Search results for: metabolism

Authors: Antonela Matana, Dubravka Brdar, Vesela Torlak, Marijana Popovic, Ivana Gunjaca, Ozren Polasek, Vesna Boraska Perica, Maja Barbalic, Ante Punda, Caroline Hayward, Tatijana Zemunik

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Parathyroid hormone (PTH) plays a critical role in the regulation of bone mineral metabolism and calcium homeostasis. Higher PTH levels are associated with heart failure, hypertension, coronary artery disease, cardiovascular mortality and poorer bone health. A twin study estimated that 60% of the variation in PTH concentrations is genetically determined. Only one GWAS of PTH concentration has been reported to date. Identified loci explained 4.5% of the variance in circulating PTH, suggesting that additional genetic variants remain undiscovered. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels in a general population. We have performed a GWAS meta-analysis on 2596 individuals originating from three Croatian cohorts: City of Split and the Islands of Korčula and Vis, within a large-scale project of “10,001 Dalmatians”. A total of 7 411 206 variants, imputed using the 1000 Genomes reference panel, with minor allele frequency ≥ 1% and Rsq ≥ 0.5 were analyzed for the association. GWAS within each data set was performed under an additive model, controlling for age, gender and relatedness. Meta-analysis was conducted using the inverse-variance fixed-effects method. Furthermore, to identify sex-specific effects, we have conducted GWAS meta-analyses analyzing males and females separately. In addition, we have performed biological pathway analysis. Four SNPs, representing one locus, reached genome-wide significance. The most significant SNP was rs11099476 on chromosome 4 (P=1.15x10-8), which explained 1.14 % of the variance in PTH. The SNP is located near the protein-coding gene RASGEF1B. Additionally, we detected suggestive association with SNPs, rs77178854 located on chromosome 2 in the DPP10 gene (P=2.46x10-7) and rs481121 located on chromosome 1 (P=3.58x10-7) near the GRIK1 gene. One of the top hits detected in the main meta-analysis, intron variant rs77178854 located within DPP10 gene, reached genome-wide significance in females (P=2.21x10-9). No single locus was identified in the meta-analysis in males. Fifteen biological pathways were functionally enriched at a P<0.01, including muscle contraction, ion homeostasis and cardiac conduction as the most significant pathways. RASGEF1B is the guanine nucleotide exchange factor, known to be associated with height, bone density, and hip. DPP10 encodes a membrane protein that is a member of the serine proteases family, which binds specific voltage-gated potassium channels and alters their expression and biophysical properties. In conclusion, we identified 2 novel loci associated with PTH levels in a general population, providing us with further insights into the genetics of this complex trait.

Keywords: general population, genome-wide association analysis, parathyroid hormone, single nucleotide polymorphisms.

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Irina Deputat, Anatoly Gribanov, Yuliya Dzhos, Alexandra Nekhoroshkova, Tatyana Yemelianova, Irina Bolshevidtseva, Irina Deryabina, Yana Kereush, Larisa Startseva, Tatyana Bagretsova, Irina Ikonnikova

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In the modern world the number of elderly people increases. Preservation of functionality of an organism in the elderly becomes very important now. During aging the higher cortical functions such as feelings, perception, attention, memory, and ideation are gradual decrease. It is expressed in the rate of information processing reduction, volume of random access memory loss, ability to training and storing of new information decrease. Perspective directions in studying of aging neurophysiological parameters are brain imaging: computer electroencephalography, neuroenergy mapping of a brain, and also methods of studying of a neurodynamic brain processes. Research aim – to study features of a brain aging in elderly people by electroencephalogram (EEG) and the DC-potential level. We examined 130 people aged 55 - 74 years that did not have psychiatric disorders and chronic states in a decompensation stage. EEG was recorded with a 128-channel GES-300 system (USA). EEG recordings are collected while the participant sits at rest with their eyes closed for 3 minutes. For a quantitative assessment of EEG we used the spectral analysis. The range was analyzed on delta (0,5–3,5 Hz), a theta - (3,5–7,0 Hz), an alpha 1-(7,0–11,0 Hz) an alpha 2-(11–13,0 Hz), beta1-(13–16,5 Hz) and beta2-(16,5–20 Hz) ranges. In each frequency range spectral power was estimated. The 12-channel hardware-software diagnostic ‘Neuroenergometr-KM’ complex was applied for registration, processing and the analysis of a brain constant potentials level. The DC-potential level registered in monopolar leads. It is revealed that the EEG of elderly people differ in higher rates of spectral power in the range delta (р < 0,01) and a theta - (р < 0,05) rhythms, especially in frontal areas in aging. By results of the comparative analysis it is noted that elderly people 60-64 aged differ in higher values of spectral power alfa-2 range in the left frontal and central areas (р < 0,05) and also higher values beta-1 range in frontal and parieto-occipital areas (р < 0,05). Study of a brain constant potential level distribution revealed increase of total energy consumption on the main areas of a brain. In frontal leads we registered the lowest values of constant potential level. Perhaps it indicates decrease in an energy metabolism in this area and difficulties of executive functions. The comparative analysis of a potential difference on the main assignments testifies to unevenness of a lateralization of a brain functions at elderly people. The results of a potential difference between right and left hemispheres testify to prevalence of the left hemisphere activity. Thus, higher rates of functional activity of a cerebral cortex are peculiar to people of early advanced age (60-64 years) that points to higher reserve opportunities of central nervous system. By 70 years there are age changes of a cerebral power exchange and level of electrogenesis of a brain which reflect deterioration of a condition of homeostatic mechanisms of self-control and the program of processing of the perceptual data current flow.

Keywords: brain, DC-potential level, EEG, elderly people

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Authors: Faheema Khan

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To investigate the ability of sensitive and tolerant genotype of Glycine max to adapt to a saline environment in a field, we examined the growth performance, water relation and activities of antioxidant enzymes in relation to photosynthetic rate, chlorophyll a fluorescence, photosynthetic pigment concentration, protein and proline in plants exposed to salt stress. Ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) were selected and grown hydroponically. After 3 days of proper germination, the seedlings were transferred to Hoagland’s solution (Hoagland and Arnon 1950). The growth chamber was maintained at a photosynthetic photon flux density of 430 μmol m−2 s−1, 14 h of light, 10 h of dark and a relative humidity of 60%. The nutrient solution was bubbled with sterile air and changed on alternate days. Ten-day-old seedlings were given seven levels of salt in the form of NaCl viz., T1 = 0 mM NaCl, T2=25 mM NaCl, T3=50 mM NaCl, T4=75 mM NaCl, T5=100 mM NaCl, T6=125 mM NaCl, T7=150 mM NaCl. The investigation showed that genotype Pusa-24, PK-416 and Pusa-20 appeared to be the most salt-sensitive. genotypes as inferred from their significantly reduced length, fresh weight and dry weight in response to the NaCl exposure. Pusa-37 appeared to be the most tolerant genotype since no significant effect of NaCl treatment on growth was found. We observed a greater decline in the photosynthetic variables like photosynthetic rate, chlorophyll fluorescence and chlorophyll content, in salt-sensitive (Pusa-24) genotype than in salt-tolerant Pusa-37 under high salinity. Numerous primers were verified on ten soybean genotypes obtained from Operon technologies among which 30 RAPD primers shown high polymorphism and genetic variation. The Jaccard’s similarity coefficient values for each pairwise comparison between cultivars were calculated and similarity coefficient matrix was constructed. The closer varieties in the cluster behaved similar in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the 10 genotypes in sub-cluster as expected from their physiological findings.Salt tolerant genotype Pusa-37, was further analysed by 2-Dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the Present study, 173 protein spots were identified. Of these, 40 proteins responsive to salinity were either up- or down-regulated in Pusa-37. Proteomic analysis in salt-tolerant genotype (Pusa-37) led to the detection of proteins involved in a variety of biological processes, such as protein synthesis (12 %), redox regulation (19 %), primary and secondary metabolism (25 %), or disease- and defence-related processes (32 %). In conclusion, the soybean plants in our study responded to salt stress by changing their protein expression pattern. The photosynthetic, biochemical and molecular study showed that there is variability in salt tolerance behaviour in soybean genotypes. Pusa-24 is the salt-sensitive and Pusa-37 is the salt-tolerant genotype. Moreover this study gives new insights into the salt-stress response in soybean and demonstrates the power of genomic and proteomic approach in plant biology studies which finally could help us in identifying the possible regulatory switches (gene/s) controlling the salt tolerant genotype of the crop plants and their possible role in defence mechanism.

