Search results for: in vitro folding

Authors: J. Vitku, L. Kolatorova, T. Chlupacova, J. Heracek, M. Hill, M. Duskova, L. Starka

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Background: 7β-hydroxy-epiandrosterone (7β–OH-EpiA) is an endogenous steroid, that has been shown to exert neuroprotective and anti-inflammatory effects in vitro as well as in animal models. However, to the best of our knowledge no information is available about concentration of this androgen metabolite in human population. The aim of the study was to measure and compare levels of 7β–OH-EpiA in men and pregnant women in different biological fluids and evaluate the relationship between 7β–OH-EpiA in men and their sperm quality. Methods: First, a sensitive isotope dilution high performance liquid chromatography-mass spectrometry method for measurement of 7β–OH-EpiA in different biological fluids was developed. Validation of the method met the requirements of FDA guidelines. Afterwards 7β–OH-EpiA in plasma and seminal plasma of 191 men with different degree of infertility (healthy men, lightly infertile men, moderately infertile men, severely infertile men) was analysed. Furthermore, the levels of 7β–OH-EpiA in plasma of 34 pregnant women in 37th week of gestation and corresponding cord plasma that reflects steroid levels in the fetus were measured. Results: Concentrations of 7β–OH-EpiA in seminal plasma were significantly higher in severely infertile men in comparison with healthy men and lightly infertile men. The same trend was observed when blood plasma was evaluated. Furthermore, plasmatic 7β –OH-EpiA negatively correlated with concentration (-0.215; p < 0.01) and total count (-0.15; p < 0.05). Seminal 7β–OH-EpiA was negatively associated with motility (-0.26; p < 0.01), progressively motile sperms (-0.233; p < 0.01) and nonprogressively motile sperms (-0.188; p < 0.05). Plasmatic 7β –OH-EpiA levels in men were generally higher in comparison with pregnant women. Levels 7β–OH-EpiA were under the lower limit of quantification (LLOQ) in majority of samples of pregnant women and cord plasma. Only 4 plasma samples of pregnant women and 7 cord blood plasma samples were above LLOQ and where in range of units of pg/ml. Conclusion: Based on available information, this is the first study measuring 7β–OH-EpiA in human samples. 7β–OH-EpiA is associated with lower sperm quality and certainly it is worth to explore its role in this field thoroughly. Interestingly, levels of 7β–OH-EpiA in pregnant women were extremely low despite the fact that steroid levels including androgens are generally higher during pregnancy. Acknowledgements: This work was supported by the project MH CR 17-30528 A from the Czech Health Research Council, MH CZ - DRO (Institute of Endocrinology - EU, 00023761) and by the MEYS CR (OP RDE, Excellent research - ENDO.CZ).

Keywords: 7β-hydroxy-epiandrosterone, steroid, sperm quality, pregnancy

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Authors: Dipanshu Sur, Ratnabali Chakravorty, Rimi Pal, Siddhartha Chatterjee, Joyshree Chaterjee, Amal Mallik

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Background: Polycystic ovary syndrome (PCOS) is a common endocrine disease in reproductive women with a complex hormonal disturbance that affects the menstrual cycle and leads to metabolic consequences in later life. Hyperandrogenaemia is noticeable features of PCOS and influence the process of folliculogenesis in women. The levels of Antimüllerian Hormone (AMH) reflect the number of pre-antral follicles and thus are a marker of oocyte pool – germinal reserve of the ovary for reproduction. Besides its utilization in IVF (In-vitro fertilization), determination of AMH may serve as an additional marker in the diagnostics of PCOS, where increased AMH levels reflect the severity of the disease. The positive correlation of serum AMH with the number of antral follicles was found also in patients with PCOS. Objective: The objective of this study was to investigate the relationship between AMH androgens and whether AMH contributes to altered folliculogenesis in non-obese women with PCOS. Methods: We designed a prospective study which included a total of 65 IVF individuals. It enrolled 26 cases of PCOS based on 2003 Rotterdam criteria and 39 ovulatory normal- non PCOS, healthy, age-matched controls. AMH levels and ovarian morphology were assessed. The relationships between AMH and androgenaemia in patients with and without PCOS were studied. Results: Mean age of PCOS patients were slightly higher than controls (32±4 and 28±3 years, respectively). AMH generally increased with antral follicle count (AFC) [P=0.001], testosterone, and luteinising hormone, and decreased with age, and serum sex hormone binding globulin (SHBG). No significant relationships were found between circulating AMH levels and BMI between PCOS and non-PCOS patients. The calculation of AMH production per antral follicle (AMH/AF) showed that there was a significant difference in median AMH/AF between PCOS and non-PCOS (P =0.001). Both PCOS and non-PCOS groups showed a very similar increase in AMH with increases in AFC, but the PCOS patients had consistently higher AMH across all AFC levels. Conclusions: These observations indicate that there is a connection between AMH and androgens levels between PCOS and non-PCOS East Indian women. Excessive granulosa cell activity may be implicated in the abnormal follicular dynamic of the syndrome. They are higher in women with PCOS and, on the other hand, very low in women with an ovarian failure.

Keywords: anti-Mullerian hormone, polycystic ovary syndrome, antral follicle count, androgens

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Authors: Nandhakumar Elumalai, Purushothaman Ayyakannu, Sachidanandam T. Panchanatham

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Background: Understanding the basic mechanisms and factors underlying the tumor growth and invasion has gained attention in recent times. The processes of angiogenesis and apoptosis are known to play a vital role in various stages of cancer. The vascular endothelial growth factor (VEGF) is well established as one of the key regulators of tumor angiogenesis while MMPs are known for their exclusive ability to degrade ECM. Objective: The present study was designed to evaluate the pro apoptotic and anti angiogenic activity of the herbal formulation Shemamruthaa. The anticancer activity of Shemamruthaa was tested in breast cancer cell line (MCF-7). Results of MTT, trypan blue and flow cytometric analysis of apoptotis suggested that Shemamruthaa can induce cytotoxicity in cancer cells, in a concentration- and time dependent manner and induce apoptosis. With these results, we further evaluated the antiangiogenic and pro-apoptotic activities of Shemamruthaa in DMBA induced mammary carcinoma in Sprague Dawley rats. Flavono tumour was induced in 8-week-old Sprague-Dawley rats by gastric intubation of 25 mg DMBA in 1ml olive oil. After 90 days of induction period, the rats were orally administered with Shemamruthaa (400 mg/kg body wt) for 45 days. Treatment with the drug SM significantly modulated the expression of p53, MMP-2, MMP-3, MMP-9 and VEGF by means of its anti angiogenic and protease inhibiting activity. Conclusion: Based on these results, it might be concluded that the formulation, Shemamruthaa, constituted of dried flowers of Hibiscus rosa-sinensis, fruits of Emblica officinalis, and honey has been found to exhibit pronounced antiproliferative and apoptotic effects. This enhanced anticancer effect of Shemamruthaa might be attributed to the synergistic action of polyphenols such as flavonoids, tannins, alkaloids, glycosides, saponins, steroids, terpenoids, vitamin C, niacin, pyrogallol, hydroxymethylfurfural, trilinolein, and other compounds present in the formulation. Collectively, these results demonstrate that Shemamruthaa holds potential to be developed as a potent chemotherapeutic agent against mammary carcinoma.

