Search results for: extracellular proteases

Authors: Faisal Almalki

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Pyran is a heterocyclic group containing oxygen that possesses a variety of pharmacological effects. Pyran is also one of the most prevalent structural subunits in natural products, such as xanthones, coumarins, flavonoids, benzopyrans, etc. Additionally demonstrating the neuroprotective properties of pyrans is the fact that this heterocycle has recently attracted the attention of scientists worldwide. Alzheimer's Disease (AD) treatment and diagnosis are two of the most critical research objectives worldwide. Increased amounts of extracellular senile plaques, intracellular neurofibrillary tangles, and a progressive shutdown of cholinergic basal forebrain neuron transmission are often related with cognitive impairment. This review highlights the various pyran scaffolds of natural and synthetic origin that are effective in the treatment of AD. For better understanding synthetic compounds are categorized as different types of pyran derivatives like chromene, flavone, xanthone, xanthene, etc. The discussion encompasses both the structure-activity correlations of these compounds as well as their activity against AD. Because of the intriguing actions that were uncovered by these pyran-based scaffolds, there is no question that they are at the forefront of the search for potential medication candidates that could treat Alzheimer's disease.

Keywords: alzheimer’s disease, pyran, coumarin, xanthone

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Authors: Michal Štros, Eva Polanská, Šárka Pospíšilová

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HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. MALDI-TOF analysis revealed that mild oxidization of HMGB1 resulted in a conformational change of the protein due to formation of an intramolecular disulphide bond by opposing Cys23 and Cys45 residues. We have demonstrated that redox state of HMGB1 could significantly modulate the ability of the protein to bind and bend DNA. We have also shown that reduced HMGB1 could easily displace histone H1 from DNA, while oxidized HMGB1 had limited capacity for H1 displacement. Using microscale thermophoresis (MST) we have further studied mechanism of HMGB1 interaction with histone H1 in free solution or when histone H1 was bound to DNA. Our MST analysis indicated that reduced HMGB1 exhibited in free solution > 1000 higher affinity of for H1 (KD ~ 4.5 nM) than oxidized HMGB1 (KD <10 M). Finally, we present a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA.

Keywords: HMGB1, histone H1, redox state, interaction, cross-linking, DNA bending, DNA end-joining, microscale thermophoresis

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Authors: Tooba N. Shamsi, Romana Perveen, Sadaf Fatima

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Protease Inhibitors (PIs) are widespread in nature, produced by animals, plants and microorganisms. They play vital role in various biological activities by keeping a check on activity of proteases. Present study aims to investigate antioxidant and anti-inflammatory properties of PPI from Cajanus cajan (CCTI) and Phaseolus limensis (LBTI). PPI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. The anti-oxidant activity was analyzed by two most common radical scavenging assays of FRAP (ferric reducing antioxidant power) and DPPH (1,1- diphenyl-2-picrylhydrazyl). Also, in-vitro anti-inflammatory activity was evaluated using albumin denaturation assay and membrane stabilization assay at different concentrations. Ascorbic acid and aspirin were used as a standards for antioxidant and anti-inflammatory assays respectively. The PPIs were also checked for antimicrobial activity against a number of bacterial strains. The CCTI and LBTI showed DPPH radical scavenging activity in a concentration–dependent manner with IC50 values 544 µg/ml and 506 µg/ml respectively comparative to ascorbic acid which was 258 µg/ml. Following FRAP assay, it was evaluated that LBTI had 87.5% and CCTI showed 84.4% antioxidant activity, taking value of standard ascorbic acid to be 100%. The PPIs also showed in-vitro anti‐inflammatory activity by inhibiting the heat induced albumin denaturation with IC50 values of 686 µg/ml and 615 µg/ml for CCTI and LBTI respectively compared to the standard (aspirin) which was 70.8 µg/ml. Red blood cells membrane stabilization with IC50 values of 641 µg/ml and 587 µg/ml for CCTI and LBTI respectively against aspirin which showed IC50 value of 70.4 µg/ml. PPIs showed antibacterial activity against 7 known strains while there was apparently no action against fungi.

Keywords: Cajanus cajan, Phaseolus limensis, Lima beans, protein protease inhibitor, antioxidant, anti-inflammatory, antimicrobial activity

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Authors: M. F. R. Zuthi, H. H. Ngo, W. S. Guo, S. S. Chen, N. C. Nguyen, L. J. Deng, T. D. C Tran

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Sustaining a desired rate of oxygen transfer for microbial activity is a matter of major concern for Biological Wastewater Treatment (MBR). The study reported in the paper was aimed at assessing the effects of microbial products on the Specific Oxygen Uptake Rate (SOUR) in a Conventional Membrane Bioreactor (CMBR) and that in a Sponge Submerged MBR (SSMBR). The production and progressive accumulation of Soluble Microbial Products (SMP) and Bound-Extracellular Polymeric Substances (BEPS) were found affecting the SOUR of the microorganisms which varied at different stages of operation of the MBR systems depending on the variable concentrations of the SMP/bEPS. The effect of bEPS on the SOUR was stronger in the SSMBR compared to that of the SMP, while relative high concentrations of SMP had adverse effects on the SOUR of the CMBR system. Of the different mathematical correlations analyzed in the study, logarithmic mathematical correlations could be established between SOUR and bEPS in SSMBR, and similar correlations could also be found between SOUR and SMP concentrations in the CMBR.