Keywords: glycine max, salt stress, RAPD, genomic and proteomic variability

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: Suikinai Nobre Santos, Ranko Gacesa, Paul F. Long, Itamar Soares de Melo

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Extremophilic microorganism are adapted to biotopes combining several stress factors (temperature, pressure, radiation, salinity and pH), which indicate the richness valuable resource for the exploitation of novel biotechnological processes and constitute unique models for investigations their biomolecules (1, 2). The above information encourages us investigate bioprospecting synthesized compounds by a noval actinomycete, designated thermotolerant Streptomyces caatingaensis CMAA 1322, isolated from sample soil tropical dry forest (Caatinga) in the Brazilian semiarid region (3-17°S and 35-45°W). This set of constrating physical and climatic factores provide the unique conditions and a diversity of well adapted species, interesting site for biotechnological purposes. Preliminary studies have shown the great potential in the production of cytotoxic, pesticidal and antimicrobial molecules (3). Thus, to extend knowledge of the genes clusters responsible for producing biosynthetic pathways of natural products in strain CMAA1322, whole-genome shotgun (WGS) DNA sequencing was performed using paired-end long sequencing with PacBio RS (Pacific Biosciences). Genomic DNA was extracted from a pure culture grown overnight on LB medium using the PureLink genomic DNA kit (Life Technologies). An approximately 3- to 20-kb-insert PacBio library was constructed and sequenced on an 8 single-molecule real-time (SMRT) cell, yielding 116,269 reads (average length, 7,446 bp), which were allocated into 18 contigs, with 142.11x coverage and N50 value of 20.548 bp (BioProject number PRJNA288757). The assembled data were analyzed by Rapid Annotations using Subsystems Technology (RAST) (4) the genome size was found to be 7.055.077 bp, comprising 6167 open reading frames (ORFs) and 413 subsystems. The G+C content was estimated to be 72 mol%. The closest-neighbors tool, available in RAST through functional comparison of the genome, revealed that strain CMAA1322 is more closely related to Streptomyces hygroscopicus ATCC 53653 (similarity score value, 537), S. violaceusniger Tu 4113 (score value, 483), S. avermitilis MA-4680 (score value, 475), S. albus J1074 (score value, 447). The Streptomyces sp. CMAA1322 genome contains 98 tRNA genes and 135 genes copies related to stress response, mainly osmotic stress (14), heat shock (16), oxidative stress (49). Functional annotation by antiSMASH version 3.0 (5) identified 41 clusters for secondary metabolites (including two clusters for lanthipeptides, ten clusters for nonribosomal peptide synthetases [NRPS], three clusters for siderophores, fourteen for polyketide synthetase [PKS], six clusters encoding a terpene, two clusters encoding a bacteriocin, and one cluster encoding a phenazine). Our work provide in comparative analyse of genome and extract produced (data no published) by lineage CMAA1322, revealing the potential of microorganisms accessed from extreme environments as Caatinga” to produce a wide range of biotechnological relevant compounds.

Keywords: caatinga, streptomyces, environmental stresses, biosynthetic pathways

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Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Imdad Ullah Khan, Yongseok Yoon, Kyeung Uk Choi, Kwang Rae Jo, Namyul Kim, Eunbee Lee, Wan Hee Kim, Oh-Kyeong Kweon

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The primary spinal cord injury (SCI) causes mechanical damage to the neurons and blood vessels. It leads to secondary SCI, which activates multiple pathological pathways, which expand neuronal damage at the injury site. It is characterized by vascular disruption, ischemia, excitotoxicity, oxidation, inflammation, and apoptotic cell death. It causes nerve demyelination and disruption of axons, which perpetuate a loss of impulse conduction through the injured spinal cord. It also leads to the production of myelin inhibitory molecules, which with a concomitant formation of an astroglial scar, impede axonal regeneration. The pivotal role regarding the neuronal necrosis is played by oxidation and inflammation. During an early stage of spinal cord injury, there occurs an abundant expression of reactive oxygen species (ROS) due to defective mitochondrial metabolism and abundant migration of phagocytes (macrophages, neutrophils). ROS cause lipid peroxidation of the cell membrane, and cell death. Abundant migration of neutrophils, macrophages, and lymphocytes collectively produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1beta (IL-1β), matrix metalloproteinase, superoxide dismutase, and myeloperoxidases which synergize neuronal apoptosis. Therefore, it is crucial to control inflammation and oxidation injury to minimize the nerve cell death during secondary spinal cord injury. Therefore, in response to oxidation and inflammation, heme oxygenase-1 (HO-1) is induced by the resident cells to ameliorate the milieu. In the meanwhile, neurotrophic factors are induced to promote neuroregeneration. However, it seems that anti-stress enzyme (HO-1) and neurotrophic factor (BDNF) do not significantly combat the pathological events during secondary spinal cord injury. Therefore, optimum healing can be induced if anti-inflammatory and neurotrophic factors are administered in a higher amount through an exogenous source. During the first experiment, the inflammation and neuroregeneration were selectively targeted. HO-1 expressing MSCs (HO-1 MSCs) and BDNF expressing MSCs (BDNF MSC) were co-transplanted in one group (combination group) of dogs with subacute spinal cord injury to selectively control the expression of inflammatory cytokines by HO-1 and induce neuroregeneration by BDNF. We compared the combination group with the HO-1 MSCs group, BDNF MSCs group, and GFP MSCs group. We found that the combination group showed significant improvement in functional recovery. It showed increased expression of neural markers and growth-associated proteins (GAP-43) than in other groups, which depicts enhanced neuroregeneration/neural sparing due to reduced expression of pro-inflammatory cytokines such as TNF-alpha, IL-6 and COX-2; and increased expression of anti-inflammatory markers such as IL-10 and HO-1. Histopathological study revealed reduced intra-parenchymal fibrosis in the injured spinal cord segment in the combination group than in other groups. Thus it was concluded that selectively targeting the inflammation and neuronal growth with the combined use of HO-1 MSCs and BDNF MSCs more favorably promote healing of the SCI. HO-1 MSCs play a role in controlling the inflammation, which favors the BDNF induced neuroregeneration at the injured spinal cord segment of dogs.