Keywords: Shemamruthaa, flavonoids, MCF-7 cell line, mammary cancer

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Authors: Jitendra Pratap, Jonathan Sivyer

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Introduction: Dual energy/Spectral CT scanning permits simultaneous acquisition of two x-ray spectra datasets and can complement radiological diagnosis by allowing tissue characterisation (e.g., uric acid vs. non-uric acid renal stones), enhancing structures (e.g. boost iodine signal to improve contrast resolution), and quantifying substances (e.g. iodine density). However, the latter showed inconsistent results between the 2 main modes of dual energy scanning (i.e. dual source vs. dual layer). Therefore, the present study aimed to determine which technology is more accurate in quantifying iodine density. Methods: Twenty vials with known concentrations of iodine solutions were made using Optiray 350 contrast media diluted in sterile water. The concentration of iodine utilised ranged from 0.1 mg/ml to 1.0mg/ml in 0.1mg/ml increments, 1.5 mg/ml to 4.5 mg/ml in 0.5mg/ml increments followed by further concentrations at 5.0 mg/ml, 7mg/ml, 10 mg/ml and 15mg/ml. The vials were scanned using Dual Energy scan mode on a Siemens Somatom Force at 80kV/Sn150kV and 100kV/Sn150kV kilovoltage pairing. The same vials were scanned using Spectral scan mode on a Philips iQon at 120kVp and 140kVp. The images were reconstructed at 5mm thickness and 5mm increment using Br40 kernel on the Siemens Force and B Filter on Philips iQon. Post-processing of the Dual Energy data was performed on vendor-specific Siemens Syngo VIA (VB40) and Philips Intellispace Portal (Ver. 12) for the Spectral data. For each vial and scan mode, the iodine concentration was measured by placing an ROI in the coronal plane. Intraclass correlation analysis was performed on both datasets. Results: The iodine concentrations were reproduced with a high degree of accuracy for Dual Layer CT scanner. Although the Dual Source images showed a greater degree of deviation in measured iodine density for all vials, the dataset acquired at 80kV/Sn150kV had a higher accuracy. Conclusion: Spectral CT scanning by the dual layer technique has higher accuracy for quantitative measurements of iodine density compared to the dual source technique.

Keywords: CT, iodine density, spectral, dual-energy

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Authors: Zsolt Szucs, Viktor Kelemen, Son Le Thai, Magdolna Csavas, Erzsebet Roth, Gyula Batta, Annelies Stevaert, Evelien Vanderlinden, Aniko Borbas, Lieve Naesens, Pal Herczegh

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The approval of modern glycopeptide antibiotics such as dalbavancin and oritavancin which have excellent activity against Gram-positive bacteria, encouraged our research group to prepare semisynthetic compounds from several members of glycopeptides by various chemical methods. Derivatives from the aglycone of ristocetin, eremomycin, vancomycin and a pseudoaglycon of teicoplanin have been synthesized in a systematic manner. Interestingly, some of the aglycoristocetin derivatives displayed noteworthy anti-influenza activity. More recently our group has been focusing on the modifications of one of the pseudoaglycons of teicoplanin. The reaction of N-ethoxycarbonyl maleimide derivatives with the primary amino function, the copper-catalysed azide-alkyne click reaction and the sulfonylation of the N-terminus were utilized to obtain systematic series of compounds. All substituents provide a more lipophilic character to the new molecules compared to the parent antibiotics, which is known to be favourable for activity against resistant bacteria. Lipoglycopeptides are also known to have antiviral properties, which has been predominantly studied on HIV by others. The structure-activity relationship study of our compounds revealed the influence of a few structural elements on biological activity. In many cases, minimal changes in lipophilicity and structure produced great differences in efficacy and cytotoxicity. In vitro experiments showed that these compounds are not only active against glycopeptide resistant Gram-positive bacteria but in several cases they prevent the infection of cell cultures by different strains of influenza viruses. This is probably related to the inhibition of the viral entry into the host cell nucleus, of which the exact mechanism is unknown. In some instances, reasonably low concentrations were sufficient to observe this effect. Several derivatives were highly cytotoxic at the same time, but some of them displayed a good selectivity index. The antiviral properties of the compounds are not restricted to influenza viruses e.g., some of them showed good activity against Human Coronavirus 229E. This work could potentially lead to the development of antiviral drugs which possess the crucial structural motifs that are needed for antiviral activity, while missing those which contribute to the antibacterial effect.

Keywords: antiviral, glycopeptide, semisynthetic, teicoplanin

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Authors: Pravina Gurjar, Bothiraja Pour, Vijay Kumbhar, Ganesh Dama

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Andrographolide (AG), a bioactive phytoconstituent, has a wider range of pharmacological action. However, due to the intestinal degradation, shows low oral bioavailability. The aim of the present work was to develop Floating In-situ gelling Gastro retentive System (FISGS) for AG in order to enhance its site specific absorption and minimize pH dependent hydrolysis in alkaline environment. Further to increase its therapeutic efficacy for peptic ulcer disease caused by H. pyroli. Gellan based floating in situ gelling system of AG were prepared by using sodium citrate and calcium carbonate. The 32 factorial designs was used to study the effect of gellan and calcium carbonate concentration (independent variables) on dependent variable such as viscosity, floating lag time and drug release. Developed system was evaluated for drug content, floating lag time, viscosity, and drug release studies. Drug content, viscosity, and floating lag time was found to be 81-99%, 67-117 Cps, and 3-5 sec, respectively. The obtained system showed good in vitro floating ability for more than 12 h using 0.1 N HCl as dissolution medium with initial burst release followed by the controlled zero order drug release up to 24 hrs. In vivo testing of FISGS of AG to rats demonstrated significant antiulcer activity that were evaluated by various parameters like pH, volume, total acidity, millimole equivalent of H+ ions/30 min, and protein content of gastric content. The densities of all the formulation batches were found to be near about 0.9 and floating duration above 12 hr. It was observed that with the increase in conc. of gellan there was increase in the viscosity of formulation but all formulations were in optimum range. The drug content of optimized batch was found to be 99.23. In histopathology study of stomach, the villi at the mucosal surface, the intercellular junction, the intestinal lumen were intact; no destruction of the epithelium, and submucosal gland in formulation treated and control group animals as compared to pure drug AG and standard ranitidine. Gellan-based in situ gastro retentive floating system could be advantageous in terms of increased bioavailability of AG to maintain an effective drug conc. in gastric fluid as well as in serum for longer period of time.

Keywords: andrographolide, floating drug delivery, in situ gelling system, gastroretentive system

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Authors: Zeina Bou Diab, Arij Daou

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The neural mechanisms of sequential behaviors are intensively studied, with songbirds a focus for learned vocal production. We are studying the premotor nucleus HVC at a nexus of multiple pathways contributing to song learning and production. The HVC consists of multiple classes of neuronal populations, each has its own cellular, electrophysiological and functional properties. During singing, a large subset of motor cortex analog-projecting HVCRA neurons emit a single 6-10 ms burst of spikes at the same time during each rendition of song, a large subset of basal ganglia-projecting HVCX neurons fire 1 to 4 bursts that are similarly time locked to vocalizations, while HVCINT neurons fire tonically at average high frequency throughout song with prominent modulations whose timing in relation to song remains unresolved. This opens the opportunity to define models relating explicit HVC circuitry to how these neurons work cooperatively to control learning and singing. We developed conductance-based Hodgkin-Huxley models for the three classes of HVC neurons (based on the ion channels previously identified from in vitro recordings) and connected them in several physiologically realistic networks (based on the known synaptic connectivity and specific glutaminergic and gabaergic pharmacology) via different architecture patterning scenarios with the aim to replicate the in vivo firing patterning behaviors. We are able, through these networks, to reproduce the in vivo behavior of each class of HVC neurons, as shown by the experimental recordings. The different network architectures developed highlight different mechanisms that might be contributing to the propagation of sequential neural activity (continuous or punctate) in the HVC and to the distinctive firing patterns that each class exhibits during singing. Examples of such possible mechanisms include: 1) post-inhibitory rebound in HVCX and their population patterns during singing, 2) different subclasses of HVCINT interacting via inhibitory-inhibitory loops, 3) mono-synaptic HVCX to HVCRA excitatory connectivity, and 4) structured many-to-one inhibitory synapses from interneurons to projection neurons, and others. Replication is only a preliminary step that must be followed by model prediction and testing.

