and processes used in the manufacturing of an Egyptian polychrome cartonnage mummy mask with the aim of dating this object and establishing trade patterns of certain materials that were used and available at the time of ancient Egypt. This anonymous-source object was held in the basement storage of the Egyptian Museum in Cairo (EMC) and has never been on display. Furthermore, there is no information available regarding its owner, provenance, date, and even the time of its possession by the museum. Moreover, the object is in a very poor condition where almost two-thirds of the mask was bent and has never received any previous conservation treatment. This research has utilized well-established multi-analytical methods to identify the considerable diversity of materials that have been used in the manufacturing of this object. These methods include Computed Tomography Scan (CT scan) to acquire detailed pictures of the inside physical structure and condition of the bended layers. Dino-Lite portable digital microscope, scanning electron microscopy with energy dispersive X-ray spectrometer (SEM-EDX), and the non-invasive imaging technique of multispectral imaging (MSI) to obtain information about the physical characteristics and condition of the painted layers and to examine the microstructure of the materials. Portable XRF Spectrometer (PXRF) and X-Ray powder diffraction (XRD) to identify mineral phases and the bulk element composition in the gilded layer, ground, and pigments; Fourier-transform infrared (FTIR) to identify organic compounds and their molecular characterization; accelerator mass spectrometry (AMS 14C) to date the object. Preliminary results suggest that there are no human remains inside the object, and the textile support is linen fibres with tabby weave 1/1 and these fibres are in a very bad condition. Several pigments have been identified, such as Egyptian blue, Magnetite, Egyptian green frit, Hematite, Calcite, and Cinnabar; moreover, the gilded layers are pure gold and the binding media in the pigments is Arabic gum and animal glue in the textile support layer.

Keywords: analytical methods, Egyptian museum, mummy mask, pigments, textile

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Authors: Navpreet Kaur, Himkusha Thakur, Nirmal Prabhakar

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The most controversial issue in crop production is the use of Organophosphate insecticides. This is evident in many reports that Organophosphate (OP) insecticides, among the broad range of pesticides are mainly involved in acute and chronic poisoning cases. OPs detection is of crucial importance for health protection, food and environmental safety. In our study, a nanocomposite of poly (3,4 ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide sheets (FTO) for the analysis of malathion OP. The -COOH functionalization of MWCNTs has been done for the covalent binding with amino groups of AChE enzyme. The use of PEDOT-MWCNT films exhibited an excellent conductivity, enables fast transfer kinetics and provided a favourable biocompatible microenvironment for AChE, for the significant malathion OP detection. The prepared biosensors were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Various optimization studies were done for different parameters including pH (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). Substrate kinetics has been performed and studied for the determination of Michaelis Menten constant. The detection limit for malathion OP was calculated to be 1 fM within the linear range 1 fM to 1 µM. The activity of inhibited AChE enzyme was restored to 98% of its original value by 2-pyridine aldoxime methiodide (2-PAM) (5 mM) treatment for 11 min. The oxime 2-PAM is able to remove malathion from the active site of AChE by means of trans-esterification reaction. The storage stability and reusability of the prepared biosensor is observed to be 30 days and seven times, respectively. The application of the developed biosensor has also been evaluated for spiked lettuce sample. Recoveries of malathion from the spiked lettuce sample ranged between 96-98%. The low detection limit obtained by the developed biosensor made them reliable, sensitive and a low cost process.

Keywords: PEDOT-MWCNT, malathion, organophosphates, acetylcholinesterase, biosensor, oxime (2-PAM)

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Authors: Thilina Ranaweera, Enes Makalic, John L. Hopper, Adrian Bickerstaffe

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Data are the primary asset of biomedical researchers, and the engine for both discovery and research translation. As the volume and complexity of research datasets increase, especially with new technologies such as large single nucleotide polymorphism (SNP) chips, so too does the requirement for software to manage, process and analyze the data. Researchers often need to execute complicated queries and conduct complex analyzes of large-scale datasets. Existing tools to analyze such data, and other types of high-dimensional data, unfortunately suffer from one or more major problems. They typically require a high level of computing expertise, are too simplistic (i.e., do not fit realistic models that allow for complex interactions), are limited by computing power, do not exploit the computing power of large-scale parallel architectures (e.g. supercomputers, GPU clusters etc.), or are limited in the types of analysis available, compounded by the fact that integrating new analysis methods is not straightforward. Solutions to these problems, such as those developed and implemented on parallel architectures, are currently available to only a relatively small portion of medical researchers with access and know-how. The past decade has seen a rapid expansion of data management systems for the medical domain. Much attention has been given to systems that manage phenotype datasets generated by medical studies. The introduction of heterogeneous genomic data for research subjects that reside in these systems has highlighted the need for substantial improvements in software architecture. To address this problem, we have developed SPARK, an enabling and translational system for medical research, leveraging existing high performance computing resources, and analysis techniques currently available or being developed. It builds these into The Ark, an open-source web-based system designed to manage medical data. SPARK provides a next-generation biomedical data management solution that is based upon a novel Micro-Service architecture and Big Data technologies. The system serves to demonstrate the applicability of Micro-Service architectures for the development of high performance computing applications. When applied to high-dimensional medical datasets such as genomic data, relational data management approaches with normalized data structures suffer from unfeasibly high execution times for basic operations such as insert (i.e. importing a GWAS dataset) and the queries that are typical of the genomics research domain. SPARK resolves these problems by incorporating non-relational NoSQL databases that have been driven by the emergence of Big Data. SPARK provides researchers across the world with user-friendly access to state-of-the-art data management and analysis tools while eliminating the need for high-level informatics and programming skills. The system will benefit health and medical research by eliminating the burden of large-scale data management, querying, cleaning, and analysis. SPARK represents a major advancement in genome research technologies, vastly reducing the burden of working with genomic datasets, and enabling cutting edge analysis approaches that have previously been out of reach for many medical researchers.