Keywords: HO-1 MSCs, BDNF MSCs, neuroregeneration, inflammation, anti-inflammation, spinal cord injury, dogs

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Authors: Kyriaki Hatziagapiou, Konstantinos Bethanis, Olti Nikola, Elias Christoforides, Eleni Koniari, Eleni Kakouri, George Lambrou, Christina Kanaka-Gantenbein

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Glioblastoma multiforme (GBM) remains a serious health challenge, as current therapeutic modalities continue to yield unsatisfactory results, with the average survival rarely exceeding 1-2 years. Natural compounds still provide some of the most promising approaches for discovering new drugs. The non-psychotropic cannabidiol (CBD) deriving from Cannabis sativa L. provides such promise. CBD is endowed with anticancer, antioxidant, and genoprotective properties as established in vitro and in in vivo experiments. CBD’s selectivity towards cancer cells and its safe profile suggest its usage in cancer therapies. However, the bioavailability of oral CBD is low due to poor aqueous solubility, erratic gastrointestinal absorption, and significant first-pass metabolism, hampering its therapeutic potential and resulting in a variable pharmacokinetic profile. In this context, CBD can take great advantage of nanomedicine-based formulation strategies. Cyclodextrins (CDs) are cyclic oligosaccharides used in the pharmaceutical industry to incorporate apolar molecules inside their hydrophobic cavity, increasing their stability, water solubility, and bioavailability or decreasing their side effects. CBD-inclusion complexes with CDs could be a good strategy to improve its properties, like solubility and stability to harness its full therapeutic potential. The current research aims to study the potential cytotoxic effect of CBD and CBD-CDs complexes CBD-RMβCD (randomly methylated β-cyclodextrin) and CBD-HPβCD (hydroxypropyl-b-CD) on the A172 glioblastoma cell line. CBD is diluted in 10% DMSO, and CBD/CDs solutions are prepared by mixing solid CBD, solid CDs, and dH2O. For the biological assays, A172 cells are incubated at a range of concentrations of CBD, CBD-RMβCD and CBD-HPβCD, RMβCD, and HPβCD (0,03125-4 mg/ml) at 24, 48, and 72 hours. Analysis of cell viability after incubation with the compounds is performed with Alamar Blue viability assay. CBD’s dilution to DMSO 10% was inadequate, as crystals are observed; thus cytotoxicity experiments are not assessed. CBD’s solubility is enhanced in the presence of both CDs. CBD/CDs exert significant cytotoxicity in a dose and time-dependent manner (p < 0.005 for exposed cells to any concentration at 48, 72, and 96 hours versus cells not exposed); as their concentration and time of exposure increases, the reduction of resazurin to resofurin decreases, indicating a reduction in cell viability. The cytotoxic effect is more pronounced in cells exposed to CBD-HPβCD for all concentrations and time-points. RMβCD and HPβCD at the highest concentration of 4 mg/ml also exerted antitumor action per se since manifesting cell growth inhibition. The results of our study could afford the basis of research regarding the use of natural products and their inclusion complexes as anticancer agents and the shift to targeted therapy with higher efficacy and limited toxicity. Acknowledgments: The research is partly funded by ΙΚΥ (State Scholarships Foundation) – Post-doc Scholarships-Partnership Agreement 2014-2020.

Keywords: cannabidiol, cyclodextrins, glioblastoma, hydroxypropyl-b-Cyclodextrin, randomly-methylated-β-cyclodextrin

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Authors: Hasmik V. Zanginyan, Gayane S. Ghazaryan, Laura M. Hovsepyan

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Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system that is induced in laboratory animals by developing an immune response against myelin epitopes. The typical clinical course is ascending palsy, which correlates with inflammation and tissue damage in the thoracolumbar spinal cord, although the optic nerves and brain (especially the subpial white matter and brainstem) are also often affected. With multiple sclerosis, there is a violation of lipid metabolism in myelin. When membrane lipids (glycosphingolipids, phospholipids) are disturbed, metabolites not only play a structural role in membranes but are also sources of secondary mediators that transmit multiple cellular signals. The purpose of this study was to investigate the effect of ganglioside as a therapeutic agent in experimental multiple sclerosis. The biological activity of a ganglioside-containing medicinal preparation (Cronassial) was evaluated in an experimental model of multiple sclerosis in laboratory animals. An experimental model of multiple sclerosis in rats was obtained by immunization with myelin basic protein (MBP), as well as homogenization of the spinal cord or brain. EAE was induced by administering a mixture of an encephalitogenic mixture (EGM) with Complete Freund’s Adjuvant. Mitochondrial fraction was isolated in a medium containing 0,25 M saccharose and 0, 01 M tris buffer, pH - 7,4, by a method of differential centrifugation on a K-24 centrifuge. Glutathione peroxidase activity was assessed by reduction reactions of hydrogen peroxide (H₂O₂) and lipid hydroperoxides (ROOH) in the presence of GSH. LPO activity was assessed by the amount of malondialdehyde (MDA) in the total homogenate and mitochondrial fraction of the spinal cord and brain of control and experimental autoimmune encephalomyelitis rats. MDA was assessed by a reaction with Thiobarbituric acid. For statistical data analysis on PNP, SPSS (Statistical Package for Social Science) package was used. The nature of the distribution of the obtained data was determined by the Kolmogorov-Smirnov criterion. The comparative analysis was performed using a nonparametric Mann-Whitney test. The differences were statistically significant when р ≤ 0,05 or р ≤ 0,01. Correlational analysis was conducted using a nonparametric Spearman test. In the work, refrigeratory centrifuge, spectrophotometer LKB Biochrom ULTROSPECII (Sweden), pH-meter PL-600 mrc (Israel), guanosine, and ATP (Sigma). The study of the process of lipid peroxidation in the total homogenate of the brain and spinal cord in experimental animals revealed an increase in the content of malonic dialdehyde. When applied, Cronassial observed normalization of lipid peroxidation processes. Reactive oxygen species, causing lipid peroxidation processes, can be toxic both for neurons and for oligodendrocytes that form myelin, causing a violation of their lipid composition. The high content of lipids in the brain and the uniqueness of their structure determines the nature of the development of LPO processes. The lipid layer of cellular and intracellular membranes performs two main functions -barrier and matrix (structural). Damage to the barrier leads to dysregulation of intracellular processes and severe disorders of cellular functions.

Keywords: experimental autoimmune encephalomyelitis, multiple sclerosis, neuroinflammation, therapy

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Authors: Priyanka Mokashi, Aparna Khanna, Nancy Pandita