Keywords: computational modeling, neural networks, temporal neural sequences, ionic currents, songbird

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Authors: Susmita Goswami, Joyeeta Mitra, Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Prabir K. Paul

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'Malbhog’, an indigenous banana variety, much prized for its flavour and delicacy suffers production losses due to Fusarium oxysporum f.sp. cubense. The pathogen enters young plants through feeder roots causing wilting of plants ultimately leading to death of plants. The pathogen spreads rapidly to other plants in the field. In eastern part of India, this variety escapes the onslaught of the pathogen when either co-cultivated or rotated with Amorphophallus campanulatus (yam). The present study provides an insight into the physiological aspect of the biocontrol by yam. In vitro application of sterile aqueous extract of yam tuber (100gm/100ml distilled water and its 1:10 and 1:100 dilutions) were mixed with PDA media which was substantially inoculated with spores of Fusarium oxysporum f.sp. cubense. The extract could significantly reduce germination of pathogen spores. Banana variety susceptible to Fusarium sp was raised in soil rite under aseptic conditions. Spores of the pathogen (106 spores/ml) were inoculated into the soil rite. The plants were spread with aqueous extract of yam. The control plants were treated with sterilized distilled water. The activity of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX) were estimated in leaves and roots at interval of 24 hours for 5 days after treatment. The incidence of wilt disease was recorded after two weeks. The results demonstrated that yam extract could induce significant activity of PAL, PPO and POX along with accumulation of phenols in both roots and leaves of banana plants. However, significantly high activity of enzymes and phenol accumulation was observed in roots. The disease incidence was significantly low in yam treated plants. The results clearly demonstrated the control of the pathogen due to induction of defense mechanism in the host by the extract. The observed control of the pathogen in the field could possibly be due to induction of such defense responses in host by exudates leached into the soil from yam tubers. Yam extract could be a potential source of environment-friendly biocide against Panama wilt of banana.

Keywords: Amorphophallus campanulatus, banana, Fusarium oxysporum f.sp. cubense, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POX)

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Authors: Yogesh Rai, Saurabh Singh, Sanjay Pandey, Dhananjay K. Sah, B. G. Roy, B. S. Dwarakanath, Anant N. Bhatt

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One of the most common signatures of highly malignant gliomas is their capacity to metabolize more glucose to lactic acid than normal brain tissues, even under normoxic conditions (Warburg effect), indicating that aerobic glycolysis is constitutively upregulated through stable genetic or epigenetic changes. However, oxidative phosphorylation (OxPhos) is also required to maintain the mitochondrial membrane potential for tumor cell survival. In the process of tumorigenesis, tumor cells during fastest growth rate exhibit both high glycolytic and high OxPhos. Therefore, metabolically reprogrammed cancer cells with combination of both aerobic glycolysis and altered OxPhos develop a robust metabolic phenotype, which confers a selective growth advantage. In our study, we grew the high glycolytic BMG-1 (glioma) cells with continuous exposure of mitochondrial uncoupler 2, 4, dinitro phenol (DNP) for 10 passages to obtain a phenotype of high glycolysis with enhanced altered OxPhos. We found that OxPhos modified BMG (OPMBMG) cells has similar growth rate and cell cycle distribution but high mitochondrial mass and functional enzymatic activity than parental cells. In in-vitro studies, OPMBMG cells showed enhanced invasion, proliferation and migration properties. Moreover, it also showed enhanced angiogenesis in matrigel plug assay. Xenografted tumors from OPMBMG cells showed reduced latent period, faster growth rate and nearly five folds reduction in the tumor take in nude mice compared to BMG-1 cells, suggesting that robust metabolic phenotype facilitates tumor formation and growth. OPMBMG cells which were found radio-resistant, showed enhanced radio-sensitization by 2-DG as compared to the parental BMG-1 cells. This study suggests that metabolic reprogramming in cancer cells enhances the potential of migration, invasion and proliferation. It also strengthens the cancer cells to escape the death processes, conferring resistance to therapeutic modalities. Our data also suggest that combining metabolic inhibitors like 2-DG with conventional therapeutic modalities can sensitize such metabolically aggressive cancer cells more than the therapies alone.

Keywords: 2-DG, BMG, DNP, OPM-BMG

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Authors: Umamaheswari Shanmugam, Silvia Ronchi, Radu Vornicu

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Software, artificial intelligence, and machine learning can improve healthcare through innovative and advanced technologies that are able to use the large amount and variety of data generated during healthcare services every day. As we read the news, over 500 machine learning or other artificial intelligence medical devices have now received FDA clearance or approval, the first ones even preceding the year 2000. One of the big advantages of these new technologies is the ability to get experience and knowledge from real-world use and to continuously improve their performance. Healthcare systems and institutions can have a great benefit because the use of advanced technologies improves the same time efficiency and efficacy of healthcare. Software-defined as a medical device, is stand-alone software that is intended to be used for patients for one or more of these specific medical intended uses: - diagnosis, prevention, monitoring, prediction, prognosis, treatment or alleviation of a disease, any other health conditions, replacing or modifying any part of a physiological or pathological process–manage the received information from in vitro specimens derived from the human samples (body) and without principal main action of its principal intended use by pharmacological, immunological or metabolic definition. Software qualified as medical devices must comply with the general safety and performance requirements applicable to medical devices. These requirements are necessary to ensure high performance and quality and also to protect patients’ safety. The evolution and the continuous improvement of software used in healthcare must take into consideration the increase in regulatory requirements, which are becoming more complex in each market. The gap between these advanced technologies and the new regulations is the biggest challenge for medical device manufacturers. Regulatory requirements can be considered a market barrier, as they can delay or obstacle the device approval, but they are necessary to ensure performance, quality, and safety, and at the same time, they can be a business opportunity if the manufacturer is able to define in advance the appropriate regulatory strategy. The abstract will provide an overview of the current regulatory framework, the evolution of the international requirements, and the standards applicable to medical device software in the potential market all over the world.

Keywords: artificial intelligence, machine learning, SaMD, regulatory, clinical evaluation, classification, international requirements, MDR, 510k, PMA, IMDRF, cyber security, health care systems.

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Authors: Tayyaba Bari, Muhammad Hamza Anjum, Samra Kanwal, Fakhera Ikram

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Osteoarthritis, a degenerative condition, affects more than 213 million individuals globally. Since articular cartilage has no or limited vessels, therefore, after deteriorating, it is unable to rejuvenate. Traditional approaches for cartilage repair, like autologous chondrocyte implantation, microfracture and cartilage transplantation are often associated with postoperative complications and lead to further degradation. Decellularized human umbilical cord has gained interest as a viable treatment for cartilage repair. Decellularization removes all cellular contents as well as debris, leaving a biologically active 3D network known as extracellular matrix (ECM). This matrix is biodegradable, non-immunogenic and provides a microenvironment for homeostasis, growth and repair. UC derived bioink function as 3D scaffolding material, not only mediates cell-matrix interactions but also adherence, proliferation and propagation of cells for 3D organoids. This study comprises different physical, chemical and biological approaches to optimize the decellularization of human umbilical cord (UC) tissues followed by the solubilization of these tissues to bioink formation. The decellularization process consisted of two cycles of freeze thaw where the umbilical cord at -20˚C was thawed at room temperature followed by dissection in small sections from 0.5 to 1cm. Similarly decellularization with ionic and non-ionic detergents Sodium dodecyl sulfate (SDS) and Triton-X 100 revealed that both concentrations of SDS i.e 0.1% and 1% were effective in complete removal of cells from the small UC tissues. The results of decellularization was further confirmed by running them on 1% agarose gel. Histological analysis revealed the efficacy of decellularization, which involves paraffin embedded samples of 4μm processed for Hematoxylin-eosin-safran and 4,6-diamidino-2-phenylindole (DAPI). ECM preservation was confirmed by Alcian Blue, and Masson’s trichrome staining on consecutive sections and images were obtained. Sulfated GAG’s content were determined by 1,9-dimethyl-methylene blue (DMMB) assay, similarly collagen quantification was done by hydroxy proline assay. This 3D bioengineered scaffold will provide a typical atmosphere as in the extracellular matrix of the tissue, which would be seeded with the mesenchymal cells to generate the desired 3D ink for in vitro and in vivo cartilage regeneration applications.

