Search results for: protein modification

Authors: Virendra Nath, Vipin Kumar

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Background: TypeII Diabetes mellitus is a foremost health problem worldwide, predisposing to increased mortality and morbidity. Undesirable effects of the current medications have prompted the researcher to develop more potential drug(s) against the disease. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptors family and take part in a vital role in the regulation of metabolic equilibrium. They can induce or repress genes associated with adipogenesis, lipid, and glucose metabolism. Aims: Investigation of PPARα/γ agonistic hits were screened by hierarchical virtual screening followed by molecular dynamics simulation and knowledge-based structure-activity relation (SAR) analysis using approved PPAR α/γ dual agonist. Methods: The PPARα/γ agonistic activity of compounds was searched by using Maestro through structure-based virtual screening and molecular dynamics (MD) simulation application. Virtual screening of nuclear-receptor ligands was done, and the binding modes with protein-ligand interactions of newer entity(s) were investigated. Further, binding energy prediction, Stability studies using molecular dynamics (MD) simulation of PPARα and γ complex was performed with the most promising hit along with the structural comparative analysis of approved PPARα/γ agonists with screened hit was done for knowledge-based SAR. Results and Discussion: The silicone chip-based approach recognized the most capable nine hits and had better predictive binding energy as compared to the reference drug compound (Tesaglitazar). In this study, the key amino acid residues of binding pockets of both targets PPARα/γ were acknowledged as essential and were found to be associated in the key interactions with the most potential dual hit (ChemDiv-3269-0443). Stability studies using molecular dynamics (MD) simulation of PPARα and γ complex was performed with the most promising hit and found root mean square deviation (RMSD) stabile around 2Å and 2.1Å, respectively. Frequency distribution data also revealed that the key residues of both proteins showed maximum contacts with a potent hit during the MD simulation of 20 nanoseconds (ns). The knowledge-based SAR studies of PPARα/γ agonists were studied using 2D structures of approved drugs like aleglitazar, tesaglitazar, etc. for successful designing and synthesis of compounds PPARγ agonistic candidates with anti-hyperlipidimic potential.

Keywords: computational, diabetes, PPAR, simulation

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Authors: Santi Maneewatchararangsri, Wanpen Chaicumpa, Patcharin Saengjaruk, Urai Chaisri

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Leptospirosis is a globally neglected disease that continues to be a significant public health and veterinary burden, with millions of cases reported each year. Early and accurate differential diagnosis of leptospirosis from other febrile illnesses and the development of a broad spectrum of leptospirosis vaccines are needed. The LipL32 outer membrane lipoprotein is a member of Leptospira adhesive matrices and has been found to exert hemolytic activity to erythrocytes in vitro. Therefore, LipL32 is regarded as a potential target for diagnosis, broad-spectrum leptospirosis vaccines, and for passive immunotherapy. In this study, we established LipL32-specific mouse monoclonal antibodies, mAbLPF1 and mAbLPF2, and their respective mouse- and humanized-engineered single chain variable fragment (ScFv). Their antibodies’ neutralizing activities against Leptospira-mediated hemolysis in vitro, and the therapeutic efficacy of mAbs against heterologous Leptospira infected hamsters were demonstrated. The epitope peptide of mAb LPF1 was mapped to a non-contiguous carboxy-terminal β-turn and amphipathic α-helix of LipL32 structure contributing to phospholipid/host cell adhesion and membrane insertion. We found that the mAbLPF2 epitope was located on the interacting loop of peptide binding groove of the LipL32 molecule responsible for interactions with host constituents. Epitope sequences are highly conserved among Leptospira spp. and are absent from the LipL32 superfamily of other microorganisms. Both epitopes are surface-exposed, readily accessible by mAbs, and immunogenic. However, they are less dominant when revealed by LipL32-specific immunoglobulins from leptospirosis-patient sera and rabbit hyperimmune serum raised by whole Leptospira. Our study also demonstrated an adhesion inhibitory activity of LipL32 protein to host membrane components and cells mediated by mAbs as well as an anti-hemolytic activity of the respective antibodies. The therapeutic antibodies, particularly the humanized-ScFv, have a potential for further development as non-drug therapeutic agent for human leptospirosis, especially in subjects allergic to antibiotics. The epitope peptides recognized by two therapeutic mAbs have potential use as tools for structure-function studies. Finally, protective peptides may be used as a target for epitope-based vaccines for control of leptospirosis.