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Diabetes mellitus is a chronic metabolic disorder which will be the 7th leading cause of death by 2030. The current line of treatment for the diabetes mellitus is oral antidiabetic drugs (biguanides, sulfonylureas, meglitinides, thiazolidinediones and alpha-glycosidase inhibitors) and insulin therapy depending upon the type 1 or type 2 diabetes mellitus. But, these treatments have their disadvantages, ranging from the developing of resistance to the drugs and adverse effects caused by them. Alternative to these synthetic agents, natural products provides a new insight for the development of more efficient and safe drugs due to their therapeutic values. Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. It is widely distributed in Asia, Africa, and South America. There are few reports on Swrtiamarin, major component of this plant for its antidiabetic activity. However, the antidiabetic activity of flavonoids from E. littorale and their mechanism of action have not yet been elucidated. Flavonoids have a positive relationship with disease prevention and can act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, adipocytes, hepatocytes and skeletal myofibers. They may exert beneficial effects in diabetes by (i) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (ii) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (iii) increasing glucose uptake in hepatocytes, skeletal muscle and white adipose tissue (iv) reducing insulin resistance, inflammation and oxidative stress. Therefore, we have isolated four flavonoid rich fractions, Fraction A (FA), Fraction B (FB), Fraction C (FC), Fraction D (FD) from crude alcoholic hot (AH) extract from E. littorale, identified by LC/MS. Total eight flavonoids were identified on the basis of fragmentation pattern. Flavonoid FA showed the presence of swertisin, isovitexin, and saponarin; FB showed genkwanin, quercetin, isovitexin, FC showed apigenin, swertisin, quercetin, 5-O-glucosylswertisin and 5-O-glucosylisoswertisin whereas FD showed the presence of swertisin. Further, these fractions were assessed for their antidiabetic activity on stimulating glucose uptake in insulin-resistant HepG2 cell line model (IR/HepG2). The results showed that FD containing C-glycoside Swertisin has significantly increased the glucose uptake rate of IR/HepG2 cells at the concentration of 10 µg/ml as compared to positive control Metformin (0.5mM) which was determined by glucose oxidase- peroxidase method. It has been reported that enhancement of glucose uptake of cells occurs due the translocation of Glut4 vesicles to cell membrane through IR/IRS1/AKT pathway. Therefore, we have studied expressions of three genes IRS1, AKT and Glut4 by real-time PCR to evaluate whether they follow the same pathway or not. It was seen that the glucose uptake rate has increased in FD treated IR/HepG2 cells due to the activation of insulin receptor substrate-1 (IRS1) followed by protein kinase B (AKT) through phosphoinositide 3-kinase (PI3K) leading to translocation of Glut 4 vesicles to cell membrane, thereby enhancing glucose uptake and insulin sensitivity of insulin resistant HepG2 cells. Hence, the up-regulation indicated the mechanism of action through which FD (Swertisin) acts as antidiabetic candidate in the treatment of type 2 diabetes mellitus.

Keywords: E. littorale, glucose transporter, glucose uptake rate, insulin resistance

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Authors: Niharika Kaushal, Minni Singh

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Citrus fruits fall into the category of those commercially grown fruits that constitute an excellent repository of phytochemicals with health-promoting properties. Fruits belonging to the citrus family, when processed by industries, produce tons of agriculture by-products in the form of peels, pulp, and seeds, which normally have no further usage and are commonly discarded. In spite of this, such residues are of paramount importance due to their richness in valuable compounds; therefore, agro-waste is considered a valuable bioresource for various purposes in the food sector. A range of biological properties, including anti-oxidative, anti-cancerous, anti-inflammatory, anti-allergenicity, and anti-aging activity, have been reported for these bioactive compounds. Taking advantage of these inexpensive residual sources requires special attention to extract bioactive compounds. Mandarin (Citrus nobilis X Citrus deliciosa) is a potential source of bioflavonoids with antioxidant properties, and it is increasingly regarded as a functional food. Despite these benefits, flavonoids suffer from a barrier of pre-systemic metabolism in gastric fluid, which impedes their effectiveness. Therefore, colloidal delivery systems can completely overcome the barrier in question. This study involved the extraction and identification of key flavonoids from mandarin biomass. Using a green chemistry approach, supercritical fluid extraction at 330 bar, temperature 40C, and co-solvent 10% ethanol was employed for extraction, and the identification of flavonoids was made by mass spectrometry. As flavonoids are concerned with a limitation, the obtained extract was encapsulated in polylactic-co-glycolic acid (PLGA) matrix using a solvent evaporation method. Additionally, the antioxidant potential was evaluated by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. A release pattern of flavonoids was observed over time using simulated gastrointestinal fluids. From the results, it was observed that the total flavonoids extracted from the mandarin biomass were estimated to be 47.3 ±1.06 mg/ml rutin equivalents as total flavonoids. In the extract, significantly, polymethoxyflavones (PMFs), tangeretin and nobiletin were identified, followed by hesperetin and naringin. The designed flavonoid-PLGA nanoparticles exhibited a particle size between 200-250nm. In addition, the bioengineered nanoparticles had a high entrapment efficiency of nearly 80.0% and maintained stability for more than a year. Flavonoid nanoparticles showed excellent antioxidant activity with an IC50 of 0.55μg/ml. Morphological studies revealed the smooth and spherical shape of nanoparticles as visualized by Field emission scanning electron microscopy (FE-SEM). Simulated gastrointestinal studies of free extract and nanoencapsulation revealed the degradation of nearly half of the flavonoids under harsh acidic conditions in the case of free extract. After encapsulation, flavonoids exhibited sustained release properties, suggesting that polymeric encapsulates are efficient carriers of flavonoids. Thus, such technology-driven and biomass-derived products form the basis for their use in the development of functional foods with improved therapeutic potential and antioxidant properties. As a result, citrus processing waste can be considered a new resource that has high value and can be used for promoting its utilization.

Keywords: citrus, agrowaste, flavonoids, nanoparticles

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Authors: Khiem M. Nguyen, Ming C. Yang

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Chloroplast pigments are extremely important during photosynthesis since they play essential roles in light absorption and energy transfer. Therefore, understanding the efficiency of chlorophyll (Chl) biosynthesis could facilitate enhancement in photo-assimilates accumulation, and ultimately, in crop yield. The Chl-deficient mutants have been used extensively to study the Chl biosynthetic pathways and the biogenesis of the photosynthetic apparatus. Rice (Oryza sativa L.) is one of the most leading food crops, serving as staple food for many parts of the world. To author’s best knowledge, Chl b–lacking rice has been found; however the molecular mechanism of Chl biosynthesis still remains unclear compared to wild-type rice. In this study, the ultrastructure analysis, photosynthetic properties, and transcriptome profile of wild-type rice (Norin No.8, N8) and its Chl b-lacking mutant (Chlorina 1, C1) were examined. The finding concluded that total Chl content and Chl b content in the C1 leaves were strongly reduced compared to N8 leaves, suggesting that reduction in the total Chl content contributes to leaf color variation at the physiological level. Plastid ultrastructure of C1 possessed abnormal thylakoid membranes with loss of starch granule, large number of vesicles, and numerous plastoglobuli. The C1 rice also exhibited thinner stacked grana, which was caused by a reduction in the number of thylakoid membranes per granum. Thus, the different Chl a/b ratio of C1 may reflect the abnormal plastid development and function. Transcriptional analysis identified 23 differentially expressed genes (DEGs) and 671 transcription factors (TFs) that were involved in Chl metabolism, chloroplast development, cell division, and photosynthesis. The transcriptome profile and DEGs revealed that the gene encoding PsbR (PSII core protein) was down-regulated, therefore suggesting that the lower in light-harvesting complex proteins are responsible for the lower photosynthetic capacity in C1. In addition, expression level of cell division protein (FtsZ) genes were significantly reduced in C1, causing chloroplast division defect. A total of 19 DEGs were identified based on KEGG pathway assignment involving Chl biosynthesis pathway. Among these DEGs, the GluTR gene was down-regulated, whereas the UROD, CPOX, and MgCH genes were up-regulated. Observation through qPCR suggested that later stages of Chl biosynthesis were enhanced in C1, whereas the early stages were inhibited. Plastid structure analysis together with transcriptomic analysis suggested that the Chl a/b ratio was amplified both by the reduction in Chl contents accumulation, owning to abnormal chloroplast development, and by the enhanced conversion of Chl b to Chl a. Moreover, the results indicated the same Chl-cycle pattern in the wild-type and C1 rice, indicating another Chl b degradation pathway. Furthermore, the results demonstrated that normal grana stacking, along with the absence of Chl b and greatly reduced levels of Chl a in C1, provide evidence to support the conclusion that other factors along with LHCII proteins are involved in grana stacking. The findings of this study provide insight into the molecular mechanisms that underlie different Chl a/b ratios in rice.