Keywords: biomedical research, genomics, information systems, software

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Authors: Sisaynew Tesfaw Admassu

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The strengthening of timber beams can be necessary for several reasons including the increase of live loads (possible in a historical building for a change of destination of use or upgrading to meet new requirements), the reduction of the resistant cross-sections following deterioration (attacks of biological agents such as fungi, and insects) or traumatic events (fires) and the excess of deflection in the members. The main purpose of strengthening an element is not merely to repair it, but also to prevent and minimize the appearance of future problems. This study did an experimental investigation on the behavior of reference and strengthened solid timber beams. The strengthening materials used in this study were CSM-450 glass fiber and steel materials for both flexural and shear strengthening techniques. Twenty-two solid timber beams of Juniperus procera (TID) species with the dimensions of 60 x 90 x 780 mm were used in the present study. The binding material to bond the strengthening materials with timber was general-purpose resin with Luperox® K10 MEKP catalyst. Three beams were used as control beams (unstrengthen beams) while the remaining nineteen beams were strengthened using the strengthening materials for flexure and shear. All the beams were tested for three points loading to failure by using a Universal Testing Machine, UTM-600kN machine. The experimental results showed that the strengthened beams performed better than the unstrengthen beams. The experimental result of flexural strengthened beams showed that the load-bearing capacity of strengthened beams increased between 16.34 – 42.55%. Four layers of Glass Fiber Reinforced polymer on the tension side of the beams was shown to be the most effective way to enhance load-bearing capacity. The strengthened beams also have an enhancement in their flexural stiffness. The stiffness of flexural strengthened beams was increased between 1.18 – 65.53% as compared to the control beams. The highest increment in stiffness has occurred on beams strengthened using 2x60 mm steel plates. The shear-strengthened beams showed a relatively small amount of performance as compared to flexural-strengthened beams; the reason is that the beams are sufficient for shear. The polyester resin used in the experimental work showed good performance in bonding agents between materials. The resin showed more effectiveness in GFRP materials than steel materials.

Keywords: heritage structures, strengthening, stiffness, adhesive, polyester resin, steel plates

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Authors: Agata Ferreira

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Rapidly increasing debates and initiatives in the area of Corporate Social Responsibility (“CSR”) have reached the world of international investment law. CSR standards that focus on the operations of multinational companies are increasingly relevant in the context of international investment policy making. In the past, the connection between CSR standards and legal framework for foreign direct investment has been largely non-existent. Recently, however, there is a growing trend of a more balance approach to rights and obligations as between investors and states under investment treaties. CSR principles join other social and environmental measures slowly being included in the investment treaties to enhance their sustainable development dimension. Issues of CSR are present on negotiation tables of new mega regional investment treaties like TTIP for example. To date, only a very few bilateral investment treaties and a handful of other international treaties with investment provisions include CSR clauses. In addition, the existing provisions tend to be of a soft type, where parties merely acknowledge importance of good corporate governance and CSR for sustainable development or generally affirm their aim to encourage enterprises to observe internationally recognised guidelines and principles of CSR. The relevant provisions often leave it up to the states to encourage enterprises operating within their territories to voluntarily incorporate CSR principles. The interaction between general non-binding CSR standards, domestic laws and policies and provisions of international investment treaties have not been tested by investment tribunals yet. The role of investment treaties in raising awareness and promoting CSR is still in its infancy. The use of CSR standards in the international investment protection regime for promotion of CSR standards, and as a tool for disciplining investors into complying with such standards, pose a number of questions and is met with resistance from investors` lobbies. Integration of these two areas, CSR and international investment law, both consisting of multilayered, diverse and often overlapping instruments is by no means an easy task. Whether international investment world is ready to embrace CSR standards or shrug them off is a matter of uncertain future. The subject however has been raised, first introductions have been made and the time will show whether the relationship between legal framework of international investment and CSR will flourish or remain dormant.