Keywords: leptospira lipl32-specific antibodies, therapeutic epitopes, epitopes characterization, immunotherapy

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Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

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Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

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Authors: Achiron Anat, Mathilda Mandel, Mayust Sue, Achiron Reuven, Gurevich Michael

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Introduction: As coronavirus disease 2019 (COVID-19) vaccination is currently spreading around the world, it is of importance to assess the ability of multiple sclerosis (MS) patients to mount an appropriate immune response to the vaccine in the context of disease-modifying treatments (DMT’s). Objectives: Evaluate immunity generated following COVID-19 vaccination in MS patients, and assess factors contributing to protective humoral and cellular immune responses in MS patients vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) virus infection. Methods: Review our recent data related to (1) the safety of PfizerBNT162b2 COVID-19 mRNA vaccine in adult MS patients; (2) the humoral post-vaccination SARS-CoV2 IgG response in MS vaccinees using anti-spike protein-based serology; and (3) the cellular immune response of memory B-cells specific for SARS-CoV-2 receptor-binding domain (RBD) and memory T-cells secreting IFN-g and/or IL-2 in response to SARS-CoV2 peptides using ELISpot/Fluorospot assays in MS patients either untreated or under treatment with fingolimod, cladribine, or ocrelizumab; (4) covariate parameters related to mounting protective immune responses. Results: COVID-19 vaccine proved safe in MS patients, and the adverse event profile was mainly characterised by pain at the injection site, fatigue, and headache. Not any increased risk of relapse activity was noted and the rate of patients with acute relapse was comparable to the relapse rate in non-vaccinated patients during the corresponding follow-up period. A mild increase in the rate of adverse events was noted in younger MS patients, among patients with lower disability, and in patients treated with DMTs. Following COVID-19 vaccination protective humoral immune response was significantly decreased in fingolimod- and ocrelizumab- treated MS patients. SARS-CoV2 specific B-cell and T-cell cellular responses were respectively decreased. Untreated MS patients and patients treated with cladribine demonstrated protective humoral and cellular immune responses, similar to healthy vaccinated subjects. Conclusions: COVID-19 BNT162b2 vaccine proved as safe for MS patients. No increased risk of relapse activity was noted post-vaccination. Although COVID-19 vaccination is new, accumulated data demonstrate differences in immune responses under various DMT’s. This knowledge can help to construct appropriate COVID-19 vaccine guidelines to ensure proper immune responses for MS patients.

Keywords: covid-19, vaccination, multiple sclerosis, IgG

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Authors: Claudio Lamilla, Andrés Santos, Vicente Llanquinao, Jocelyn Hermosilla, Leticia Barrientos

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The industry in general is very interested in improving and optimizing industrial processes in order to reduce the costs involved in obtaining raw materials and production. Thus, an interesting and cost-effective alternative is the incorporation of bioactive metabolites in such processes, being an example of this enzymes which catalyze efficiently a large number of enzymatic reactions of industrial and biotechnological interest. In the search for new sources of these active metabolites, Antarctica is one of the least explored places on our planet where the most drastic cold conditions, salinity, UVA-UVB and liquid water available are present, features that have shaped all life in this very harsh environment, especially bacteria that live in different Antarctic ecosystems, which have had to develop different strategies to adapt to these conditions, producing unique biochemical strategies. In this work the production of cellulolytic enzymes of seven bacterial strains isolated from marine sediments at different sites in the Antarctic was evaluated. Isolation of the strains was performed using serial dilutions in the culture medium at M115°C. The identification of the strains was performed using universal primers (27F and 1492R). The enzyme activity assays were performed on R2A medium, carboxy methyl cellulose (CMC)was added as substrate. Degradation of the substrate was revealed by adding Lugol. The results show that four of the tested strains produce enzymes which degrade CMC substrate. The molecular identifications, showed that these bacteria belong to the genus Streptomyces and Pseudoalteromonas, being Streptomyces strain who showed the highest activity. Only some bacteria in marine sediments have the ability to produce these enzymes, perhaps due to their greater adaptability to degrade at temperatures bordering zero degrees Celsius, some algae that are abundant in this environment and have cellulose as the main structure. The discovery of new enzymes adapted to cold is of great industrial interest, especially for paper, textiles, detergents, biofuels, food and agriculture. These enzymes represent 8% of industrial demand worldwide and is expected to increase their demand in the coming years. Mainly in the paper and food industry are required in extraction processes starch, protein and juices, as well as the animal feed industry where treating vegetables and grains helps improve the nutritional value of the food, all this clearly puts Antarctic microorganisms and their enzymes specifically as a potential contribution to industry and the novel biotechnological applications.