Keywords: umbilical cord, 3d printing, bioink, tissue engineering, cartilage regeneration

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Authors: William E. Bauta, Jennifer Rebeles, Reggie Jacob

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Porphyrins are noteworthy in biomedical science for their cancer tissue accumulation and photophysical properties. The preferential accumulation of some porphyrins in cancerous tissue has been known for many years. This, combined with their characteristic photophysical and photochemical properties, including their strong fluorescence and their ability to generate reactive oxygen species in vivo upon laser irradiation, has led to much research into the application of porphyrins as cancer diagnostic and therapeutic agents. Porphyrins have been used as dyes to detect cancer cells both in vivo and, less commonly, in vitro. In one example, human sputum samples from lung cancer patients and patients without the disease were dissociated and stained with the porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-porphine). Cells were analyzed by flow cytometry. Cancer samples were identified by their higher TCPP fluorescence intensity relative to the no-cancer controls. However, quantitative analysis of fluorescence in cell suspensions stained with multiple fluorophores requires particles stained with each of the individual fluorophores as controls. Fluorescent control particles must be compatible in size with flow cytometer fluidics and have favorable hydrodynamic properties in suspension. They must also display fluorescence comparable to the cells of interest and be stable upon storage amine-functionalized spherical polystyrene beads in the 5 to 20-micron diameter range that was reacted with TCPP and EDC in aqueous pH six buffer overnight to form amide bonds. Beads were isolated by centrifugation and tested by flow cytometry. The 10-micron amine-functionalized beads displayed the best combination of fluorescence intensity and hydrodynamic properties, such as lack of clumping and remaining in suspension during the experiment. These beads were further optimized by varying the stoichiometry of EDC and TCPP relative to the amine. The reaction was accompanied by the formation of a TCPP-related particulate, which was removed, after bead centrifugation, using a microfiltration process. The resultant TCPP-functionalized beads were compatible with flow cytometry conditions and displayed a fluorescence comparable to that of stained cells, which allowed their use as fluorescence standards. The beads were stable in refrigerated storage in the dark for more than eight months. This work demonstrates the first preparation of porphyrin-functionalized flow cytometry control beads.

Keywords: tetraaryl porphyrin, polystyrene beads, flow cytometry, peptide coupling

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Authors: Ahmed. Abdel Bary, Omneya. Khowessah, Mojahed. al-jamrah

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Introduction: The major challenge with the design of oral dosage forms lies with their poor bioavailability. The most frequent causes of low oral bioavailability are attributed to poor solubility and low permeability. The aim of this study was to develop solid nanodispersed tablet formulation of Glimepiride for the enhancement of the solubility and bioavailability. Methodology: Solid nanodispersions of Glimepiride (GMP) were prepared using two different ratios of 2 different carriers, namely; PEG6000, pluronic F127, and by adopting two different techniques, namely; solvent evaporation technique and fusion technique. A full factorial design of 2 3 was adopted to investigate the influence of formulation variables on the prepared nanodispersion properties. The best chosen formula of nanodispersed powder was formulated into tablets by direct compression. The Differential Scanning Calorimetry (DSC) analysis and Fourier Transform Infra-Red (FTIR) analysis were conducted for the thermal behavior and surface structure characterization, respectively. The zeta potential and particle size analysis of the prepared glimepiride nanodispersions was determined. The prepared solid nanodispersions and solid nanodispersed tablets of GMP were evaluated in terms of pre-compression and post-compression parameters, respectively. Results: The DSC and FTIR studies revealed that there was no interaction between GMP and all the excipients used. Based on the resulted values of different pre-compression parameters, the prepared solid nanodispersions powder blends showed poor to excellent flow properties. The resulted values of the other evaluated pre-compression parameters of the prepared solid nanodispersion were within the limits of pharmacopoeia. The drug content of the prepared nanodispersions ranged from 89.6 ± 0.3 % to 99.9± 0.5% with particle size ranged from 111.5 nm to 492.3 nm and the resulted zeta potential (ζ ) values of the prepared GMP-solid nanodispersion formulae (F1-F8) ranged from -8.28±3.62 mV to -78±11.4 mV. The in-vitro dissolution studies of the prepared solid nanodispersed tablets of GMP concluded that GMP- pluronic F127 combinations (F8), exhibited the best extent of drug release, compared to other formulations, and to the marketed product. One way ANOVA for the percent of drug released from the prepared GMP-nanodispersion formulae (F1- F8) after 20 and 60 minutes showed significant differences between the percent of drug released from different GMP-nanodispersed tablet formulae (F1- F8), (P<0.05). Conclusion: Preparation of glimepiride as nanodispersed particles proven to be a promising tool for enhancing the poor solubility of glimepiride.

Keywords: glimepiride, solid Nanodispersion, nanodispersed tablets, poorly water soluble drugs

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Authors: Adebisi Adunola Demehin, Wanlaya Thamnarak, Jaruwan Chatwichien, Chatchakorn Eurtivong, Kiattawee Choowongkomon, Somsak Ruchirawat, Nopporn Thasana

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein involved in cellular signalling processes and, its aberrant activity is crucial in the development of many cancers such as lung cancer. Selaginellaceae are fern allies that have long been used in Chinese traditional medicine to treat various cancer types, especially lung cancer. Biflavonoids, the major secondary metabolites in Selaginellaceae, have numerous pharmacological activities, including anti-cancer and anti-inflammatory. For instance, amentoflavone induces a cytotoxic effect in the human NSCLC cell line via the inhibition of PARP-1. However, to the best of our knowledge, there are no studies on biflavonoids as EGFR inhibitors. Thus, this study aims to investigate the EGFR inhibitory activities of biflavonoids isolated from Selaginella siamensis and Selaginella bryopteris. Amentoflavone, tetrahydroamentoflavone, sciadopitysin, robustaflavone, robustaflavone-4-methylether, delicaflavone, and chrysocauloflavone were isolated from the ethyl-acetate extract of the whole plants. The structures were determined using NMR spectroscopy and mass spectrometry. In vitro study was conducted to evaluate their cytotoxicity against A549, HEPG2, and T47D human cancer cell lines using the MTT assay. In addition, a target-based assay was performed to investigate their EGFR inhibitory activity using the kinase inhibition assay. Finally, a molecular docking study was conducted to predict the binding modes of the compounds. Robustaflavone-4-methylether and delicaflavone showed the best cytotoxic activity on all the cell lines with IC50 (µM) values of 18.9 ± 2.1 and 22.7 ± 3.3 on A549, respectively. Of these biflavonoids, delicaflavone showed the most potent EGFR inhibitory activity with an 84% relative inhibition at 0.02 nM using erlotinib as a positive control. Robustaflavone-4-methylether showed a 78% inhibition at 0.15 nM. The docking scores obtained from the molecular docking study correlated with the kinase inhibition assay. Robustaflavone-4-methylether and delicaflavone had a docking score of 72.0 and 86.5, respectively. The inhibitory activity of delicaflavone seemed to be linked with the C2”=C3” and 3-O-4”’ linkage pattern. Thus, this study suggests that the structural features of these compounds could serve as a basis for developing new EGFR-TK inhibitors.

Keywords: anticancer, biflavonoids, EGFR, molecular docking, Selaginellaceae

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Authors: O. Antypenko, I. Vasilieva, S. Kovalenko

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Investigations for new effective and less toxic antimicrobials agents are always up-to-date. The tetrazole derivatives are quite interesting objects as for synthesis as well as for pharmacological screening. Thus, some derivatives of tetrazole demonstrated antimicrobial activity, namely 5-phenyl-tetrazolo[1,5-c]quinazoline was effective one against Staphylococcus aureus and Esherichia faecalis (MIC = 250 mg/L). Besides, investigation of the 9-bromo(chloro)-5-morpholin(piperidine)-4-yl-tetrazolo[1,5-c]quinazoline’s antimicrobial activity against Esherichia coli and Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus revealed that sensitivity of Gram-positive bacteria to the compounds was higher than that of Gram-negative bacteria. So, our previously synthesized, 31 derivatives of 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas were decided to test for their in vitro antibacterial activity against Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Enterobacter aerogenes, Enterococcus faecalis ATCC 29212), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, Klebsiella pneumoniae 68) and antifungal properties against Candida albicans ATCC 885653. Agar-diffusion method was used for determination of the preliminary activity compared to well-known reference antimicrobials. All the compounds were dissolved in DMSO at a concentration of 100 μg/disk, using inhibition zone diameter (IZD, mm) as a measure for the antimicrobial activity. The most active turned to be 3 structures, that inhibited several bacterial strains: 1-ethyl-3-(5-fluoro-2-(1H-tetrazol-5-yl)phenyl)urea (1), 1-(4-bromo-2-(1H-tetrazol-5-yl)-phenyl)-3-(4-(trifluoromethyl)phenyl)urea (2) and 1-(4-chloro-2-(1H-tetrazol-5-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (3). IZM (mm) was 40 (Escherichia coli), 25 (Klebsiella pneumonia) for compound 1; 12 (Pseudomonas aeruginosa), 15 (Staphylococcus aureus), 10 (Enterococcus faecalis) for compound 2; 25 (Staphylococcus aureus), 15 (Enterococcus faecalis) for compound 3. The most sensitive to the activity of the substances were Gram-negative bacteria Pseudomonas aeruginosa. While none of compound effected on Candida albicans. Speaking about, reference drugs: Amikacin (30 µg/disk) showed 27 and Ceftazide (30 µg/disk) 25 against Pseudomonas aeruginosa. That is, unfortunately, higher than studied 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas. Obtained results will be used for further purposeful optimization of the leading compounds in the more effective antimicrobials because of the ever-mounting problem of microorganism’s resistance.