Keywords: corporate social responsibility, foreign direct investment, investment treaties, sustainable development

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Authors: Mohamed Moussaoui, Mouna Baassi, Soukayna Baammi, Hatim Soufi, Mohammed Salah, Rachid Daoud, Achraf EL Allali, Mohammed Elalaoui Belghiti, Said Belaaouad

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The present study aims to investigate the quantitative structure-activity relationship (QSAR) of a series of Thiazole derivatives reported as anticancer agents (hepatocellular carcinoma), using principally the electronic descriptors calculated by the density functional theory (DFT) method and by applying the multiple linear regression method. The developed model showed good statistical parameters (R²= 0.725, R²ₐ𝒹ⱼ= 0.653, MSE = 0.060, R²ₜₑₛₜ= 0.827, Q²𝒸ᵥ = 0.536). The energy of the highest occupied molecular orbital (EHOMO) orbital, electronic energy (TE), shape coefficient (I), number of rotatable bonds (NROT), and index of refraction (n) were revealed to be the main descriptors influencing the anti-cancer activity. Additional Thiazole derivatives were then designed and their activities and pharmacokinetic properties were predicted using the validated QSAR model. These designed molecules underwent evaluation through molecular docking (MD) and molecular dynamic (MD) simulations, with binding affinity calculated using the MMPBSA script according to a 100 ns simulation trajectory. This process aimed to study both their affinity and stability towards Cyclin-Dependent Kinase 2 (CDK2), a target protein for cancer disease treatment. The research concluded by identifying four CDK2 inhibitors - A1, A3, A5, and A6 - displaying satisfactory pharmacokinetic properties. MDs results indicated that the designed compound A5 remained stable in the active center of the CDK2 protein, suggesting its potential as an effective inhibitor for the treatment of hepatocellular carcinoma. The findings of this study could contribute significantly to the development of effective CDK2 inhibitors.

Keywords: QSAR, ADMET, Thiazole, anticancer, molecular docking, molecular dynamic simulations, MMPBSA calculation

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Authors: Ramin Mehdiabadi, Neda Menbari, Mohammad Nazir Menbari

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As a pressing public health concern, breast cancer stands as the predominant oncological diagnosis and principal cause of cancer-related mortality among women globally, accounting for 11.7% of new cancer incidences and 6.9% of cancer-related deaths. The annual figures indicate that approximately 230,480 women are diagnosed with breast cancer in the United States alone, with 39,520 succumbing to the disease. While developed economies have reported a deceleration in both incidence and mortality rates across various forms of cancer, including breast cancer, emerging and low-income economies manifest a contrary escalation, largely attributable to lifestyle-mediated risk factors such as tobacco usage, physical inactivity, and high caloric intake. Breast cancer is distinctly characterized by molecular heterogeneity, manifesting in specific subtypes delineated by biomarkers—Estrogen Receptors (ER), Progesterone Receptors (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). These subtypes, comprising Luminal A, Luminal B, HER2-enriched, triple-negative/basal-like, and normal-like, necessitate nuanced, subtype-specific therapeutic regimens, thereby challenging the applicability of generalized treatment protocols. Within this molecular complexity, the transcription factor Forkhead Box M1 (FoxM1) has garnered attention as a significant driver of cellular proliferation, tumorigenesis, metastatic progression, and treatment resistance in a spectrum of human malignancies, including breast cancer. Concurrently, microRNAs (miRs), specifically miR-216b-5p, have been identified as post-transcriptional gene expression regulators and potential tumor suppressors. The overarching objective of this academic investigation is to explicate the multifaceted interrelationship between FoxM1 and miR-216b-5p across the disparate molecular subtypes of breast cancer. Employing a methodologically rigorous, interdisciplinary research design that incorporates cutting-edge molecular biology techniques, sophisticated bioinformatics analytics, and exhaustive meta-analyses of extant clinical data, this scholarly endeavor aims to unveil novel biomarker-specific therapeutic pathways. By doing so, this research is positioned to make a seminal contribution to the advancement of personalized, efficacious, and minimally toxic treatment paradigms, thus profoundly impacting the global efforts to ameliorate the burden of breast cancer.