Keywords: Chl-deficient mutant, grana stacked, photosynthesis, RNA-Seq, transcriptomic analysis

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Authors: E. Locatelli, I. Monaco, R. C. Martin, Y. Li, R. Pini, M. Chiariello, M. Comes Franchini

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Despite the many advantages of application of nanomaterials in the field of nanomedicine, increasing concerns have been expressed on their potential adverse effects on human health. There is urgency for novel green strategies toward novel materials with enhanced biocompatibility using safe reagents. Photothermal ablation therapy, which exploits localized heat increase of a few degrees to kill cancer cells, has appeared recently as a non-invasive and highly efficient therapy against various cancer types; anyway new agents able to generate hyperthermia when irradiated are needed and must have precise biocompatibility in order to avoid damage to healthy tissues and prevent toxicity. Recently, there has been increasing interest in magnesium as a biomaterial: it is the fourth most abundant cation in the human body, and it is essential for human metabolism. However magnesium nanoparticles (Mg NPs) have had limited diffusion due to the high reduction potential of magnesium cations, which makes NPs synthesis challenging. Herein, we report the synthesis of Mg NPs and their surface functionalization for the obtainment of a stable and biocompatible nanomaterial suitable for photothermal ablation therapy against cancer. We synthesized the Mg crystals by reducing MgCl2 with metallic lithium and exploiting naphthalene as an electron carrier: the lithium–naphthalene complex acts as the real reducing agent. Firstly, the nanocrystal particles were coated with the ligand 12-ethoxy ester dodecanehydroxamic acid, and then entrapped into water-dispersible polymeric micelles (PMs) made of the FDA-approved PLGA-b-PEG-COOH copolymer using the oil-in-water emulsion technique. Lately, we developed a more straightforward methodology by introducing chitosan, a highly biocompatible natural product, at the beginning of the process, simultaneously using lithium–naphthalene complex, thus having a one-pot procedure for the formation and surface modification of MgNPs. The obtained MgNPs were purified and fully characterized, showing diameters in the range of 50-300 nm. Notably, when coated with chitosan the particles remained stable as dry powder for more than 10 months. We proved the possibility of generating a temperature rise of a few to several degrees once MgNPs were illuminated using a 810 nm diode laser operating in continuous wave mode: the temperature rise resulted significant (0-15 °C) and concentration dependent. We then investigated potential cytotoxicity of the MgNPs: we used HN13 epithelial cells, derived from a head and neck squamous cell carcinoma and the hepa1-6 cell line, derived from hepatocellular carcinoma and very low toxicity was observed for both nanosystems. Finally, in vivo photothermal therapy was performed on xenograft hepa1-6 tumor bearing mice: the animals were treated with MgNPs coated with chitosan and showed no sign of suffering after the injection. After 12 hours the tumor was exposed to near-infrared laser light. The results clearly showed an extensive damage to tumor tissue after only 2 minutes of laser irradiation at 3Wcm-1, while no damage was reported when the tumor was treated with the laser and saline alone in control group. Despite the lower photothermal efficiency of Mg with respect to Au NPs, we consider MgNPs a promising, safe and green candidate for future clinical translations.

Keywords: chitosan, magnesium nanoparticles, nanomedicine, photothermal therapy

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Authors: Patricia Shirley Almeida Prado, Liz Brito, Selma Paixão Argollo, Gracie Moreira, Leticia Matos Sobrinho

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Diffuse idiopathic skeletal hyperostosis (DISH) is a degenerative bone disease also known as Forestier´s disease and ankylosing hyperostosis of the spine is characterized by a tendency toward ossification of half the anterior longitudinal spinal ligament without intervertebral disc disease. DISH is not considered to be osteoarthritis, although the two conditions commonly occur together. Diagnostic criteria include fusion of at least four vertebrae by bony bridges arising from the anterolateral aspect of the vertebral bodies. These vertebral bodies have a 'dripping candle wax' appearance, also can be seen periosteal new bone formation on the anterior surface of the vertebral bodies and there is no ankylosis at zygoapophyseal facet joint. Clinically, patients with DISH tend to be asymptomatic some patients mention moderate pain and stiffness in upper back. This disease is more common in man, uncommon in patients younger than 50 years and rare in patients under 40 years old. In modern populations, DISH is found in association with obesity, (type II) diabetes; abnormal vitamin A metabolism and also associated with higher levels of serum uric acid. There is also some association between the increase of risk of stroke or other cerebrovascular disease. The DISH condition can be confused with Heterotopic Ossification, what is the bone formation in the soft tissues as the result of trauma, wounding, surgery, burnings, prolonged immobility and some central nervous system disorder. All these conditions have been described extensively as myositis ossificans which can be confused with the fibrodysplasia (myositis) ossificans progressive. As in the DISH symptomatology it can be asymptomatic or extensive enough to impair joint function. A third confusion osteoarthritis disease that can bring confusion are the enthesopathies that occur in the entire skeleton being common on the ischial tuberosities, iliac crests, patellae, and calcaneus. Ankylosis of the sacroiliac joint by bony bridges may also be found. CASE 1: this case is skeletal remains presenting skull, some vertebrae and scapulae. This case remains unidentified and due to lack of bone remains. Sex, age and ancestry profile was compromised, however the DISH pathognomonic findings and diagnostic helps to estimate sex and age characteristics. Moreover to presenting DISH these skeletal remains also showed some bone alterations and non-metrics as fusion of the first vertebrae with occipital bone, maxillae and palatine torus and scapular foramen on the right scapulae. CASE 2: this skeleton remains shows an extensive bone heterotopic ossification on the great trochanter area of left femur, right fibula showed a healed fracture in its body however in its inteosseous crest there is an extensive bone growth, also in the Ilium at the region of inferior gluteal line can be observed some pronounced bone growth and the skull presented a pronounced mandibular, maxillary and palatine torus. Despite all these pronounced heterotopic ossification the whole skeleton presents moderate bone overgrowth that is not linked with aging, since the skeleton belongs to a young unidentified individual. The appropriate osteopathological diagnosis support the human identification process through medical reports and also assist with epidemiological data that can strengthen vulnerable anthropological estimates.

Keywords: bone disease, DISH, human identification, human remains

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

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Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

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Authors: Rajeswari Raguraman, Sowmya Parameswaran, Krishnakumar Subramanian, Jagat Kanwar, Rupinder Kanwar