Keywords: microbial products, microbial activity, specific oxygen uptake rate, membrane bioreactor

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Authors: Benarous Khedidja, Yousfi Mohamed

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Infections caused by Candida species manifest in a number of diseases, including candidemia, vulvovaginal candidiasis, endocarditis, and peritonitis. These Candida species have been reported to have lipolytic activity by secretion of lipolytic enzymes such as esterases, lipases and phospholipases. These Extracellular hydrolytic enzymes seem to play an important role in Candida overgrowth. Candidiasis is commonly treated with antimycotics such as clotrimazole and nystatin, which bind to a major component of the fungal cell membrane (ergosterol). This binding forms pores in the membrane that lead to death of the fungus. Due to their secondary effects, scientists have thought of another treatment basing on lipase inhibition but we haven’t found any lipase inhibitors used as candidiasis treatment. In this work, we are interested to lipases inhibitors such as alkaloids as another candidiasis treatment. In the first part, we have proceeded to optimize the alkaloid structures and protein 3D structure using Hyperchem software. Secondly, we have docked inhibitors using Genetic algorithm with GOLD software. The results have shown ten possibilities of binding inhibitor to Candida rugosa lipase (CRL) but only one possibility has been accepted depending on the weakest binding energy.

Keywords: marrubiin, candida rugosa lipase, docking, gold

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Authors: Natasa Ilic, Alisa Gruden-Movsesijan, Maja Kosanovic, Sofija Glamoclija, Marina Bekic, Ljiljana Sofronic-Milosavljevic, Sergej Tomic

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As a very promising candidate for modulation of immune response in the sense of biasing the inflammatory towards an anti-inflammatory type of response, Trichinella spiralis infection was shown to successfully alleviate the severity of experimental autoimmune encephalomyelitis, the animal model of human disease multiple sclerosis. This effect is achieved via its excretory-secretory muscle larvae (ES L1) products which affect the maturation status and function of dendritic cells (DCs) by inducing the tolerogenic status of DCs, which leads to the mitigation of the Th1 type of response and the activation of a regulatory type of immune response both in vitro and in vivo. ES L1 alone or via treated DCs successfully mitigated EAE in the same manner as the infection itself. On the other hand, it has been shown that T. spiralis infection slows down the tumour growth and significantly reduces the tumour size in the model of mouse melanoma, while ES L1 possesses a pro-apoptotic and anti-survival effect on melanoma cells in vitro. Hence, although the mechanisms still need to be revealed, T. spiralis infection and its ES L1 products have a bit of controversial potential to modulate both inflammatory diseases and malignancies. The recent discovery of T. spiralis extracellular vesicles (TsEVs) suggested that the induction of complex regulation of the immune response requires simultaneous delivery of different signals in nano-sized packages. This study aimed to explore whether TsEVs bare the similar potential as ES L1 to influence the status of DCs in initiation, progression and regulation of immune response, but also to investigate the effect of both ES L1 and TsEVs on myeloid derived suppressor cells (MDSC) which present the regular tumour tissue environment. TsEVs were enriched from the conditioned medium of T. spiralis muscle larvae by differential centrifugation and used for the treatment of human monocyte-derived DCs and MDSC. On DCs, TsEVs induced low expression of HLA DR and CD40, moderate CD83 and CD86, and increased expression of ILT3 and CCR7 on treated DCs, i.e., they induced tolerogenic DCs. Such DCs possess the capacity to polarize T cell immune response towards regulatory type, with an increased proportion of IL-10 and TGF-β producing cells, similarly to ES L1. These findings indicated that the ability of TsEVs to induce tolerogenic DCs favoring anti-inflammatory responses may be helpful in coping with diseases that involve Th1/Th17-, but also Th2-mediated inflammation. In MDSC in vitro model, although both ES L1 and TsEVs had the same impact on MDSC phenotype i.e., they acted suppressive, ES L1 treated MDSC, unlike TsEVs treated ones, induced T cell response characterized by the increased RoRγT and IFN-γ, while the proportion of regulatory cells was decreased followed by the decrease in IL-10 and TGF-β positive cells proportion within this population. These findings indicate the interesting ability of ES L1 to modulate T cells response via MDSC towards pro-inflamatory type, suggesting that, unlike TsEVs which consistently demonstrate the suppresive effect on inflammatory response, it could be used also for the development of new approaches aimed for the treatment of malignant diseases. Acknowledgment: This work was funded by the Promis project – Nano-MDCS-Thera, Science Fund, Republic of Serbia.