Keywords: breast cancer, fox m1, microRNAs, mir-216b-5p, gene expression

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Authors: Eman M. Sarhan, Doaa A. Ghareeb, Gabriella Ortore, Amr A. Amara, Mohamed M. El-Sayed

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Drug salting and nanoparticle-based drug delivery formulations are considered to be an effective means for rendering the hydrophobic drugs’ nano-scale dispersion in aqueous media, and thus circumventing the pitfalls of their poor solubility as well as enhancing their membrane permeability. The current study aims to increase the bioavailability of quaternary ammonium berberine through acid salting and biodegradable bovine serum albumin (BSA)-based nanoparticulate drug formulation. Berberine hydroxide (BBR-OH) that was chemically synthesized by alkalization of the commercially available berberine hydrochloride (BBR-HCl) was then acidified to get Di-berberine sulfate (BBR)₂SO₄. The purified crystals were spectrally characterized. The desolvation technique was optimized for the preparation of size-controlled BSA-BBR-HCl, BSA-BBR-OH, and BSA-(BBR)₂SO₄ nanoparticles. Particle size, zeta potential, drug release, encapsulation efficiency, Fourier transform infrared spectroscopy (FTIR), tandem MS-MS spectroscopy, energy-dispersive X-ray spectroscopy (EDX), scanning and transmitting electron microscopic examination (SEM, TEM), in vitro bioactivity, and in silico drug-polymer interaction were determined. BSA (PDB ID; 4OR0) protonation state at different pH values was predicted using Amber12 molecular dynamic simulation. Then blind docking was performed using Lamarkian genetic algorithm (LGA) through AutoDock4.2 software. Results proved the purity and the size-controlled synthesis of berberine-BSA-nanoparticles. The possible binding poses, hydrophobic and hydrophilic interactions of berberine on BSA at different pH values were predicted. Antioxidant, anti-hemolytic, and cell differentiated ability of tested drugs and their nano-formulations were evaluated. Thus, drug salting and the potentially effective albumin berberine nanoparticle formulations can be successfully developed using a well-optimized desolvation technique and exhibiting better in vitro cellular bioavailability.

Keywords: berberine, BSA, BBR-OH, BBR-HCl, BSA-BBR-HCl, BSA-BBR-OH, (BBR)₂SO₄, BSA-(BBR)₂SO₄, FTIR, AutoDock4.2 Software, Lamarkian genetic algorithm, SEM, TEM, EDX

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Authors: Habtamu Abera Goshu, Ping Yan

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Copy number variation (CNV) is a significant marker of the genetic and phenotypic diversity among individuals that accounts for complex quantitative traits of phenotype and diseases via modulating gene dosage, position effects, alteration of downstream pathways, modification of chromosome structure, and position within the nucleus and disrupting coding regions in the genome. Associating copy number variations (CNVs) with growth and gene expression are a powerful approach for identifying genomic characteristics that contribute to phenotypic and genotypic variation. A previous study using next-generation sequencing illustrated that the choline kinase beta (CHKB), Krüpple-like factor 6 (KLF6), glypican 1(GPC1), and cholinergic receptor muscarinic 3 (CHRM3) genes reside within copy number variable regions (CNVRs) of yak populations that overlap with quantitative trait loci (QTLs) of meat quality and growth. As a result, this research aimed to determine the association of CNVs of the KLF6, CHKB, GPC1, and CHRM3 genes with growth traits in the Datong yak breed. The association between the CNV types of the KLF6, CHKB, GPC1, and CHRM3 genes and the growth traits in the Datong yak breed was determined by one-way analysis of variance (ANOVA) using SPSS software. The CNV types were classified as a loss (a copy number of 0 or 1), gain (a copy number >2), and normal (a copy number of 2) relative to the reference gene, BTF3 in the 387 individuals of Datong yak. These results indicated that the normal CNV types of the CHKB and GPC1 genes were significantly (P<0.05) associated with high body length, height and weight, and chest girth in six-month-old and five-year-old Datong yaks. On the other hand, the loss CNV types of the KLF6 gene is significantly (P<0.05) associated with body weight and length and chest girth at six-month-old and five-year-old Datong yaks. In the contrary, the gain CNV type of the CHRM3 gene is highly (P<0.05) associated with body weight, length, height, and chest girth in six-month-old and five-year-old. This work provides the first observation of the biological role of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in the Datong yak breed and might, therefore, provide a novel opportunity to utilize data on CNVs in designing molecular markers for the selection of animal breeding programs for larger populations of various yak breeds. Therefore, we hypothesized that this study provided inclusive information on the application of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in growth traits in Datong yaks and its possible function in bovine species.