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Background: Tumor microenvironment has been implicated in several cancers to regulate cell growth, invasion and metastasis culminating in outcome of therapy. Tumor stroma consists of multiple cell types that are in constant cross-talk with the tumor cells to favour a pro-tumorigenic environment. Not much is known about the existence of tumor microenvironment in the pediatric intraocular malignancy, Retinoblastoma (RB). In the present study, we aim to understand the multiple stromal cellular subtypes and tumor stromal interactions expressed in RB tumors. Materials and Methods: Immunohistochemistry for stromal cell markers CD31, CD68, alpha-smooth muscle (α-SMA), vimentin and glial fibrillary acidic protein (GFAP) was performed on formalin fixed paraffin embedded tissues sections of RB (n=12). The differential expression of stromal target molecules; fibroblast activation protein (FAP), tenascin-C (TNC), osteopontin (SPP1), bone marrow stromal antigen 2 (BST2), stromal derived factor 2 and 4 (SDF2 and SDF4) in primary RB tumors (n=20) and normal retina (n=5) was studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. The differential expression was correlated with the histopathological features of RB. The interaction between RB cell lines (Weri-Rb-1, NCC-RbC-51) and Bone marrow stromal cells (BMSC) was also studied using direct co-culture and indirect co-culture methods. The functional effect of the co-culture methods on the RB cells was evaluated by invasion and proliferation assays. Global gene expression was studied by using Affymetrix 3’ IVT microarray. Pathway prediction was performed using KEGG and the key molecules were validated using qRT-PCR. Results: The immunohistochemistry revealed the presence of several stromal cell types such as endothelial cells (CD31+;Vim+/-); macrophages (CD68+;Vim+/-); Fibroblasts (Vim+; CD31-;CD68- );myofibroblasts (α-SMA+/ Vim+) and invading retinal astrocytes/ differentiated retinal glia (GFAP+; Vim+). A characteristic distribution of these stromal cell types was observed in the tumor microenvironment, with endothelial cells predominantly seen in blood vessels and macrophages near actively proliferating tumor or necrotic areas. Retinal astrocytes and glia were predominant near the optic nerve regions in invasive tumors with sparse distribution in tumor foci. Fibroblasts were widely distributed with rare evidence of myofibroblasts in the tumor. Both gene and protein expression revealed statistically significant (P<0.05) up-regulation of FAP, TNC and BST2 in primary RB tumors compared to the normal retina. Co-culture of BMSC with RB cells promoted invasion and proliferation of RB cells in direct and indirect contact methods respectively. Direct co-culture of RB cell lines with BMSC resulted in gene expression changes in ECM-receptor interaction, focal adhesion, IL-8 and TGF-β signaling pathways associated with cancer. In contrast, various metabolic pathways such a glucose, fructose and amino acid metabolism were significantly altered under the indirect co-culture condition. Conclusion: The study suggests that the close interaction between RB cells and the stroma might be involved in RB tumor invasion and progression which is likely to be mediated by ECM-receptor interactions and secretory factors. Targeting the tumor stroma would be an attractive option for redesigning treatment strategies for RB.

Keywords: gene expression profiles, retinoblastoma, stromal cells, tumor microenvironment

Procedia PDF Downloads 354

Authors: Piacentini Diego, Della Rovere Federica, Fattorini Laura, Lanni Francesca, Cittadini Martina, Altamura Maria Maddalena, Falasca Giuseppina

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Plants have evolved sophisticated mechanisms to cope with environmental cues. Changes in intracellular content and distribution of phytohormones, such as the auxin indole-3-acetic acid (IAA), have been involved in morphogenic adaptation to environmental stresses. In addition to phytohormones, plants can rely on a plethora of small signal molecules able to promptly sense and transduce the stress signals, resulting in morpho/physiological responses thanks also to their capacity to modulate the levels/distribution/reception of most hormones. Among these signaling molecules, nitrogen monoxide (nitric oxide – NO) is a critical component in several plant acclimation strategies to both biotic and abiotic stresses. Depending on its levels, NO increases plant adaptation by enhancing the enzymatic or non-enzymatic antioxidant systems or by acting as a direct scavenger of reactive oxygen/nitrogen (ROS/RNS) species produced during the stress. In addition, exogenous applications of NO-specific donor compounds showed the involvement of the signal molecule in auxin metabolism, transport, and signaling, under both physiological and stress conditions. However, the complex mechanisms underlying NO action in interacting with phytohormones, such as auxins, during metal stress responses are still poorly understood and need to be better investigated. Emphasis must be placed on the response of the root system since it is the first plant organ system to be exposed to metal soil pollution. The monocot Oryza sativa L. (rice) has been chosen given its importance as a stable food for some 4 billion people worldwide. In addition, increasing evidence has shown that rice is often grown in contaminated paddy soils with high levels of heavy metal cadmium (Cd) and metalloid arsenic (As). The facility through which these metals are taken up by rice roots and transported to the aerial organs up to the edible caryopses makes rice one of the most relevant sources of these pollutants for humans. This study aimed to evaluate if NO has a mitigatory activity in the roots of rice seedlings against Cd or As toxicity and to understand if this activity requires interactions with auxin. Our results show that exogenous treatments with the NO-donor SNP alleviate the stress induced by Cd, but not by As, in in-vitro-grown rice seedlings through increased intracellular root NO levels. The damages induced by the pollutants include root growth inhibition, root histological alterations and ROS (H2O2, O2●ˉ), and RNS (ONOOˉ) production. Also, SNP treatments mitigate both the root increase in root IAA levels and the IAA alteration in distribution monitored by the OsDR5::GUS system due to the toxic metal exposure. Notably, the SNP-induced mitigation of the IAA homeostasis altered by the pollutants does not involve changes in the expression of OsYUCCA1 and ASA2 IAA-biosynthetic genes. Taken together, the results highlight a mitigating role of NO in the rice root system, which is pollutant-specific, and involves the interaction of the signal molecule with both IAA and brassinosteroids at different (i.e., transport, levels, distribution) and multiple levels (i.e., transcriptional/post-translational levels). The research is supported by Progetti Ateneo Sapienza University of Rome, grant number: RG120172B773D1FF

Keywords: arsenic, auxin, cadmium, nitric oxide, rice, root system

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Authors: Dilek Ozturk, Narin Ozturk, Zeliha Pala Kara, Engin Kaptan, Serap Sancar Bas, Nurten Ozsoy, Alper Okyar

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Most physiological processes oscillate in a rhythmic manner in mammals including metabolism and energy homeostasis, locomotor activity, hormone secretion, immune and endocrine system functions. Endocrine body rhythms are tightly regulated by the circadian timing system. The hypothalamic-pituitary-thyroid (HPT) axis is under circadian control at multiple levels from hypothalamus to thyroid gland. Since circadian timing system controls a variety of biological functions in mammals, circadian rhythms of biological functions may modify the drug tolerability/toxicity depending on the dosing time. Selective mTOR (mammalian target of rapamycin) inhibitor everolimus is an immunosuppressant and anticancer agent that is active against many cancers. It was also found to be active in medullary thyroid cancer. The aim of this study was to investigate the dosing time-dependent toxicity of everolimus on the thyroid gland and hormones in mice. Healthy C57BL/6J mice were synchronized with 12h:12h Light-Dark cycle (LD12:12, with Zeitgeber Time 0 – ZT0 – corresponding to Light onset). Everolimus was administered to male (5 mg/kg/day) and female mice (15 mg/kg/day) orally at ZT1-rest period- and ZT13-activity period- for 4 weeks; body weight loss, clinical signs and possible changes in serum thyroid hormone levels (TSH and free T4) were examined. Histological alterations in the thyroid gland were evaluated according to the following criteria: follicular size, colloid density and viscidity, height of the follicular epithelium and the presence of necrotic cells. The statistical significance between differences was analyzed with ANOVA. Study findings included everolimus-related diarrhea, decreased activity, decreased body weight gains, alterations in serum TSH levels, and histopathological changes in thyroid gland. Decreases in mean body weight gains were more evident in mice treated at ZT1 as compared to ZT13 (p < 0.001, for both sexes). Control tissue sections of thyroid glands exhibited well-organized histoarchitecture when compared to everolimus-treated groups. Everolimus caused histopathological alterations in thyroid glands in male (5 mg/kg, slightly) and female mice (15 mg/kg; p < 0.01 for both ZT as compared to their controls) irrespective of dosing-time. TSH levels were slightly decreased upon everolimus treatment at ZT13 in both males and females. Conversely, increases in TSH levels were observed when everolimus treated at ZT1 in both males (5 mg/kg; p < 0.05) and females (15 mg/kg; slightly). No statistically significant alterations in serum free T4 levels were observed. TSH and free T4 is clinically important thyroid hormones since a number of disease states have been linked to alterations in these hormones. Serum free T4 levels within the normal ranges in the presence of abnormal serum TSH levels in everolimus treated mice may suggest subclinical thyroid disease which may have repercussions on the cardiovascular system, as well as on other organs and systems. Our study has revealed the histological damage on thyroid gland induced by subacute everolimus administration, this effect was irrespective of dosing time. However, based on the body weight changes and clinical signs upon everolimus treatment, tolerability for the drug was best following dosing at ZT13 in both male and females. Yet, effects of everolimus on thyroid functions may deserve further studies regarding their clinical importance and chronotoxicity.