Keywords: dendritic cells, myeloid derived suppressor cells, immunomodulation, Trichinella spiralis

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Authors: Arifur Rahman, Ardeshir Amirkhani, Honghua Hu, Mark Molloy, Karen Vickery

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Introduction and Objectives: Staphylococcus aureus and coagulase-negative staphylococci comprises approximately 65% of infections associated with medical devices and are well known for their biofilm formatting ability. Biofilm-related infections are extremely difficult to eradicate owing to their high tolerance to antibiotics and host immune defences. Currently, there is no efficient method for early biofilm detection. A better understanding to enable detection of biofilm specific proteins in vitro and in vivo can be achieved by studying planktonic and different growth phases of biofilms using a proteome analysis approach. Our goal was to construct a reference map of planktonic and biofilm associated proteins of S. aureus. Methods: S. aureus reference strain (ATCC 25923) was used to grow 24 hours planktonic, 3-day wet biofilm (3DWB), and 12-day wet biofilm (12DWB). Bacteria were grown in tryptic soy broth (TSB) liquid medium. Planktonic growth was used late logarithmic bacteria, and the Centres for Disease Control (CDC) biofilm reactor was used to grow 3 days, and 12-day hydrated biofilms, respectively. Samples were subjected to reduction, alkylation and digestion steps prior to Multiplex labelling using Tandem Mass Tag (TMT) 10-plex reagent (Thermo Fisher Scientific). The labelled samples were pooled and fractionated by high pH RP-HPLC which followed by loading of the fractions on a nanoflow UPLC system (Eksigent UPLC system, AB SCIEX). Mass spectrometry (MS) data were collected on an Orbitrap Elite (Thermo Fisher Scientific) Mass Spectrometer. Protein identification and relative quantitation of protein levels were performed using Proteome Discoverer (version 1.3, Thermo Fisher Scientific). After the extraction of protein ratios with Proteome Discoverer, additional processing, and statistical analysis was done using the TMTPrePro R package. Results and Discussion: The present study showed that a considerable proteomic difference exists among planktonic and biofilms from S. aureus. We identified 1636 total extracellular secreted proteins, of which 350 and 137 proteins of 3DWB and 12DWB showed significant abundance variation from planktonic preparation, respectively. Of these, simultaneous up-regulation in between 3DWB and 12DWB proteins such as extracellular matrix-binding protein ebh, enolase, transketolase, triosephosphate isomerase, chaperonin, peptidase, pyruvate kinase, hydrolase, aminotransferase, ribosomal protein, acetyl-CoA acetyltransferase, DNA gyrase subunit A, glycine glycyltransferase and others we found in this biofilm producer. On the contrary, simultaneous down-regulation in between 3DWB and 12DWB proteins such as alpha and delta-hemolysin, lipoteichoic acid synthase, enterotoxin I, serine protease, lipase, clumping factor B, regulatory protein Spx, phosphoglucomutase, and others also we found in this biofilm producer. In addition, we also identified a big percentage of hypothetical proteins including unique proteins. Therefore, a comprehensive knowledge of planktonic and biofilm associated proteins identified by S. aureus will provide a basis for future studies on the development of vaccines and diagnostic biomarkers. Conclusions: In this study, we constructed an initial reference map of planktonic and various growth phase of biofilm associated proteins which might be helpful to diagnose biofilm associated infections.

Keywords: bacterial biofilms, CDC bioreactor, S. aureus, mass spectrometry, TMT

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Authors: Merry Meryam Martgrita, Marselina Irasonia Tan

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One of several aggresive cancer is cancer that overexpress c-erbB2 receptor along with the expression of estrogen receptor. Components of extracellular matrix play an important role to increase cancer cells proliferation, migration and invasion. Both components can affect cancer development by regulating the signal transduction pathways in cancer cells. In recent research, SKOV-3 ovarian cancer cell line, that overexpress c-erbB2 receptor was cultured on type IV collagen and treated with estradiol-17β, to reveal the role of both components on RNA and protein level of c-erbB2 receptor. In this research we found a modulation phenomena of increasing and decreasing of c-erbB2 RNA level and a stabilisation phenomena of c-erbB2 protein expression due to estradiol-17β and type IV collagen. It seemed that estradiol-17β has an important role to increase c-erbB2 transcription and the stability of c-erbB2 protein expression. Type IV collagen has an opposite role. It blocked c-erbB2 transcription when it bound to integrin receptor in SKOV-3 cells.

Keywords: c-erbB2, estradiol-17β, SKOV-3, type IV collagen

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Authors: Ihn Sook Jeong, Eun Joo Lee, Jae Hyung Kim, Gun Ho Kim, Young Jun Hwang

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This study investigated intravenous (IV) infiltration using bioelectrical impedance for 27 hospitalized patients in a long-term care hospital. Impedance parameters showed significant differences before and after infiltration as follows. First, the resistance (R) after infiltration significantly decreased compared to the initial resistance. This indicates that the IV solution flowing from the vein due to infiltration accumulates in the extracellular fluid (ECF). Second, the relative resistance at 50 kHz was 0.94 ± 0.07 in 9 subjects without infiltration and was 0.75 ± 0.12 in 18 subjects with infiltration. Third, the magnitude of the reactance (Xc) decreased after infiltration. This is because IV solution and blood components released from the vein tend to aggregate in the cell membrane (and acts analogously to the linear/parallel circuit), thereby increasing the capacitance (Cm) of the cell membrane and reducing the magnitude of reactance. Finally, the data points plotted in the R-Xc graph were distributed on the upper right before infiltration but on the lower left after infiltration. This indicates that the infiltration caused accumulation of fluid or blood components in the epidermal and subcutaneous tissues, resulting in reduced resistance and reactance, thereby lowering integrity of the cell membrane. Our findings suggest that bioelectrical impedance is an effective method for detection of infiltration in a noninvasive and quantitative manner.