Keywords: Copy number variation, growth traits, yak, genes

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Authors: Yee-Seul So, Guiseppe Ianiri, Alex Idnurm, Yong-Sun Bahn

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Tor1 is a serine/threonine protein kinase that is widely conserved across eukaryotic species. Tor1 was first identified in Saccharomyces cerevisiae as a target of rapamycin (TOR). The TOR pathway has been implicated in regulating cellular responses to nutrients, proliferation, translation, transcription, autophagy, and ribosome biogenesis. Here we identified two homologues of S. cerevisiae Tor proteins, CNAG_06642 (Tor1) and CNAG_05220 (Tlk1, TOR-like kinase 1), in Cryptococcus neoformans causing a life-threatening fungal meningoencephalitis. Both Tor1 and Tlk1 have rapamycin-binding (RB) domains but Tlk1 has truncated RB form. To study the TOR-signaling pathway in the fungal pathogen, we attempt to construct the tor1Δ and tlk1Δ mutants and phenotypically analyze them. Although we failed to construct the tor1Δ mutant, we successfully construct the tlk1Δ mutant. The tlk1Δ mutant does not exhibit any discernable phenotypes, suggesting that Tlk1 is dispensable in C. neoformans. The essentiality of TOR1 is independently confirmed by constructing the TOR1 promoter replacement strain by using a copper transporter 4 (CTR4) promoter and the TOR1/tor1 heterozygous mutant in diploid C. neoformans strain background followed by sporulation analysis. To further analyze the function of Tor1, we construct TOR1 overexpression mutant using a constitutively active histone H3 in C. neoformans. We find that the Tor1 overexpression mutant is resistant to rapamycin but the tlk1Δ mutant does not exhibit any altered resistance to rapamycin, further confirming that Tor1, but not Tlk1, is critical for TOR signaling. Furthermore, we found that Tor1 is involved in response to diverse stresses, including genotoxic stress, oxidative stress, thermo-stress, antifungal drug treatment, and production of melanin. To identify any TOR-related transcription factors, we screened C. neoformans transcription factor library that we constructed in our previous study and identified several potential downstream factors of Tor1, including Atf1, Crg1 and Bzp3. In conclusion, the current study provides insight into the role of the TOR signaling pathway in human fungal pathogens as well as C. neoformans.

Keywords: fungal pathogen, serine/threonine kinase, target of rapamycin, transcription factor

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

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Authors: Zelal Polat, Şebnem Harsa, Semra Ülkü

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Lactic acid exists in nature optically in two forms, L(+), D(-)-lactic acid, and has been used in food, leather, textile, pharmaceutical and cosmetic industries. Moreover, L(+)-lactic acid constitutes the raw material for the production of poly-L-lactic acid which is used in biomedical applications. Microbially produced lactic acid was aimed to be recovered from the fermentation media efficiently and economically. Among the various downstream operations, ion exchange chromatography is highly selective and yields a low cost product recovery within a short period of time. In this project, Lactobacillus casei NRRL B-441 was used for the production of L(+)-lactic acid from whey by fermentation at pH 5.5 and 37°C that took 12 hours. The product concentration was 50 g/l with 100% L(+)-lactic acid content. Next, the suitable resin was selected due to its high sorption capacity with rapid equilibrium behavior. Dowex marathon WBA, weakly basic anion exchanger in OH form reached the equilibrium in 15 minutes. The batch adsorption experiments were done approximately at pH 7.0 and 30°C and sampling was continued for 20 hours. Furthermore, the effect of temperature and pH was investigated and their influence was found to be unimportant. All the adsorption/desorption experiments were applied to both model lactic acid and biomass free fermentation broth. The ion exchange equilibria of lactic acid and L(+)-lactic acid in fermentation broth on Dowex marathon WBA was explained by Langmuir isotherm. The maximum exchange capacity (qm) for model lactic acid was 0.25 g La/g wet resin and for fermentation broth 0.04 g La/g wet resin. The equilibrium loading and exchange efficiency of L(+)-lactic acid in fermentation broth were reduced as a result of competition by other ionic species. The competing ions inhibit the binding of L(+)-lactic acid to the free sites of ion exchanger. Moreover, column operations were applied to recover adsorbed lactic acid from the ion exchanger. 2.0 M HCl was the suitable eluting agent to recover the bound L(+)-lactic acid with a flowrate of 1 ml/min at ambient temperature. About 95% of bound L(+)-lactic acid was recovered from Dowex marathon WBA. The equilibrium was reached within 15 minutes. The aim of this project was to investigate the purification of L(+)-lactic acid with ion exchange method from fermentation broth. The additional goals were to investigate the end product purity, to obtain new data on the adsorption/desorption behaviours of lactic acid and applicability of the system in industrial usage.

Keywords: fermentation, ion exchange, lactic acid, purification, whey

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Authors: Nur Iman Subono, Meidi Kosandi