Keywords: antimicrobial, antifungal, compounds, 1-(2-(1H-tetrazol-5-yl)-R1-phenyl)-3-R2-phenyl(ethyl)ureas

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Authors: Daphne Meza, Louie Abejar, David A. Rubenstein, Wei Yin

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Endothelial cell (ECs) morphology and function is highly impacted by the mechanical stresses these cells experience in vivo. Any change in the mechanical environment can trigger pathological EC responses. A detailed understanding of EC morphological response and function upon subjection to individual and simultaneous mechanical stimuli is needed for advancement in mechanobiology and preventive medicine. To investigate this, a programmable device capable of simultaneously applying physiological fluid shear stress (FSS) and cyclic strain (CS) has been developed, characterized and validated. Its validation was performed both experimentally, through tracer tracking, and theoretically, through the use of a computational fluid dynamics model. The effectiveness of the device was evaluated through EC morphology changes under mechanical loading conditions. Changes in cell morphology were evaluated through: cell and nucleus elongation, cell alignment and junctional actin production. The results demonstrated that the combined FSS-CS stimulation induced visible changes in EC morphology. Upon simultaneous fluid shear stress and biaxial tensile strain stimulation, cells were elongated and generally aligned with the flow direction, with stress fibers highlighted along the cell junctions. The concurrent stimulation from shear stress and biaxial cyclic stretch led to a significant increase in cell elongation compared to untreated cells. This, however, was significantly lower than that induced by shear stress alone, indicating that the biaxial tensile strain may counteract the elongating effect of shear stress to maintain the shape of ECs. A similar trend was seen in alignment, where the alignment induced by the concurrent application of shear stress and cyclic stretch fell in between that induced by shear stress and tensile stretch alone, indicating the opposite role shear stress and tensile strain may play in cell alignment. Junctional actin accumulation was increased upon shear stress alone or simultaneously with tensile stretch. Tensile stretch alone did not change junctional actin accumulation, indicating the dominant role of shear stress in damaging EC junctions. These results demonstrate that the shearing-stretching device is capable of applying well characterized dynamic shear stress and tensile strain to cultured ECs. Using this device, EC response to altered mechanical environment in vivo can be characterized in vitro.

Keywords: cyclic stretch, endothelial cells, fluid shear stress, vascular biology

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Authors: Aurea C. L. Lacerda, Paulo R. H. Moreno, Bruna M. P. Vianna, Cristina H. R. Serra, Airton Martin, André R. Baby, Vladi O. Consiglieri, Telma M. Kaneko

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The modified Franz cell is the most widely used model for in vitro permeation studies, however it still presents some disadvantages. Thus, some alternative methods have been developed such as Skin PAMPA, which is a bio- artificial membrane that has been applied for skin penetration estimation of xenobiotics based on HT permeability model consisting. Skin PAMPA greatest advantage is to carry out more tests, in a fast and inexpensive way. The membrane system mimics the stratum corneum characteristics, which is the primary skin barrier. The barrier properties are given by corneocytes embedded in a multilamellar lipid matrix. This layer is the main penetration route through the paracellular permeation pathway and it consists of a mixture of cholesterol, ceramides, and fatty acids as the dominant components. However, there is no consensus on the membrane composition. The objective of this work was to compare the performance among different bio-artificial membranes for studying the permeation in skin PAMPA system. Material and methods: In order to mimetize the lipid composition`s present in the human stratum corneum six membranes were developed. The membrane composition was equimolar mixture of cholesterol, ceramides 1-O-C18:1, C22, and C20, plus fatty acids C20 and C24. The membrane integrity assay was based on the transport of Brilliant Cresyl Blue, which has a low permeability; and Lucifer Yellow with very poor permeability and should effectively be completely rejected. The membrane characterization was performed using Confocal Laser Raman Spectroscopy, using stabilized laser at 785 nm with 10 second integration time and 2 accumulations. The membrane behaviour results on the PAMPA system were statistically evaluated and all of the compositions have shown integrity and permeability. The confocal Raman spectra were obtained in the region of 800-1200 cm-1 that is associated with the C-C stretches of the carbon scaffold from the stratum corneum lipids showed similar pattern for all the membranes. The ceramides, long chain fatty acids and cholesterol in equimolar ratio permitted to obtain lipid mixtures with self-organization capability, similar to that occurring into the stratum corneum. Conclusion: The artificial biological membranes studied for Skin PAMPA showed to be similar and with comparable properties to the stratum corneum.

Keywords: bio-artificial membranes, comparison, confocal Raman, skin PAMPA

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Authors: Carolina S. Monteiro, Eugénia Pinto, Miguel A. Faria, Sara C. Cunha

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Rice (Oryza sativa) production plays a vital role in reducing hunger and poverty and assumes particular importance in low-income and developing countries. Rice is a sensitive plant, and production occurs strictly where suitable temperature and water conditions are found. Climatic changes are likely to affect worldwide, and some models have predicted increased temperatures, variations in atmospheric CO₂ concentrations and modification in precipitation patterns. Therefore, the ongoing climatic changes threaten rice production by increasing biotic and abiotic stress factors, and crops will grow in different environmental conditions in the following years. Around the world, the effects will be regional and can be detrimental or advantageous depending on the region. Mediterranean zones have been identified as possible hot spots, where dramatic temperature changes, modifications of CO₂ levels, and rainfall patterns are predicted. The actual estimated atmospheric CO₂ concentration is around 400 ppm, and it is predicted that it can reach up to 1000–1200 ppm, which can lead to a temperature increase of 2–4 °C. Alongside, rainfall patterns are also expected to change, with more extreme wet/dry episodes taking place. As a result, it could increase the migration of pathogens, and a shift in the occurrence of mycotoxins, concerning their types and concentrations, is expected. Mycotoxigenic spoilage fungi can colonize the crops and be present in all rice food chain supplies, especially Penicillium species, mainly resulting in ochratoxin A (OTA) contamination. In this scenario, the objectives of the present study are evaluating the effect of temperature (20 vs. 25 °C), CO₂ (400 vs. 1000 ppm), and water stress (0.93 vs 0.95 water activity) on growth and OTA production by a Penicillium nordicum strain in vitro on rice-based media and when colonizing layers of raw rice. Results demonstrate the effect of temperature, CO₂ and drought on the OTA production in a rice-based environment, thus contributing to the development of mycotoxins predictive models in climate change scenarios. As a result, improving mycotoxins' surveillance and monitoring systems, whose occurrence can be more frequent due to climatic changes, seems relevant and necessary. The development of prediction models for hazard contaminants presents in foods highly sensitive to climatic changes, such as mycotoxins, in the highly probable new agricultural scenarios is of paramount importance.