Keywords: intravenous infiltration, impedance, parameters, resistance, reactance

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Authors: Chao Wu

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Thioredoxin reductase 2 is a mitochondrial enzyme that belongs to the cellular defense against oxidative stress. We deleted mitochondrial Txnrd2 in a KrasG12D-driven pancreatic tumor model. Despite an initial increase in precursor lesions, tumor incidence decreased significantly. We isolated cancer cell lines from these genetically engineered mice and observed an impaired proliferation and colony formation. Reactive Oxygen Species, as determined by DCF fluorescence, were increased. We detected a higher mitochondrial copy number in Txnrd2-deficient cells (KTP). However, measurement of mitochondrial bioenergetics showed no impairment of mitochondrial function and comparable O₂-consumption and extracellular acidification rates. In addition, the mitochondrial complex composition was affected in Txnrd2 deleted cell lines. To gain better insight into the role of Txnrd2, we deleted Txnrd2 in clones from parental KrasG12D cell lines using Crispr/Cas9 technology. The deletion was confirmed by western blot and activity assay. Interestingly, and in line with previous RNA expression analysis, we saw changes in EMT markers in Txnrd2 deleted cell lines and control cell lines. This might help us explain the reduced tumor incidence in KrasG12D; Txnrd2∆panc mice.

Keywords: PDAC, TXNRD2, epithelial-to-mesenchymal-transition, ROS

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Authors: Habib Onsori, Somayeh Akrami, Mohammad Rahmati

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Deafness is the most common sensory disorder with the frequency of 1/1000 in many populations. Mutations in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 are associated with congenital hearing loss. Approximately 80% of congenital hearing loss cases are recessively inherited and 15% dominantly inherited. Mutations of the GJB2 gene, encoding gap junction protein Connexin 26 (Cx26), are the most common cause of hereditary congenital hearing loss in many countries. This report presents two cases of different mutations from Iranian patients with bilateral hearing loss. DNA studies were performed for the GJB2 gene by PCR and sequencing methods. In one of them, direct sequencing of the gene showed a heterozygous T→C transition at nucleotide 604 resulting in a cysteine to arginine amino acid substitution at codon 202 (C202R) in the fourth extracellular domain (TM4) of the protein. The analyses indicate that the C202R mutation appeared de novo in the proband with a possible dominant effect (GenBank: KF 638275). In the other one, DNA sequencing revealed a compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL). So screening the mutations for hearing loss individuals referring to genetics counseling centers before marriage and or pregnancy is recommended.

Keywords: CX26, deafness, GJB2, mutation

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Authors: Dina Helmy El-Ghonemy, Thanaa Hamed Ali

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The aim of this study was to purify and characterize a keratinolytic enzyme produced by Aspergillus sp. DHE7 cultured in basal medium containing chicken feather as substrate. The enzyme was purified through ammonium sulfate saturation of 60%, followed by gel filtration chromatography in Sephadex G-100, with a 16.4-purification fold and recovery yield of 52.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme is a monomeric enzyme with an apparent molecular mass of 30 kDa — the purified keratinase of Aspergillus sp. DHE7 exhibited activity in a broad range of pH (7- 9) and temperature (40℃-60℃) profiles with an optimal activity at pH eight and 50℃. The keratinolytic activity was inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride and ethylenediaminetetraacetate, while no reduction of activity was detected by the addition of dimethyl sulfoxide (DMSO). Bivalent cations, Ca²⁺ and Mn²⁺, were able to greatly enhance the activity of keratinase by 125.7% and 194.8%, respectively, when used at one mM final concentration. On the other hand, Cu²⁺ and Hg²⁺ inhibited the enzyme activity, which might be indicative of essential vicinal sulfhydryl groups of the enzyme for productive catalysis. Furthermore, the purified keratinase showed significant stability and compatibility against the tested commercial detergents at 37ºC. Therefore, these results suggested that the purified keratinase from Aspergillus sp. DHE7 may have potential use in the detergent industry and should be of interest in the processing of poultry feather waste.

Keywords: Aspergillus sp. DHE7, biochemical characterization, keratinase, purification, waste management

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Authors: Abdelaziz Messis, Azzeddine Bettache, Nawel Boucherba, Said Benallaoua, Mouloud Kecha

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Actinobacteria are of special biotechnological interest since they are known to produce chemically diverse compounds with a wide range of biological activity. This distinct clade of Gram-positve bacteria include some of the key antibiotic producers and are also sources of several bioactive compounds, established commercially a newly filamentous bacteria was recovered from Tikjda forest soil (Algeria) for its high antifungal activity against various pathogenic and phytopathogenic fungi. The nucleotide sequence of the 16S rRNA gene (1454 pb) of Streptomyces sp. TKJ2 exhibited close similarity (99 %) with other Streptomyces16S rRNA genes. Antifungal metabolite production of Streptomyces sp TKJ2 was evaluated using six different fermentation media. The extracellular products contained potent antifungal agents. Antifungal protein produced by Streptomyces sp. TKJ2 on PCA medium has been purified by ammonium sulfate precipitation, SPE column chromatography and high-performance liquid chromatography in a reverse-phase column. The UV chromatograms of the active fractions obtained at 214 nm by NanoLC-ESI-MS/MS have different molecular weights. The F20 Peptidic fraction obtained from culture filtrat of Streptomyces sp. TKJ2 precipitated at 30% of ammonium sulfate was selected for analysis by infusion ESI-MS which yielded a singly charged ion mass of 437.17 Da.