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This paper examines the role of regional cooperation in the Association of Southeast Asian Nations (ASEAN) in the fight against human trafficking for Indonesia. Many among scholars suggest that regional cooperation is necessary for combating human trafficking for its transnational and organized character as a crime against humanity. ASEAN members have been collectively active in responding transnational security issues with series of talks and collaboration agreement since early 2000s. Lately in 2015, ASEAN agreed on ASEAN Convention against Trafficking in Persons, particularly Women and Children (ACTIP) that requires each member to collaborate in information sharing and providing effective safeguard and protection of victims. Yet, the frequency of human trafficking crime occurrence remains high and tend to increase in Indonesian in 2017-2018. The objective of this paper is to examine the effectiveness and success of ACTIP implementation in the fight against human trafficking in Indonesia. Based on two years of research (2017-2018) in three provinces with the largest number of victims in Indonesia, this paper shows the tendency of persisting crime despite the implementation of regional and national anti-trafficking policies. The research was conducted by archive study, literature study, discourse analysis, and depth interviews with local government officials, police, prosecutors, victims, and traffickers. This paper argues that the relative success of ASEAN in establishing convention at the high-level meetings has not been followed with the success in its implementation in the society. Three main factors have contributed to the ineffectiveness of the agreements, i.e. (1) ASEAN institutional arrangement as a collection of sovereign states instead of supranational organization with binding authority; (2) the lack of commitment of ASEAN sovereign member-states to the agreements; and (3) the complexity and variety of the nature of the crime in each member-state. In effect, these factors have contributed to generating the regionalism paradox in ASEAN where states tend to revert to national policies instead of seeking regional collective solution.

Keywords: human trafficking, transnational security, regionalism, anti trafficking policy

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Authors: Y. Saylan, F. Yılmaz, A. Denizli

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Rheumatoid arthritis (RA) which is the most common autoimmune disorder of the body's own immune system attacking healthy cells. RA has both articular and systemic effects.Until now romatiod factor (RF) assay is used the most commonly diagnosed RA but it is not specific. Anti-cyclic citrullinated peptide (anti-CCP) antibodies are IgG autoantibodies which recognize citrullinated peptides and offer improved specificity in early diagnosis of RA compared to RF. Anti-CCP antibodies have specificity for the diagnosis of RA from 91 to 98% and the sensitivity rate of 41-68%. Molecularly imprinted polymers (MIP) are materials that are easy to prepare, less expensive, stable have a talent for molecular recognition and also can be manufactured in large quantities with good reproducibility. Molecular recognition-based adsorption techniques have received much attention in several fields because of their high selectivity for target molecules. Quartz crystal microbalance (QCM) is an effective, simple, inexpensive approach mass changes that can be converted into an electrical signal. The applications for specific determination of chemical substances or biomolecules, crystal electrodes, cover by the thin films for bind or adsorption of molecules. In this study, we have focused our attention on combining of molecular imprinting into nanofilms and QCM nanosensor approaches and producing QCM nanosensor for anti-CCP, chosen as a model protein, using anti-CCP imprinted nanofilms. For this aim, anti-CCP imprinted QCM nanosensor was characterized by Fourier transform infrared spectroscopy, atomic force microscopy, contact angle measurements and ellipsometry. The non-imprinted nanosensor was also prepared to evaluate the selectivity of the imprinted nanosensor. Anti-CCP imprinted QCM nanosensor was tested for real-time detection of anti-CCP from aqueous solution. The kinetic and affinity studies were determined by using anti-CCP solutions with different concentrations. The responses related with mass shifts (Δm) and frequency shifts (Δf) were used to evaluate adsorption properties and to calculate binding (Ka) and dissociation (Kd) constants. To show the selectivity of the anti-CCP imprinted QCM nanosensor, competitive adsorption of anti-CCP and IgM was investigated.The results indicate that anti-CCP imprinted QCM nanosensor has a higher adsorption capabilities for anti-CCP than for IgM, due to selective cavities in the polymer structure.

Keywords: anti-CCP, molecular imprinting, nanosensor, rheumatoid arthritis, QCM

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Authors: Einat E. Peles, Shaul Schreiber, Miriam Adelson