Keywords: circadian rhythm, chronotoxicity, everolimus, thyroid gland, thyroid hormones

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Authors: Narin Ozturk, Xiao-Mei Li, Sylvie Giachetti, Francis Levi, Alper Okyar

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P-glycoprotein (P-gp, MDR1, ABCB1) is a transmembrane protein acting as an ATP-dependent efflux pump and functions as a biological barrier by extruding drugs and xenobiotics out of cells in healthy tissues especially in intestines, liver and brain as well as in tumor cells. The circadian timing system controls a variety of biological functions in mammals including xenobiotic metabolism and detoxification, proliferation and cell cycle events, and may affect pharmacokinetics, toxicity and efficacy of drugs. Selective mTOR (mammalian target of rapamycin) inhibitor everolimus is an immunosuppressant and anticancer drug that is active against many cancers, and its pharmacokinetics depend on P-gp. The aim of this study was to investigate the dosing time-dependent toxicity of everolimus with respect to the intestinal P-gp expression rhythms in mdr1a::Luc mice using Real Time-Biolumicorder (RT-BIO) System. Mdr1a::Luc male mice were synchronized with 12 h of Light and 12 h of Dark (LD12:12, with Zeitgeber Time 0 – ZT0 – corresponding Light onset). After 1-week baseline recordings, everolimus (5 mg/kg/day x 14 days) was administered orally at ZT1-resting period- and ZT13-activity period- to mdr1a::Luc mice singly housed in an innovative monitoring device, Real Time-Biolumicorder units which let us monitor real-time and long-term gene expression in freely moving mice. D-luciferin (1.5 mg/mL) was dissolved in drinking water. Mouse intestinal mdr1a::Luc oscillation profile reflecting P-gp gene expression and locomotor activity pattern were recorded every minute with the photomultiplier tube and infrared sensor respectively. General behavior and clinical signs were monitored, and body weight was measured every day as an index of toxicity. Drug-induced body weight change was expressed relative to body weight on the initial treatment day. Statistical significance of differences between groups was validated with ANOVA. Circadian rhythms were validated with Cosinor Analysis. Everolimus toxicity changed as a function of drug timing, which was least following dosing at ZT13, near the onset of the activity span in male mice. Mean body weight loss was nearly twice as large in mice treated with 5 mg/kg everolimus at ZT1 as compared to ZT13 (8.9% vs. 5.4%; ANOVA, p < 0.001). Based on the body weight loss and clinical signs upon everolimus treatment, tolerability for the drug was best following dosing at ZT13. Both rest-activity and mdr1a::Luc expression displayed stable 24-h periodic rhythms before everolimus and in both vehicle-treated controls. Real-time bioluminescence pattern of mdr1a revealed a circadian rhythm with a 24-h period with an acrophase at ZT16 (Cosinor, p < 0.001). Mdr1a expression remained rhythmic in everolimus-treated mice, whereas down-regulation was observed in P-gp expression in 2 of 4 mice. The study identified the circadian pattern of intestinal P-gp expression with an unprecedented precision. The circadian timing depending on the P-gp expression rhythms may play a crucial role in the tolerability/toxicity of everolimus. The circadian changes in mdr1a genes deserve further studies regarding their relevance for in vitro and in vivo chronotolerance of mdr1a-transported anticancer drugs. Chronotherapy with P-gp-effluxed anticancer drugs could then be applied according to their rhythmic patterns in host and tumor to jointly maximize treatment efficacy and minimize toxicity.

Keywords: circadian rhythm, chronotoxicity, everolimus, mdr1a::Luc mice, p-glycoprotein

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Authors: Marcela Parra-Vargas, Ana Sandoval-Rodriguez, Roberto Rodriguez-Echevarria, Jose Dominguez-Rosales, Juan Armendariz-Borunda

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Non-alcoholic fatty liver disease (NAFLD) is characterized by an excess of hepatic lipids, and it is to author’s best knowledge, the most prevalent chronic liver disorder. Anthocyanin-rich food consumption is linked to health benefits in metabolic disorders associated with obesity and NAFLD, although the precise functional role of anthocyanidin delphinidin (Dp) has yet to be established. The aim of this study was to investigate the effect of the Dp in NAFLD metabolic alterations by evaluating prevention or amelioration of hepatic lipid accumulation, as well as molecular mechanisms in two experimental obesity-related models of NALFD. In vitro: HepG2 cells were incubated with sodium palmitate (PA, 1 mM) to induce lipotoxic damage, and concomitantly treated with Dp (180 uM) for 24 h. Subsequently, total lipid accumulation was measured by colorimetric staining with Oil Red O, and total intrahepatic triglycerides were determined by an enzymatic assay. To assess molecular mechanisms, cells were pre-treated with PA for 24 h and then exposed to Dp for 1 h. In vivo: four-week-old male C57BL/6Nhsd mice were allocated in two main groups. Mice were fed with standard diet (control) or high-fat and high-carbohydrate diet (45% fat, HFD) for 16 wk to induce NAFLD. Then HFD was divided into subgroups: one treated orally with Dp (15 mg/kg bw, HFD-Dp) every day for 4 wk, while HFD group treated with vehicle (DMSO). Weight and fasting glucose were recorded weekly, while dietary ingestion was measured daily. Insulin tolerance test was performed at the end of treatment. Liver histology was evaluated with H&E and Masson’s trichrome stain. RT-PCR was used to evaluate gene expression and Western Blot to determine levels of protein in both experimental models. Parametric data were analyzed with one-way ANOVA and Tukey’s post-hoc test. Kruskal-Wallis and Mann-Whitney U test for non-parametric data, and P < 0.5 were considered significant. Dp prevented hepatic lipid accumulation by PA in HepG2 hepatocytes. Furthermore, Dp down-regulated gene expression of SREBP1c, FAS, and CPT1a without modifying AMPK phosphorylation levels. In vivo, Dp oral administration did not ameliorate lipid metabolic alterations raised by HFD. Adiposity, dietary ingestion, fasting glucose, and insulin sensitivity after Dp treatment remained similar to HFD group. Histological analysis showed hepatic damage in HFD groups and no differences between HFD and HFD-Dp groups were found. Hepatic gene expression of ACC and FAS were not altered by HFD. SREBP1c was similar in both HFD and HFD-Dp groups. No significant changes were observed in SREBP1c, ACC, and FAS adipose tissue gene expression by HFD or Dp treatment. Additionally, immunoblotting analysis revealed no changes in pathway SIRT1-LKB-AMPK and PPAR alpha by both HFD groups compared to control. In conclusion, the antioxidant Dp may provoke beneficial effects in the prevention of hepatic lipid accumulation. Nevertheless, the oral dose administrated in mice that simulated the total intake of anthocyanins consumed daily by humans has no effect as a treatment on hepatic lipid metabolic alterations and histological abnormalities associated with exposure to chronic HFD. A healthy lifestyle with regular intake of antioxidants such as anthocyanins may prevent metabolic alterations in NAFLD.