Keywords: actinobacteria, antifungal protein, chromatography, Streptomyces

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Authors: Ayodeji O. Falade, Leonard V. Mabinya, Uchechukwu U. Nwodo, Anthony I. Okoh

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Given the high-utility of peroxidase, its production in large amount is of utmost importance. Over the years, actinomycetes have been the major peroxidase-producing bacteria. Consequently, other classes of bacteria with peroxidase production potentials are underexplored. This study, therefore, sought to enhance peroxidase production by a Raoultella species, a new ligninolytic proteobacteria strain, by determining the optimum culture conditions (initial pH, incubation temperature and agitation speed) for peroxidase production under submerged fermentation using the classical process of one variable at a time and supplementing the fermentation medium with some lignin model and inorganic nitrogen compounds. Subsequently, the time-course assay was carried out under optimized conditions. Then, some agricultural residues were valorized for peroxidase production under solid state fermentation. Peroxidase production was optimal at initial pH 5, incubation temperature of 35 °C and agitation speed of 150 rpm with guaiacol and ammonium chloride as the best inducer and nitrogen supplement respectively. Peroxidase production by the Raoultella species was optimal at 72 h with specific productivity of 16.48 ± 0.89 U mg⁻¹. A simultaneous production of a non-peroxide dependent extracellular enzyme which suggests probable laccase production was observed with specific productivity of 13.63 ± 0.45 U mg⁻¹ while sawdust gave the best peroxidase yield under solid state fermentation. In conclusion, peroxidase production by the Raoultella species was increased by 3.40-fold.

Keywords: enzyme production, ligninolytic bacteria, peroxidase, proteobacteria

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Authors: Jma Hannan, Prawej Ansari, Yasser H. A. Abdel-Wahab, Peter R. Flatt

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Spirulina platensis (SP, Blue-green algae) have been accepted as a supplement for the treatment of pre and post-diabetes. The present study investigated the effects of butanol fraction from ethanol extract of S. platensis on insulin release from BRIN BD11 cells, isolated mouse islets, and perfused rat pancreas, as well as glucose homeostasis in type 2 diabetic rats and their molecular pathways. In a dose-dependent manner, S. platensis increased insulin release from mouse islets and pancreatic β-cells. The extract also elevated insulin release in perfused rat pancreas. Glucose, isobutylmethylxanthine, tolbutamide, and a depolarizing concentration of KCl significantly potentiated insulin release from BRIN BD11 cells. The effect of diazoxide and verapamil, as well as the absence of extracellular Ca2+ showed a reduction in insulin secretion. When administered orally together with glucose (2.5g/kg bw), S. platensis extract improved fasting and postprandial blood glucose in type 2 diabetes. These data suggest that the anti-diabetic activity of S. platensis is partly mediated by insulin secretion via the KATP channel-dependent pathway/the intracellular cAMP pathway.

Keywords: Insulin, glucose, S. platensis, type 2 diabetes, medicinal plants

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Authors: Olena Grygorieva

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Nowadays mechanisms of thymocytes emigration from the thymus to the periphery are investigated actively. We have proposed a hypothesis of thymocytes’ migration from the thymus through lymphatic vessels during periodical short-term local edema. By morphological, hystochemical methods we have examined quantity of lymphocytes, epitelioreticulocytes, mast cells, blood and lymphatic vessels in morpho-functional areas of rats’ thymuses during the first week after birth in 4 hours interval. In newborn and beginning from 8 hour after birth every 12 hours specific density of the thymus, absolute quantity of microcirculatory vessels, especially of lymphatic ones, lymphcyte-epithelial index, quantity of mast cells and their degranulative forms increase. Structure of extracellular matrix, intrathymical microenvironment and lymphocytes’ adhesive properties change. Absolute quantity of small lymphocytes in thymic cortex changes wavy. All these changes are straightly expressed from 0 till 2, from 12 till 16, from 108 till 120 hours of postnatal life. During this periods paravasal lymphatic vessels are stuffed with lymphocytes, i.e. discrete migration of lymphocytes from the thymus occurs. After rapid edema reduction, quantity of lymphatic vessels decrease, they become empty. Therefore, in the thymus of newborn periodical short-term local edema is observed, on its top discrete migration of lymphocytes from the thymus occurs.