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Background: Since established more than 50 years ago, methadone maintenance treatment (MMT) is the most effective treatment for opioid addiction, a chronic relapsing brain disorder that became an epidemic in western societies. Treatment includes daily individual optimal medication methadone dose (a long acting mu opioid receptor full agonist), accompanied with psychosocial therapy. It is well established that the longer retention in treatment the better outcome and survival occur. It reduces the likelihood to infectious diseases and overdose death that associated with drug injecting, enhanced social rehabilitation and eliminate criminal activity, and lead to healthy productive life. Aim: To evaluate predictors for long term retention in treatment we analyzed our prospective follow up of a major MMT clinic affiliated to a big tertiary medical center. Population Methods: Between June 25, 1993, and June 24, 2016, all 889 patients ( ≥ 18y) who ever admitted to the clinic were prospectively followed-up until May 2017. Duration in treatment from the first admission until the patient quit treatment or until the end of follow-up (24 years) was taken for calculating cumulative retention in treatment using survival analyses (Kaplan Meier) with log-rank and Cox regression for multivariate analyses. Results: Of the 889 patients, 25.2% were females who admitted to treatment at younger age (35.0 ± 7.9 vs. 40.6 ± 9.8, p < .0005), but started opioid usage at same age (22.3 ± 6.9). In addition to opioid use, on admission to MMT 58.5% had positive urine for benzodiazepines, 25% to cocaine, 12.4% to cannabis and 6.9% to amphetamines. Hepatitis C antibody tested positive in 55%, and HIV in 7.8% of the patients and 40%. Of all patients, 75.7% stayed at least one year in treatment, and of them, 67.7% stopped opioid usage (based on urine tests), and a net reduction observed in all other substance abuse (proportion of those who stopped minus proportion of those who have started). Long term retention up to 24 years was 8.0 years (95% Confidence Interval (CI) 7.4-8.6). Predictors for longer retention in treatment (Cox regression) were being older on admission ( ≥ 30y) Odds Ratio (OR) =1.4 (CI 1.1-1.8), not abusing opioids after one year OR=1.8 (CI 1.5-2.1), not abusing benzodiazepine after one year OR=1.7 (CI 1.4-2.1) and treating with methadone dose ≥ 100mg/day OR =1.8 (CI 1.5-2.3). Conclusions: Treating and following patients over 24 years indicate success of two main outcomes, high rate of retention after one year (75.7%) and high proportion of opiate abuse cessation (67.7%). As expected, longer cumulative retention was associated with patients treated with high adequate methadone dose that successfully result in opioid cessation. Based on these findings, in order to reduce morbidity and mortality, we find the establishment of more MMT clinics within a general hospital, a most urgent necessity.

Keywords: methadone maintenance treatment, epidemic, opioids, retention

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Authors: Priyanka Bhowmik, Subrata Majumdar, Debprasad Chattopadhyay

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Background: Cervical cancer is the third major cause of cancer in women and the second most frequent cause of cancer related deaths causing 300,000 deaths annually worldwide. Evasion of immune response by Human Papilloma Virus (HPV), the key contributing factor behind cancer and pre-cancerous lesions of the uterine cervix, makes immunotherapy a necessity to treat this disease. Objective: A Heat killed fraction of Mycobacterium indicus pranii (MIP), a non-pathogenic Mycobacterium has been shown to exhibit cytotoxic effects on different cancer cells, including human cervical carcinoma cell line HeLa. However, the underlying mechanisms remain unknown. The aim of this study is to decipher the mechanism of MIP induced HeLa cell death. Methods: The cytotoxicity of Mycobacterium indicus pranii against HeLa cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and Propidium iodide (PI) staining. The assessment of reactive oxygen species (ROS) generation and cell cycle analysis were measured by flow cytometry. The expression of apoptosis associated genes was analyzed by real time PCR. Result: MIP could inhibit the proliferation of HeLa cell in a time and dose dependent manner but caused minor damage to normal cells. The induction of apoptosis was confirmed by the cell surface presentation of phosphatidyl serine, DNA fragmentation, and mitochondrial damage. MIP caused very early (as early as 30 minutes) transcriptional activation of p53, followed by a higher activation (32 fold) at 24 hours suggesting prime importance of p53 in MIP-induced apoptosis in HeLa cell. The up regulation of p53 dependent pro-apoptotic genes Bax, Bak, PUMA, and Noxa followed a lag phase that was required for the transcriptional p53 program. MIP also caused the transcriptional up regulation of Toll like receptor 2 and 4 after 30 minutes of MIP treatment suggesting recognition of MIP by toll like receptors. Moreover, MIP caused the inhibition of expression of HPV anti apoptotic gene E6, which is known to interfere with p53/PUMA/Bax apoptotic cascade. This inhibition might have played a role in transcriptional up regulation of PUMA and subsequently apoptosis. ROS was generated transiently which was concomitant with the highest transcription activation of p53 suggesting a plausible feedback loop network of p53 and ROS in the apoptosis of HeLa cells. Scavenger of ROS, such as N-acetyl-L-cysteine, decreased apoptosis suggesting ROS is an important effector of MIP induced apoptosis. Conclusion: Taken together, MIP possesses full potential to be a novel therapeutic agent in the clinical treatment of cervical cancer.

Keywords: cancer, mycobacterium, immunity, immunotherapy.

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Authors: Farahnaz Sadat Golestan Hashemi, Mohd Razi Ismail, Mohd Y. Rafii

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A huge revolution has emerged in genome engineering by the discovery of CRISPR (clustered regularly interspaced palindromic repeats) and CRISPR-associated system genes (Cas) in bacteria. The function of type II Streptococcus pyogenes (Sp) CRISPR/Cas9 system has been confirmed in various species. Other S. thermophilus (St) CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been also reported for preventing phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. The S. thermophilus CRISPR3-Cas system also acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed in the CRISPR1-Cas system. It is worth mentioning, for the effective DNA cleavage activity, RNA-guided Cas9 orthologs require their own specific PAM (protospacer adjacent motif) sequences. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of target site for the three orthogonals of Cas9 protein, a well-organized procedure will be required for high-throughput and accurate mining of possible target sites in a large genomic dataset. Consequently, we created a reliable procedure to explore potential gRNA sequences for type I (Streptococcus thermophiles), II (Streptococcus pyogenes), and III (Streptococcus thermophiles) CRISPR/Cas systems. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows: i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. The output of such procedure highlights the power of comparative genome mining for different CRISPR/Cas systems. This could yield a repertoire of Cas9 variants with expanded capabilities of gRNA design, and will pave the way for further advance genome and epigenome engineering.