Keywords: climate changes, ochratoxin A, penicillium, rice

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Authors: Melanie Ostermann, Eivind Birkeland, Ying Xue, Alexander Sauter, Mihaela R. Cimpan

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Background: New reliable and robust techniques to assess biological effects of nanomaterials (NMs) in vitro are needed to speed up safety analysis and to identify key physicochemical parameters of NMs, which are responsible for their acute cytotoxicity. The central aim of this study was to validate and evaluate the applicability and reliability of an impedance-based flow cytometry (IFC) technique for the high-throughput screening of NMs. Methods: Eight inorganic NMs from the European Commission Joint Research Centre Repository were used: NM-302 and NM-300k (Ag: 200 nm rods and 16.7 nm spheres, respectively), NM-200 and NM- 203 (SiO₂: 18.3 nm and 24.7 nm amorphous, respectively), NM-100 and NM-101 (TiO₂: 100 nm and 6 nm anatase, respectively), and NM-110 and NM-111 (ZnO: 147 nm and 141 nm, respectively). The aim was to assess the biological effects of these materials on human monoblastoid (U937) cells. Dispersions of NMs were prepared as described in the NANOGENOTOX dispersion protocol and cells were exposed to NMs at relevant concentrations (2, 10, 20, 50, and 100 µg/mL) for 24 hrs. The change in electrical impedance was measured at 0.5, 2, 6, and 12 MHz using the IFC AmphaZ30 (Amphasys AG, Switzerland). A traditional toxicity assay, Trypan Blue Dye Exclusion assay, and dark-field microscopy were used to validate the IFC method. Results: Spherical Ag particles (NM-300K) showed the highest toxic effect on U937 cells followed by ZnO (NM-111 ≥ NM-110) particles. Silica particles were moderate to non-toxic at all used concentrations under these conditions. A higher toxic effect was seen with smaller sized TiO2 particles (NM-101) compared to their larger analogues (NM-100). No interferences between the IFC and the used NMs were seen. Uptake and internalization of NMs were observed after 24 hours exposure, confirming actual NM-cell interactions. Conclusion: Results collected with the IFC demonstrate the applicability of this method for rapid nanotoxicity assessment, which proved to be less prone to nano-related interference issues compared to some traditional toxicity assays. Furthermore, this label-free and novel technique shows good potential for up-scaling in directions of an automated high-throughput screening and for future NM toxicity assessment. This work was supported by the EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584), the Research Council of Norway, project NorNANoREG (239199/O70), the EuroNanoMed II 'GEMN' project (246672), and the UH-Nett Vest project.

Keywords: cytotoxicity, high-throughput, impedance, nanomaterials

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Authors: Adnan Bekhit, Eskedar Lodebo, Ariaya Hymete, Hanan Ragab, Alaa El-Din Bekhit

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Malaria and leishmaniasis have emerged as serious universal health problems throughout history of mankind. According to the WHO 2008 malarial report, half of the world population is at risk of malarial infection with an estimate of 1 million deaths occurring annually mainly in the African region. Furthermore, 12-15 million people are infected with Leishmaniasis worldwide. Despite the continuous introduction of a large number of agents for the treatment of malaria, there is still unmet medical needs due to the emergence of resistance. Resistance has occurred for almost all therapeutic agents approved for the treatment of malaria. Accordingly, it was the aim of this work to design and synthesis a group of antimalarial-antileshmanial agents that would show inhibitory activity against chloroquine-resistant strain of Plasmodium falciparum. The synthesized compounds were designed to contain a pyrazolylpyrazoline moiety having an aromatic group (p-tolyl or p-chlorophenyl) at N1-position of one pyrazoline ring due to the reports of promising activities of such compounds. A formyl or acyl substituent was introduced at the N1-position of the other pyrazoline ring, to investigate the effect of bulkiness of acyl substituents at this position. The synthesized compounds were evaluated for their in-vivo antimalarial activity against Plasmodium berghei infected mice at dose levels of 20 and 30 mg/Kg. the two most active compounds were evaluated for their antimalarial activity against chloroquin-resistant strain (RKL9) of Plasmodium falciparum. In addition, the synthesized compounds were tested for their in-vitro antileshmanial activity against Leishmania aethiopica promastigotes and amastigotes. For both antimalarial and antileishmanial activities, compounds having an N1-p-tolyl group at the first pyrazoline ring did not require bulkiness at the second pyrazoline ring nitrogen where the compound bearing an acetyl group proved to be the most active of the whole series. On the other hand, bulkiness at the N1-position of the second pyazoline ring was necessary in case of compounds carrying the p-chlorophenyl group, where the two derivatives having an N1-butanoyl and an N1-benzoyl moieties at the second pyrazoline showed the best activity. Furthermore, the toxicity of the active compounds were tested and were proved to be non-toxic at 125, 250 and 500 mg/Kg. In addition, docking of the most active compound (having a p-tolyl group at the first pyrazoline-N and an acetyl moiety on the other pyrazoline-N) was performed against dihydrofolate reductase enzyme.

Keywords: pyrazoline derivatives, in-vivo antimalarial activity, docking, dihydrofolate reductase

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Authors: Virag Vass, Ilona Bereczki, Erzsebet Szabo, Nora Debreczeni, Aniko Borbas, Pal Herczegh, Arpad Tosaki

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Hydrogen sulfide (H₂S) is a toxic gas, but it is produced by certain tissues in a small quantity. According to earlier studies, ibuprofen and H₂S has a protective effect against damaging heart tissue caused by ischemia-reperfusion. Recently, we have been investigating the effect of a new water-soluble H₂S releasing ibuprofen molecule administered after artificially generated ischemia-reperfusion on isolated rat hearts. The H₂S releasing property of the new ibuprofen derivative was investigated in vitro in medium derived from heart endothelial cell isolation at two concentrations. The ex vivo examinations were carried out on rat hearts. Rats were anesthetized with an intraperitoneal injection of ketamine, xylazine, and heparin. After thoracotomy, hearts were excised and placed into ice-cold perfusion buffer. Perfusion of hearts was conducted in Langendorff mode via the cannulated aorta. In our experiments, we studied the dose-effect of the H₂S releasing molecule in Langendorff-perfused hearts with the application of gradually increasing concentration of the compound (0- 20 µM). The H₂S releasing ibuprofen derivative was applied before the ischemia for 10 minutes. H₂S concentration was measured with an H₂S detecting electrochemical sensor from the coronary effluent solution. The 10 µM concentration was chosen for further experiments when the treatment with this solution was occurred after the ischemia. The release of H₂S is occurred by the hydrolyzing enzymes that are present in the heart endothelial cells. The protective effect of the new H₂S releasing ibuprofen molecule can be confirmed by the infarct sizes of hearts using the Triphenyl-tetrazolium chloride (TTC) staining method. Furthermore, we aimed to define the effect of the H₂S releasing ibuprofen derivative on autophagic and apoptotic processes in damaged hearts after investigating the molecular markers of these events by western blotting and immunohistochemistry techniques. Our further studies will include the examination of LC3I/II, p62, Beclin1, caspase-3, and other apoptotic molecules. We hope that confirming the protective effect of new H₂S releasing ibuprofen molecule will open a new possibility for the development of more effective cardioprotective agents with exerting fewer side effects. Acknowledgment: This study was supported by the grants of NKFIH- K-124719 and the European Union and the State of Hungary co- financed by the European Social Fund in the framework of GINOP- 2.3.2-15-2016-00043.

Keywords: autophagy, hydrogen sulfide, ibuprofen, ischemia, reperfusion

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Authors: Vivek Kumar Gupta, Kripi Vohra, Monika Gupta

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Pulses have been a vital ingredient of the balanced human diet in India. Lentil (Lens culinaris Medikus or Lens esculenta Moench.) is a common legume known since biblical times. Lentil seeds, with or without hulls, are cooked as dhal and this has been the main dish for millennia in the South Asian region. Oxidative stress can damage lipids, proteins, enzymes, carbohydrates and DNA in cells and tissues, resulting in membrane damage, fragmentation or random cross linking of molecules like DNA, enzymes and structural proteins and even lead to cell death induced by DNA fragmentation and lipid peroxidation. These consequences of oxidative stress construct the molecular basis in the development of cancer, neurodegenerative disorders, cardiovascular diseases, diabetes and autoimmune. The aim of the present work is to assess the antioxidant potential of the peteroleum ether, acetone, methanol and water extract of the Lens esculenta seeds. In vitro antioxidant assessment of the extracts was carried out using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging activity, reducing power assay. The quantitative estimation of total phenolic content, total flavonoid content in extracts and in plant material, total saponin content, total alkaloid content, crude fibre content, total volatile content, fat content and mucilage content in drug material was also carried out. Though all the extracts exhibited dose dependent reducing power activity the acetone extract was found to possess significant hydrogen donating ability in DPPH (45.83%-93.13%) and hydroxyl radical scavenging system (28.7%-46.41%) than the peteroleum ether, methanol and water extracts. Total phenolic content in the acetone and methanol extract was found to be 608 and 188 mg gallic acid equivalent of phenol/g of sample respectively. Total flavonoid content of acetone and methanol extract was found to be 128 and 30.6 mg quercetin equivalent/g of sample respectively. It is evident that acetone extract of Lentil seeds possess high levels of polyphenolics and flavonoids that could be utilized as antioxidants and neutraceuticals.