Keywords: lymphocytes, lymphatic vessels, mast cells, thymus

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Authors: E. Roshan Ara Begum, K. Bhavani, K. Subachitra, C. Kirthika, R. Shenbagarathai

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In recent years, there has been a growing concern for the production of chitosan blend nanofibrous scaffold for its favorable physicochemical properties which mimic the native extracellular matrix (ECM) both morphologically and chemically. Therefore, this study focused on production of β-chitosan(β-Cts) and Poly(vinyl alcohol)(PVA) blend nanofibrous scaffold by electrospinning. β-Cts was extracted from the squid pen waste of locally available squid variety Loligo duvauceli (Indian Squid). To the best of our knowledge, there are no reports on nanofibers preparation from the extracted β-Cts. Both the β-Cts and PVA polymers were mixed in two different proportions (30:70 and 40:60 respectively. The electrospun nanofibrous scaffolds were characterized by SEM, swelling property, in vitro enzymatic degradation, and hemo, biocompatibility properties. β-Cts/PVA nanofibers scaffolds had an average fiber diameter of 120 to 550nm.Among the two different β-Cts/PVA blends nanofibers the β-Cts/PVA (40:60) blend fibers demonstrated favourable tissue engineering properties. The β-Cts/PVA (40:60) blend nanofibers exhibited a swelling ratio of 36 ± 2.5% with mass loss percentage of 20 ± 2.71% after 4 weeks of degradation. It has exhibited good hemocompatible properties. HEK-293(Human Embryonic Kidney) cells lines were able to adhere and proliferate well in the β-Cts/PVA blends nanofibers. All these results indicated that electrospun β-Cts/PVA blends nanofibers are a suitable scaffold to be used for tissue engineering purposes.

Keywords: β-chitosan, electrospinning, nanofibers, poly(vinyl alcohol) (PVA)

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Authors: Husain S. Yawer, Vasim Raja Panwar, Nidhi Priya

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The objective of this study is to evaluate and compare the role of nano-gelatin and Bioengineered Scaffolds on the attachment, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSCs). Tooth decay and early fall have each been one of the most prevailing dental disorders which cause physical and emotional suffering and compromise the patient's quality of life. The design of novel scaffolding materials will be based on mimicking the architecture of natural dental extracellular matrix which may provide as in vivo environments for proper cell growth. This methodology will involve the combination of nano-fibred gelatin as well as biodegradable hydrogel based tooth scaffold. We have measured and optimized the Dental Pulp Stem Cells growth profile in cultures carried out on collagen-coated plastic surface, however, for tissue regeneration study, we aim to develop an enhanced microenvironment for stem cell growth and dental tissue regeneration. We believe biomimetic cell adhesion and scaffolds might provide a near in vivo growth environment for proper growth and differentiation of human DPSCs, which further help in dentin/pulp tissue regeneration.

Keywords: nano-gelatin, stem cells, dental pulp, scaffold

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Authors: Kyle Phillips, Ndiko Ludidi

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Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22 which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extracellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of up-regulation and silencing of the maize RD22 gene on the tolerance of maize to drought. The potential role of the maize RD22 gene in maize drought tolerance can be used as a tool to improve food security.

Keywords: abscisic acid, drought-responsive cis-acting elements, maize drought tolerance, RD22

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Authors: Oktira Roka Aji, Maelita R. Moeis, Ihsanawati, Ernawati A. Giri-Rachman

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Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains and extensively drug-resistant strains have become a major public concern. One of the potential candidates for drug target is the cytoplasmic domain of PhoR Histidine Kinase, a part of the Two Component System (TCS) PhoR-PhoP in Mycobacterium tuberculosis (Mtb). TCS PhoR-PhoP relay extracellular signal to control the expression of 114 virulent associated genes in Mtb. The 3D structure of PhoR cytoplasmic domain is needed to screen novel drugs using structure based drug discovery. The PhoR cytoplasmic domain from Mtb H37Rv was overexpressed in E. coli BL21(DE3), then purified using IMAC Ni-NTA Agarose his-tag affinity column and DEAE-ion exchange column chromatography. The molecular weight of the purified protein was estimated to be 37 kDa after SDS-PAGE analysis. This sample was used for pre-crystallization screening by applying sitting drop vapor diffusion method using Natrix (HR2-116) 48 solutions crystal screen kit at 25ºC. Needle-like crystals were observed after the seventh day of incubation in test solution No.47 (0.1 M KCl, 0.01 M MgCl2.6H2O, 0.05 M Tris-Cl pH 8.5, 30% v/v PEG 4000). Further testing is required for confirming the crystal.

Keywords: tuberculosis, two component system, histidine kinase, needle-like crystals

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Authors: Osama M. Darwesh, Sahar H. Hassan, Abd El-Raheem R. El-Shanshoury, Shawky Z. Sabae

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Nanotechnology as multidisciplinary technology is growing rapidly with important applications in several sectors. Also, nanobiotechnology is known for the use of microorganisms for the synthesis of targeted nanoparticles. The present study deals with the green synthesis of silver nanoparticles using aquatic bacteria and the development of a biogenic nanocomposite for environmental applications. Twenty morphologically different colonies were isolated from the collected water samples from eight different locations at the Rosetta branch of the Nile Delta, Egypt. The obtained results illustrated that the most effective bacterial isolate (produced the higher amount of AgNPs after 24 h of incubation time) is isolate R3. Bacillus tequilensis was the strongest extracellular bio-manufactory of AgNPs. Biosynthesized nanoparticles had a spherical shape with a mean diameter of 2.74 to 28.4 nm. The antimicrobial activity of silver nanoparticles against many pathogenic microbes indicated that the produced AgNPs had high activity against all tested multi-antibiotic resistant pathogens. Also, the stabilized prepared AgNPs-SA nanocomposite has greater catalytic activity for the decolourization of some dyes like Methylene blue (MB) and Crystal violet. Such results represent a promising stage for producing eco-friendly, cost-effective, and easy-to-handle devices for the bioremediation of contaminated industrial wastewater.