Keywords: CRISPR/Cas systems, gRNA mining, Streptococcus pyogenes, Streptococcus thermophiles

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Authors: G. Krishnamoorthy, S. Anandhakumar

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The effect of D-Lysine (D-Lys) on collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide(NHS) initiated cross linking using experimental and modelling tools are evaluated. The results of the Coll-D-Lys-EDC/NHS scaffold also indicate an increase in the tensile strength (TS), percentage of elongation (% E), denaturation temperature (Td), and decrease the decomposition rate compared to L-Lys-EDC/NHS. Scanning electron microscopic (SEM) and atomic force microscopic (AFM) analyses revealed a well ordered with properly oriented and well-aligned structure of scaffold. The D-Lys stabilizes the scaffold against degradation by collagenase than L-Lys. The cell assay showed more than 98% fibroblast viability (NIH3T3) and improved cell adhesions, protein adsorption after 72h of culture when compared with native scaffold. Cell attachment after 74h was robust, with cytoskeletal analysis showing that the attached cells were aligned along the fibers assuming a spindle-shape appearance, despite, gene expression analyses revealed no apparent alterations in mRNA levels, although cell proliferation was not adversely affected. D-Lysine (D-Lys) plays a pivotal role in the self-assembly and conformation of collagen fibrils. The D-Lys assisted EDC/NHS initiated cross-linking induces the formation of an carboxamide by the activation of the side chain -COOH group, followed by aminolysis of the O-iso acylurea intermediates by the -NH2 groups are directly joined via an isopeptides bond. This leads to the formation of intra- and inter-helical cross links. Modeling studies indicated that D-Lys bind with collagen-like peptide (CLP) through multiple H-bonding and hydrophobic interactions. Orientational changes in collagenase on CLP-D-Lys are observed which may decrease its accessibility to degradation and stabilize CLP against the action of the former. D-Lys has lowest binding energy and improved fibrillar-assembly and staggered alignment without the undesired structural stiffness and aggregations. The proteolytic machinery is not well equipped to deal with Coll-D-Lys than Coll-L-Lys scaffold. The information derived from the present study could help in designing collagenolytically stable heterochiral collagen based scaffold for biomedical applications.

Keywords: collagen, collagenase, collagen like peptide, D-lysine, heterochiral collagen scaffold

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Authors: Akhila Jose

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The cement industry is one of the major contributors to the global warming due to the release of greenhouse gases. The primary binder in conventional concrete is Ordinary Portland cement (OPC) and billions of tons are produced annually all over the world. An alternative binding material to OPC is needed to reduce the environmental impact caused during the cement manufacturing process. Geopolymer concrete is an ideal material to substitute cement-based binder. Geopolymer is an inorganic alumino-silicate polymer. Geopolymer Concrete (GPC) is formed by the polymerization of aluminates and silicates formed by the reaction of solid aluminosilicates with alkali hydroxides or alkali silicates. Various Industrial bye- products like Fly Ash (FA), Rice Husk Ash (RHA), Ground granulated Blast Furnace Slag (GGBFS), Silica Fume (SF), Red mud (RM) etc. are rich in aluminates and silicates. Using by-products from other industries reduces the carbon dioxide emission and thus giving a sustainable way of reducing greenhouse gas emissions and also a way to dispose the huge wastes generated from the major industries like thermal plants, steel plants, etc. The earlier research about geopolymer were focused on heat cured fly ash based precast members and this limited its applications. The heat curing mechanism itself is highly cumbersome and costly even though they possess high compressive strength, low drying shrinkage and creep, and good resistance to sulphate and acid environments. GPC having comparable strength and durability characteristics of OPC were able to develop under ambient cured conditions is the solution making it a sustainable alternative in future. In this paper an attempt has been made to review and compare the feasibility of ambient cured GPC over heat cured geopolymer concrete with respect to strength and serviceability characteristics. The variation on the behavior of structural members is also reviewed to identify the research gaps for future development of ambient cured geopolymer concrete. The comparison and analysis of studies showed that GPC most importantly ambient cured type has a comparable behavior with respect to OPC based concrete in terms strength and durability criteria.

Keywords: geopolymer concrete, oven heated, durability properties, mechanical properties

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