Keywords: anthocyanins, antioxidants, delphinidin, non-alcoholic fatty liver disease, obesity

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Authors: Kuladip Jana

Abstract:

Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

Abstract:

Cardiac hypertrophy arises in response to persistent increases in hemodynamic loads. In comparison, diabetic cardiomyopathy is defined by an abnormal myocardial changes without other cardiac-related risk factors. Pathological cardiac hypertrophy and myocardial remodeling are hallmarks of cardiovascular diseases and are risk factors for heart failure. The transcription factor forkhead box class O (FOXOs) can protect heart tissue by hostile oxidative stress and stimulating apoptosis and autophagy. FOXO proteins, as sensitive elements and mediators in response to environmental changes, have been revealed to prevent and inverse cardiac hypertrophy. FOXOs are inhibited by insulin and are critical mediators of insulin action. Insulin deficiency and uncontrolled diabetes lead to a catabolic state. FOXO1 acts downstream of the insulin-dependent pathways, which are dysregulated in diabetes. It regulates cardiomyocyte hypertrophy downstream of IGF1R/PI3K/Akt activation, which are critical regulators of cardiac hypertrophy. The complex network of signaling pathways comprising insulin/IGF-1 signaling, AMPK, JNK, and Sirtuins regulate the development of cardiovascular dysfunction by modulating the activity of FOXOs. Insulin receptors and IGF1R act via the PI3k/Akt and the MAPK/ERK pathways. Activation of Akt in response to insulin or IGF-1 induces phosphorylation of FOXOs. Increased protein synthesis induced by activation of the IGF-I/Akt/mTOR signaling pathway leads to hypertrophy. This pathway and the myostatin/Smad pathway are potent negative muscle development regulators. In cardiac muscle, insulin receptor substrates (IRS)-1 or IRS-2 activates the Akt signaling pathway and inactivate FOXO1. Under metabolic stress, p38 MAPK promotes degradation of IRS-1 and IRS-2 in cardiac myocytes and activates FOXO1, leading to cardiomyopathy. Sirt1 and FOXO1 interaction play an essential role in starvation-induced autophagy in cardiac metabolism. Inhibition of Angiotensin-II induced cardiomyocyte hypertrophy is associated with reduced FOXO1 acetylation and activation of Sirt1. The NF-κB, ERK, and FOXOs are de-acetylated by SIRT1. De-acetylation of FOXO1 induces the expression of genes involved in autophagy and stimulates autophagy flux. Therefore, under metabolic stress, FOXO1 can cause diabetic cardiomyopathy. The overexpression of FOXO1 leads to decreased cardiomyocyte size and suppresses cardiac hypertrophy through inhibition of the calcineurin–NFAT pathway. Diabetes mellitus is associated with elevation of O-GlcNAcylation. Some of its binding partners regulate the substrate selectivity of O-GlcNAc transferase (OGT). O-GlcNAcylation of essential contractile proteins may inhibit protein-protein interactions, reduce calcium sensitivity, and modulate contractile function. Uridine diphosphate (UDP)-GlcNAc is the obligatory substrate of OGT, which catalyzes a reversible post-translational protein modification. The increase of O-GlcNAcylation is accompanied by impaired cardiac hypertrophy in diabetic hearts. Inhibition of O-GlcNAcylation blocks activation of ERK1/2 and hypertrophic growth. O-GlcNAc modification on NFAT is required for its translocation from the cytosol to the nucleus, where NFAT stimulates the transcription of various hypertrophic genes. Inhibition of O-GlcNAcylation dampens NFAT-induced cardiac hypertrophic growth. Transcriptional activity of FOXO1 is enriched by improved O-GlcNAcylation upon high glucose stimulation or OGT overexpression. In diabetic conditions, the modification of FOXO1 by O-GlcNAc is promoted in cardiac troponin I and myosin light chain 2. Therefore targeting O-GlcNAcylation represents a potential therapeutic option to prevent hypertrophy in the diabetic heart.

Keywords: diabetes, cardiac hypertrophy, O-GlcNAcylation, FOXO1, Akt, PI3K, AMPK, insulin

Procedia PDF Downloads 81

Authors: Magdy I. A. Alshourbagi

Abstract:

Background: The National Institute for Deafness and Communication Disorders defines idiopathic sudden sensorineural hearing loss as the idiopathic loss of hearing of at least 30 dB across 3 contiguous frequencies occurring within 3 days.The most common clinical presentation involves an individual experiencing a sudden unilateral hearing loss, tinnitus, a sensation of aural fullness and vertigo. The etiologies and pathologies of ISSNHL remain unclear. Several pathophysiological mechanisms have been described including: vascular occlusion, viral infections, labyrinthine membrane breaks, immune associated disease, abnormal cochlear stress response, trauma, abnormal tissue growth, toxins, ototoxic drugs and cochlear membrane damage. The rationale for the use of hyperbaric oxygen to treat ISSHL is supported by an understanding of the high metabolism and paucity of vascularity to the cochlea. The cochlea and the structures within it require a high oxygen supply. The direct vascular supply, particularly to the organ of Corti, is minimal. Tissue oxygenation to the structures within the cochlea occurs via oxygen diffusion from cochlear capillary networks into the perilymph and the cortilymph. . The perilymph is the primary oxygen source for these intracochlear structures. Unfortunately, perilymph oxygen tension is decreased significantly in patients with ISSHL. To achieve a consistent rise of perilymph oxygen content, the arterial-perilymphatic oxygen concentration difference must be extremely high. This can be restored with hyperbaric oxygen therapy. Subject and Methods: A 37 year old man was presented at the clinic with a five days history of muffled hearing and tinnitus of the right ear. Symptoms were sudden onset, with no associated pain, dizziness or otorrhea and no past history of hearing problems or medical illness. Family history was negative. Physical examination was normal. Otologic examination revealed normal tympanic membranes bilaterally, with no evidence of cerumen or middle ear effusion. Tuning fork examination showed positive Rinne test bilaterally but with lateralization of Weber test to the left side, indicating right ear sensorineural hearing loss. Audiometric analysis confirmed sensorineural hearing loss across all frequencies of about 70- dB in the right ear. Routine lab work were all within normal limits. Clinical diagnosis of idiopathic sudden sensorineural hearing loss of the right ear was made and the patient began a medical treatment (corticosteroid, vasodilator and HBO therapy). The recommended treatment profile consists of 100% O2 at 2.5 atmospheres absolute for 60 minutes daily (six days per week) for 40 treatments .The optimal number of HBOT treatments will vary, depending on the severity and duration of symptomatology and the response to treatment. Results: As HBOT is not yet a standard for idiopathic sudden sensorineural hearing loss, it was introduced to this patient as an adjuvant therapy. The HBOT program was scheduled for 40 sessions, we used a 12-seat multi place chamber for the HBOT, which was started at day seven after the hearing loss onset. After the tenth session of HBOT, improvement of both hearing (by audiogram) and tinnitus was obtained in the affected ear (right). Conclusions: In conclusion, HBOT may be used for idiopathic sudden sensorineural hearing loss as an adjuvant therapy. It may promote oxygenation to the inner ear apparatus and revive hearing ability. Patients who fail to respond to oral and intratympanic steroids may benefit from this treatment. Further investigation is warranted, including animal studies to understand the molecular and histopathological aspects of HBOT and randomized control clinical studies.

Keywords: idiopathic sudden sensorineural hearing loss (issnhl), hyperbaric oxygen therapy (hbot), the decibel (db), oxygen (o2)

Procedia PDF Downloads 400