Keywords: bioremediation, AgNPs, AgNPs-SA nanocomposite, Bacillus tequilensis, nanobiotechnology

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Authors: Abtehal Y. Anaas, Mohd. Nazmi Bin Abd. Manap

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Meat Tenderness is one of the most important factors affecting consumers' assessment of meat quality. Variation in meat tenderness is genetically controlled and varies among breeds, and it is also influenced by environmental factors that can affect its creation during rigor mortis and postmortem. The final postmortem meat tenderization relies on the extent of proteolysis of myofibrillar proteins caused by the endogenous activity of the proteolytic calpain system. This calpain system includes different calcium-dependent cysteine proteases, and an inhibitor, calpastatin. It is widely accepted that in farm animals including chickens, the μ-calpain gene (CAPN1) is a physiological candidate gene for meat tenderness. This study aimed to identify the association of single nucleotide polymorphism (SNP) markers in the CAPN1 gene with the tenderness of chicken breast meat from two Malaysian native and commercial broiler breed crosses. Ten, five months old native chickens and ten, 42 days commercial broilers were collected from the local market and breast muscles were removed two hours after slaughter, packed separately in plastic bags and kept at -20ºC for 24 h. The tenderness phenotype for all chickens’ breast meats was determined by Warner-Bratzler Shear Force (WBSF). Thawing and cooking losses were also measured in the same breast samples before using in WBSF determination. Polymerase chain reaction (PCR) was used to identify the previously reported C7198A and G9950A SNPs in the CAPN1 gene and assess their associations with meat tenderness in the two breeds. The broiler breast meat showed lower shear force values and lower thawing loss rates than the native chickens (p<0.05), whereas there were similar in the rates of cooking loss. The study confirms some previous results that the markers CAPN1 C7198A and G9950A were not significantly associated with the variation in meat tenderness in chickens. Therefore, further study is needed to confirm the functional molecular mechanism of these SNPs and evaluate their associations in different chicken populations.

Keywords: CAPNl, chicken, meat tenderness, meat quality, SNPs

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Authors: Barbora Chmelova, Radek Sachl

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Gangliosides are amphipathic membrane lipids. Due to the composition of bulky oligosaccharide chains containing one or more sialic acids linked to the hydrophobic ceramide base, gangliosides are classified among glycosphingolipids. This unique structure induces a high self-aggregating tendency of gangliosides and, therefore, the formation of nanoscopic clusters called nanodomains. Gangliosides are preferentially present in an extracellular membrane leaflet of all human tissues and thus have an impact on a huge number of biological processes, such as intercellular communication, cell signalling, membrane trafficking, and regulation of receptor activity. Defects in their metabolism, impairment of proper ganglioside function, or changes in their organization lead to serious health conditions such as Alzheimer´s and Parkinson´s diseases, autoimmune diseases, tumour growth, etc. This work mainly focuses on ganglioside organization into nanodomains and their dynamics within the plasma membrane. Current research investigates static ganglioside nanodomains characterization; nevertheless, the information about their diffusion is missing. In our study, fluorescence correlation spectroscopy is implemented together with stimulated emission depletion (STED-FCS), which combines the diffraction-unlimited spatial resolution with high temporal resolution. By comparison of the experiments performed on model vesicles containing 4 % of either GM1, GM2, or GM3 and Monte Carlo simulations of diffusion on the plasma membrane, the description of ganglioside clustering, diffusion of nanodomains, and even diffusion of ganglioside molecules inside investigated nanodomains are described.

Keywords: gangliosides, nanodomains, STED-FCS, flourescence microscopy, membrane diffusion

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Authors: Ji Hye Im, Nguyen T. T. Phuong, Keon Wook Kang

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Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Keywords: TAMR-MCF-7, CREB, estrogen receptor, aromatase

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Authors: Hassan Mohammadi Khujin

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Tissue engineering is the science of tissues and complex organs creation using scaffolds, cells and biologically active components. Most cells require scaffolds to grow and proliferate. These temporary support structures for tissue regeneration are later replaced with extracellular matrix produced inside the body. Recent advances in additive manufacturing methods allow production of highly porous, complex three dimensional scaffolds suitable for cell growth and proliferation. The current paper investigates the mechanical properties, including elastic modulus and compressive strength, as well as fluid flow dynamics, including permeability and flow-induced shear stress of scaffolds with four triply periodic minimal surface (TPMS) configurations, namely the Schwarz primitive, the Schwarz diamond, the gyroid, and the Neovius structures. Higher porosity in all scaffold types resulted in lower mechanical properties. The permeability of the scaffolds was determined using Darcy's law with reference to geometrical parameters and the pressure drop derived from the computational fluid dynamics (CFD) analysis. Higher porosity enhanced permeability and reduced wall shear stress in all scaffold designs.