Keywords: antioxidant, flavanoids, Lens esculenta, polyphenols

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Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

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Authors: Rema Vazhappilly, Tapas Das

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Oleic acid is a cis unsaturated fatty acid and is known to be a partially essential fatty acid due to its limited endogenous synthesis during pregnancy and lactation. Previous studies have demonstrated the role of oleic acid in neuronal differentiation and brain phospholipid synthesis. These evidences indicate a major role for oleic acid in learning and memory. Interestingly, oleic acid has been shown to enhance hippocampal long term potentiation (LTP), the physiological correlate of long term synaptic plasticity. However the effect of oleic acid on short term synaptic plasticity has not been investigated. Short term potentiation (STP) is the physiological correlate of short term synaptic plasticity which is the key underlying molecular mechanism of short term memory and neuronal information processing. STP in the hippocampal CA1 region has been known to require the activation of N-methyl-D-aspartate receptors (NMDARs). The NMDAR dependent hippocampal STP as a potential mechanism for short term memory has been a subject of intense interest for the past few years. Therefore in the present study the effect of oleic acid on NMDAR dependent hippocampal STP was determined in mouse hippocampal slices (in vitro) using Multi-electrode array system. STP was induced by weak tetanic Stimulation (one train of 100 Hz stimulations for 0.1s) of the Schaffer collaterals of CA1 region of the hippocampus in slices treated with different concentrations of oleic acid in presence or absence of NMDAR antagonist D-AP5 (30 µM) . Oleic acid at 20 (mean increase in fEPSP amplitude = ~135 % Vs. Control = 100%; P<0.001) and 30 µM (mean increase in fEPSP amplitude = ~ 280% Vs. Control = 100%); P<0.001) significantly enhanced the STP following weak tetanic stimulation. Lower oleic acid concentrations at 10 µM did not modify the hippocampal STP induced by weak tetanic stimulation. The hippocampal STP induced by weak tetanic stimulation was completely blocked by the NMDA receptor antagonist D-AP5 (30µM) in both oleic acid and control treated hippocampal slices. This lead to the conclusion that the hippocampal STP elicited by weak tetanic stimulation and enhanced by oleic acid was NMDAR dependent. Together these findings suggest that oleic acid may enhance the short term memory and neuronal information processing through the modulation of NMDAR dependent hippocampal short-term synaptic plasticity. In conclusion this study suggests the possible role of oleic acid to prevent the short term memory loss and impaired neuronal function throughout development.

Keywords: oleic acid, short-term potentiation, memory, field excitatory post synaptic potentials, NMDA receptor

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Authors: Maged El-Sayed Mohamed

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Plant tissue culture practices are presented nowadays as the most promising substitute to a whole plant in the terms of secondary metabolites production. They offer the advantages of high production, tunability and they have less effect on plant ecosystems. Lantana camara is a weed, which is common all over the world as an ornamental plant. Weeds can adapt to any type of soil and climate due to their rich cellular machinery for secondary metabolites’ production. This characteristic is found in Lantana camara as a plant of very rich diversity of secondary metabolites with no dominant class of compounds. Aim: This trait has encouraged the author to develop tissue culture experiments for Lantana camara to be a platform for production and manipulation of secondary metabolites through biotransformation. Methodology: The plant was collected in its flowering stage in September 2014, from which explants were prepared from shoot tip, auxiliary bud and leaf. Different types of culture media were tried as well as four phytohormones and their combinations; NAA, 2,4-D, BAP and kinetin. Explants were grown in dark or in 12 hours dark and light cycles at 25°C. A metabolic profile for the produced callus was made and then compared to the whole plant profile. The metabolic profile was made using GC-MS for volatile constituents (extracted by n-hexane) and by HPLC-MS and capillary electrophoresis-mass spectrometry (CE-MS) for non-volatile constituents (extracted by ethanol and water). Results: The best conditions for the callus induction was achieved using MS media supplied with 30 gm sucrose and NAA/BAP (1:0.2 mg/L). Initiation of callus was favoured by incubation in dark for 20 day. The callus produced under these conditions showed yellow colour, which changed to brownish after 30 days. The rate of callus growth was high, expressed in the callus diameter, which reached to 1.15±0.2 cm in 30 days; however, the induction of callus delayed for 15 days. The metabolic profile for both volatile and non-volatile constituents of callus showed more simple background metabolites than the whole plant with two new (unresolved) peaks in the callus’ nonvolatile constituents’ chromatogram. Conclusion: Lantana camara callus production can be itself a source of new secondary metabolites and could be used for biotransformation studies due to its simple metabolic background, which allow easy identification of newly formed metabolites. The callus production gathered the simple metabolic background with the rich cellular secondary metabolite machinery of the plant, which could be elicited to produce valuable medicinally active products.

Keywords: capillary electrophoresis-mass spectrometry, gas chromatography, metabolic profile, plant tissue culture

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Authors: Benjawan Saengwichian, Alasdair E. Charles, Philip J. Hyde

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Magnetically controlled growing rods (MCGRs) have been used to stabilise and correct spinal curvature in children to support non-invasive scoliosis adjustment. Although the encapsulated driving components are intended to be isolated from body fluid contact, in vivo corrosion was observed on these components due to sealing mechanism damage. Consequently, a corrosion circuit is created with the body fluids, resulting in malfunction of the lengthening mechanism. Particularly, the chloride ions in blood plasma or cerebrospinal fluid (CSF) may corrode the MCGR alloys, possibly resulting in metal ion release in long-term use. However, there is no data available on the corrosion resistance of spinal implant alloys in CSF. In this study, an in vitro immersion configuration was designed to simulate in vivo corrosion of 440C SS-Ti6Al4V couples. The 440C stainless steel (SS) was heat-treated to investigate the effect of tempering temperature on intergranular corrosion (IGC), while crevice and galvanic corrosion were studied by limiting the clearance of dissimilar couples. Tests were carried out in a neutral artificial cerebrospinal fluid (ACSF) and phosphate-buffered saline (PBS) under aeration and deaeration for 2 months. The composition of the passive films and metal ion release were analysed. The effect of galvanic coupling, pH, dissolved oxygen and anion species on corrosion rates and corrosion mechanisms are discussed based on quantitative and qualitative measurements. The results suggest that ACSF is more aggressive than PBS due to the combination of aggressive chlorides and sulphate anions, while phosphate in PBS acts as an inhibitor to delay corrosion. The presence of Vivianite on the SS surface in PBS lowered the corrosion rate (CR) more than 5 times for aeration and nearly 2 times for deaeration, compared with ACSF. The CR of 440C is dependent on passive film properties varied by tempering temperature and anion species. Although the CR of Ti6Al4V is insignificant, it tends to release more Ti ions in deaerated ACSF than under aeration, about 6 µg/L. It seems the crevice-like design has more effect on macroscopic corrosion than combining the dissimilar couple, whereas IGC is dominantly observed on sensitized microstructure.