Keywords: highly porous scaffolds, tissue engineering, finite elements analysis, CFD analysis

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Authors: Sree Vennela P., V. Samyuktha Bhardwaj

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Hyponatremia is an electrolyte disturbance in which the sodium ion concentration in the serum is lower than normal. Sodium is the dominant extracellular cation (positive ion) and cannot freely cross from the interstitial space through the cell membrane, into the cell. Its homeostasis (stability of concentration) inside the cell is vital to the normal function of any cell. Normal serum sodium levels are between 135 and 145 mEq/L. Hyponatremia is defined as a serum level of less than 135 mEq/L and is considered severe when the serum level is below 125 mEq/L. In the vast majority of cases, Hyponatremia occurs as a result of excess body water diluting the serum sodium (salt level in the blood). Hyponatremia is often a complication of other medical illnesses in which excess water accumulates in the body at a higher rate than can be excreted (for example in congestive heart failure, syndrome of inappropriate antidiuretic hormone, SIADH, or polydipsia). Sometimes it may be a result of over-hydration (drinking too much water).Lack of sodium (salt) is very rarely the cause of Hyponatremia, although it can promote Hyponatremia indirectly. In particular, sodium loss can lead to a state of volume depletion (loss of blood volume in the body), with volume depletion serving as a signal for the release of ADH (anti-diuretic hormone). As a result of ADH-stimulated water retention (too much water in the body), blood sodium becomes diluted and Hyponatremia results.

Keywords: Tolvaptan, hyponatremia, syndrome of insufficient anti diuretic hormone (SIADH), euvolemic hyponatremia

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Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

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Authors: Mehak Munjal, Raj Kishore Sharma, Gurmeet Singh

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Microbial Fuel Cells (MFCs) are an alternative sustainable approach that utilize bacteria present in waste water as a bio-catalyst for the production of energy. It is a promising growing technology with minimal requirement for chemical supplements. Here electrode material plays a vital role in its performance. The present study represents CoFe2O4 spinel as a novel anode material in the MFC. It not only improve the bacterial metabolics but also enhance the power output. Generally, biocompatible conductive carbon paper/cloth, graphite and stainless steel are utilised as anode in MFCs. However, these materials lack electrochemical activity for anodic microbial reaction. Therefore, we developed CoFe2O4 on graphite sheet which enhanced the anodic charge transfer process. Redox pair in CoFe2O4 helped in improvement of extracellular electron transfer, thereby enhancing the performance. The physical characterizations (FT-IR, XRD, Raman) and electrochemical measurements demonstrate the strong interaction with E.coli bacteria and thus providing an excellent power density i.e. 1850 mW/m2 .The maximum anode half -cell potential is measured to be 0.65V. Therefore, use of noble metal free anodic material further decrease the cost and the long term cell stability makes it an effective material for practical applications.

Keywords: microbial fuel cell, cobalt ferrite, E. coli, bioelectricity

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Authors: Laura Ulitzky, Dougbeh-Chris Nyan, Manuel M. Lafer, Erica Silberstein, Nicoleta Cehan, Deborah R. Taylor

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Hepatitis C virus (HCV)-associated hepatocellular carcinoma, fibrosis and cirrhosis are more frequent in men and postmenopausal women than in premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol (estrogen) plays an innate role in preventing viral infection and liver disease. Estrogen classically acts through nuclear estrogen receptors or, alternatively, through the membrane-bound G-protein-coupled estrogen receptor (GPR30 or GPER). We observed a marked decrease in detectable virus when HCV-infected human hepatoma cells were treated with estrogen. The effect was mimicked by both Tamoxifen (Tam) and G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. Through GPR30, estrogen-mediated the down-regulation of occludin; a tight junction protein and HCV receptor, by promoting activation of matrix metalloproteinases (MMPs). Activated MMP-9 was secreted in response to estrogen, cleaving occludin in the extracellular Domain D, the motif required for HCV entry and spread. This pathway gives new insight into a novel innate immune pathway and the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.

Keywords: HCV, estrogen, occludin, MMPs

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Authors: B. Kodedova, E. Filova, M. Kralovic, E. Amler

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Vitiligo is the most common depigmentation disorder of the skin characterized by loss of melanocyte in the epidermis that leads to white lesions. One of the possible treatments is autologous transplantation of melanocytes. Biodegradable electrospun polymeric nanofibers provide good mechanical properties and could serve as suitable scaffold for epithelial cells cultivation and follow up transplantation. Moreover the microarchitecture of nanofibers mimics the structure of extracellular matrix and its porosity allows nutrients and waste exchange. The aim of this work was to develop biocompatible and biodegradable polymeric scaffolds suitable for autologous melanocytes transplantation. Electrospun polyvinyl alcohol (PVA) nanofibers were modified by cold methane plasma to lower their hydrofility and to achieve better adhesion, proliferation and viability of the murine melanocyte (Melan-a). Cells were seeded on the modified scaffolds and their adhesion, metabolic activity, proliferation and melanin synthesis was evaluated and compared to non-modified scaffolds. Results clearly indicate that cold methane plasma modified PVA nanofibers are suitable for melanocyte cultivation and may be future candidate for vitiligo treatment. Furthermore, the nanofibers can be functionalized with various bioactive substances, for enhancement of the melanocyte proliferation, melanogenesis or healing and regenerative processes. The project was supported by the Ministry of Education, Youth and Sports NPU I: LO1309 and by Grant Agency of Charles University (grant No. 1228214).

Keywords: melanocyte, nanofibers, polyvinyl alcohol, plasma modification

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