Keywords: cerebrospinal fluid, crevice corrosion, intergranular corrosion, magnetically controlled growing rods

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Authors: Pao-Huei Chen, Shu-Mei Lin, Yih-Ming Weng, Zer-Ran Yu, Be-Jen Wang

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Pancreatic α-amylase and intestinal α-glucosidase are the critical enzymes for the breakdown of complex carbohydrates into di- or mono-saccharide, which play an important role in modulating postprandial blood sugars. Angiotensin converting enzyme (ACE) converts inactive angiotensin-I into active angiotensin-II, which subsequently increase blood pressure through triggering vasoconstriction and aldosterone secretion. Thus, inhibition of carbohydrate-digestion enzymes and ACE will help the management of blood glucose and blood pressure, respectively. Studies showed Pleurotus citrinopileatus (PC), an edible mushroom and commonly cultured in oriental countries, exerted anticancer, immune improving, antioxidative, hypoglycemic and hypolipidemic effects. Previous studies also showed various molecular weights (MW) fractioned from extracts may affect biological activities due to varying contents of bioactive components. Thus, the objective of this study is to investigate the in vitro antioxidant, hypoglycemic and hypotenstive effects and distribution of active compounds of various MWs of cold water extract from P. citrinopileatus (CWEPC). CWEPC was fractioned into four various MW fractions, PC-I (＜1 kDa), PC-II (1-3.5 kDa), PC-III (3.5-10 kDa), and PC-IV (＞10 kDa), using an ultrafiltration system. The physiological activities, including antioxidant activities, the inhibition capabilities of pancreatic α-amylase, intestinal α-glucosidase, and hypertension-linked ACE, and the active components, including polysaccharides, protein, and phenolic contents, of CWEPC and four fractions were determined. The results showed that fractions with lower MW exerted a higher antioxidant activity (p<0.05), which was positively correlated to the levels of total phenols. In contrast, the inhibition effects on the activities of α-amylase, α-glucosidase, and ACE of PC-IV fraction were significantly higher than CWEPC and the other three low MW fractions (< 10 kDa), which was more related to protein contents. The inhibition capability of CWEPC and PC-IV on α-amylase activity was 1/13.4 to 1/2.7 relative to that of acarbose (positive control), respectively. However, the inhibitory ability of PC-IV on α-glucosidase (IC50 = 0.5 mg/mL) was significantly higher than acarbose (IC50 = 1.7 mg/mL). Kinetic data revealed that PC-IV fraction followed a non-competitive inhibition on α-glucosidase activity. In conclusion, the distribution of various bioactive components contribute to the functions of different MW fractions on oxidative stress prevention, and blood pressure and glucose modulation.

Keywords: α-Amylase, angiotensin converting enzyme, α-Glucosidase, Pleurotus citrinopileatus

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Authors: Ieva Bruzauskaite, Jovile Raudoniute, Karina Poliakovaite, Danguole Zabulyte, Daiva Bironaite, Ruta Aldonyte

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Introduction: Air pollution is a growing global problem which has been shown to be responsible for various adverse health outcomes. Immunotoxicity, such as dysregulated inflammation, has been proposed as one of the main mechanisms in air pollution-associated diseases. Chronic obstructive pulmonary disease (COPD) is among major morbidity and mortality causes worldwide and is characterized by persistent airflow limitation caused by the small airways disease (obstructive bronchiolitis) and irreversible parenchymal destruction (emphysema). Exact pathways explaining the air pollution induced and mediated disease states are still not clear. However, modern societies understand dangers of polluted air, seek to mitigate such effects and are in need for reliable biomarkers of air pollution. We hypothesise that post-translational modifications of structural proteins, e.g. citrullination, might be a good candidate biomarker. Thus, we have designed this study, where mice were exposed to diesel exhaust and the ongoing protein modifications and inflammation in lungs and other tissues were assessed. Materials And Methods: To assess the effects of diesel exhaust a in vivo study was designed. Mice (n=10) were subjected to everyday 2-hour exposure to diesel exhaust for 14 days. Control mice were treated the same way without diesel exhaust. The effects within lung and other tissues were assessed by immunohistochemistry of formalin-fixed and paraffin-embedded tissues. Levels of inflammation and citrullination related markers were investigated. Levels of parenchymal damage were also measured. Results: In vivo study corroborates our own data from in vitro and reveals diesel exhaust initiated inflammatory shift and modulation of lung peptidyl arginine deiminase 4 (PAD4), citrullination associated enzyme, levels. In addition, high levels of citrulline were observed in exposed lung tissue sections co-localising with increased parenchymal destruction. Conclusions: Subacute exposure to diesel exhaust renders mice lungs inflammatory and modifies certain structural proteins. Such structural changes of proteins may pave a pathways to lost/gain function of affected molecules and also propagate autoimmune processes within the lung and systemically.

Keywords: air pollution, citrullination, in vivo, lungs

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Authors: Choupani Andisheh, Temucin Elif Sevval, Bediz Bekir

Abstract:

Due to their various advantages compared to many other drug delivery systems such as hypodermic injections and oral medications, microneedle arrays (MNAs) are a promising drug delivery system. To achieve enhanced performance of the MN, it is crucial to develop numerical models, optimization methods, and simulations. Accordingly, in this work, the optimized design of dissolvable MNAs, as well as their manufacturing, is investigated. For this purpose, a mechanical model of a single MN, having the geometry of an obelisk, is developed using commercial finite element software. The model considers the condition in which the MN is under pressure at the tip caused by the reaction force when penetrating the skin. Then, a multi-objective optimization based on non-dominated sorting genetic algorithm II (NSGA-II) is performed to obtain geometrical properties such as needle width, tip (apex) angle, and base fillet radius. The objective of the optimization study is to reach a painless and effortless penetration into the skin along with minimizing its mechanical failures caused by the maximum stress occurring throughout the structure. Based on the obtained optimal design parameters, master (male) molds are then fabricated from PMMA using a mechanical micromachining process. This fabrication method is selected mainly due to the geometry capability, production speed, production cost, and the variety of materials that can be used. Then to remove any chip residues, the master molds are cleaned using ultrasonic cleaning. These fabricated master molds can then be used repeatedly to fabricate Polydimethylsiloxane (PDMS) production (female) molds through a micro-molding approach. Finally, Polyvinylpyrrolidone (PVP) as a dissolvable polymer is cast into the production molds under vacuum to produce the dissolvable MNAs. This fabrication methodology can also be used to fabricate MNAs that include bioactive cargo. To characterize and demonstrate the performance of the fabricated needles, (i) scanning electron microscope images are taken to show the accuracy of the fabricated geometries, and (ii) in-vitro piercing tests are performed on artificial skin. It is shown that optimized MN geometries can be precisely fabricated using the presented fabrication methodology and the fabricated MNAs effectively pierce the skin without failure.

Keywords: microneedle, microneedle array fabrication, micro-manufacturing structural optimization, finite element analysis

Procedia PDF Downloads 113

Authors: Nicholas Fletcher, Zachary Houston, Yongmei Zhao, Christopher Howard, Kristofer Thurecht

Abstract:

One promising approach to enhance nanomedicine therapeutic efficacy is to include a targeting agent, such as an antibody, to increase accumulation at the tumor site. However, the application of such targeted nanomedicines remains limited, in part due to difficulties involved with biomolecule conjugation to synthetic nanomaterials. One approach recently developed to overcome this has been to engineer bispecific antibodies (BsAbs) with dual specificity, whereby one portion binds to methoxy polyethyleneglycol (mPEG) epitopes present on synthetic nanomedicines, while the other binds to molecular disease markers of interest. In this way, noncovalent complexes of nanomedicine core, comprising a hyperbranched polymer (HBP) of primarily mPEG, decorated with targeting ligands are able to be produced by simple mixing. Further work in this area has now demonstrated such complexes targeting the breast cancer marker epidermal growth factor receptor (EGFR) to show enhanced binding to tumor cells both in vitro and in vivo. Indeed the enhanced accumulation at the tumor site resulted in improved therapeutic outcomes compared to untargeted nanomedicines and free chemotherapeutics. The current work on these BsAb-HBP conjugates focuses on further probing antibody-nanomaterial interactions and demonstrating broad applicability to a range of cancer types. Herein are reported BsAb-HBP materials targeted towards prostate-specific membrane antigen (PSMA) and study of their behavior in vivo using ⁸⁹Zr positron emission tomography (PET) in a dual-tumor prostate cancer xenograft model. In this model mice bearing both PSMA+ and PSMA- tumors allow for PET imaging to discriminate between nonspecific and targeted uptake in tumors, and better quantify the increased accumulation following BsAb conjugation. Also examined is the potential for formation of these targeted complexes in situ following injection of individual components? The aim of this approach being to avoid undesirable clearance of proteinaceous complexes upon injection limiting available therapeutic. Ultimately these results demonstrate BsAb functionalized nanomaterials as a powerful and versatile approach for producing targeted nanomedicines for a variety of cancers.

Keywords: bioengineering, cancer, nanomedicine, polymer chemistry

Procedia PDF Downloads 141