Keywords: antarctic, bacteria, biotechnological, cellulolytic enzymes

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Authors: Kusmartono, Siti Chuzaemi, Hartutik dan Mashudi

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Two studies were conducted involving an in vitro (Expt 1) and in vivo (Expt 2) measurements. Expt 1. aimed to evaluate effects of adding CT extracts on gas production and efficiency of microbial protein synthesis (EMPS), Expt 2 aimed to evaluate effects of supplementing shrub/tree leaves as CT source on feed consumption, digestibility, N retention, body weight gain and dressing percentage of growing sheep fed on elephant grass (EG) as a basal diet.Ten shrub and tree leaves used as CT sources were wild sunflower (Tithonia diversifolia), mulberry (Morus macroura), cassava (Manihot utilissima), avicienna (Avicennia marina), calliandra (Calliandra calothyrsus), sesbania (Sesbania grandiflora), acacia (acacia vilosa), glyricidia (Glyricidia sepium), jackfruit (Artocarpus heterophyllus), moringa (Moringa oleifera). The treatments applied in Expt 1 were: T1=Elephant grass (60%)+concentrate (40%); T2 = T1 + CT (3% DM); T3= T2 + PEG; T4 = T1 + CT (3.5% DM); T5 = T4 + PEG; T6 = T1 + CT (4% DM) and T7 = T6 + PEG. Data obtained were analysed using Randomized Block Design. Statistical analyses showed that treatments significanty affected (P<0.05) total gas production and EMPS. The lowest values of total gas production (45.9 ml/500 mg DM) and highest value of EMPS (64.6 g/kg BOTR) were observed in the treatment T4 (3.5% CT from cassava leave extract). Based on this result it was concluded that this treatment was the best and was chosen for further investigation using in vivo method. The treatmets applied for in vivo trial were: T1 = EG (60%) + concentrate (40%); T2 = T1 + dried cassava leave (equivalent to 3.5% CT); T3 = T2 + PEG. 18 growing sheep aging of 8-9 months and weighing of 23.67kg ± 1.23 were used in Expt 2. Results of in vivo study showed that treatments significanty affected (P<0.05) nutrients intake and digestibility (DM, OM and CP). N retention for sheep receiving treatment T2 were significantly higher (P<0.05; 15.6 g/d) than T1 (9.1 g/d) and T3 (8.53 g/d). Similar results were obtained for daily weight gain where T2 were the highest (62.79 g/d), followed by T1 (51.9 g/d) and T3 (52.85 g/d). Dressing percentage of T2 was the highest (51.54%) followed by T1 (49.61%) and T3 (49.32%). It can be concluded that adding adding dried cassava leaves did not reduce palatability due to CT, but rather increased OM digestibility and hence feed consumption was improved. N retention was increased due to the action of CT in the cassava leaves and this may have explained a higher input of N into duodenum which was further led to higer daily weight gain and dressing percentage.

Keywords: in vitro gas production, sheep, shrub and tree leaves, condensed tannin

Procedia PDF Downloads 237

Authors: Irene Carmagnola, Valeria Chiono, Sandra Pacharra, Jochen Salber, Sean McMahon, Chris Lovell, Pooja Basnett, Barbara Lukasiewicz, Ipsita Roy, Xiang Zhang, Gianluca Ciardelli

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Thrombosis and restenosis after stenting procedure can be prevented by promoting fast stent wall endothelialisation. It is well known that surface functionalisation with antifouling molecules combining with extracellular matrix proteins is a promising strategy to design biomimetic surfaces able to promote fast endothelialization. In particular, REDV has gained much attention for the ability to enhance rapid endothelialization due to its specific affinity with endothelial cells (ECs). In this work, a two-step plasma treatment was performed to polymerize a thin layer of acrylic acid, used to subsequently graft PEGylated-REDV and polyethylene glycol (PEG) at different molar ratio with the aim to selectively promote endothelial cell adhesion avoiding platelet activation. PEGylate-REDV was provided by Biomatik and it is formed by 6 PEG monomer repetitions (Chempep Inc.), with an NH2 terminal group. PEG polymers were purchased from Chempep Inc. with two different chain lengths: m-PEG6-NH2 (295.4 Da) with 6 monomer repetitions and m-PEG12-NH2 (559.7 Da) with 12 monomer repetitions. Plasma activation was obtained by operating at 50W power, 5 min of treatment and at an Ar flow rate of 20 sccm. Pure acrylic acid (99%, AAc) vapors were diluted in Ar (flow = 20 sccm) and polymerized by a pulsed plasma discharge applying a discharge RF power of 200 W, a duty cycle of 10% (on time = 10 ms, off time = 90 ms) for 10 min. After plasma treatment, samples were dipped into an 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) solution (ratio 4:1, pH 5.5) for 1 h at 4°C and subsequently dipped in PEGylate-REDV and PEGylate-REDV:PEG solutions at different molar ratio (100 μg/mL in PBS) for 20 h at room temperature. Surface modification was characterized through physico-chemical analyses and in vitro cell tests. PEGylated-REDV peptide and PEG were successfully bound to the carboxylic groups that are formed on the polymer surface after plasma reaction. FTIR-ATR spectroscopy, X -ray Photoelectron Spectroscopy (XPS) and contact angle measurement gave a clear indication of the presence of the grafted molecules. The use of PEG as a spacer allowed for an increase in wettability of the surface, and the effect was more evident by increasing the amount of PEG. Endothelial cells adhered and spread well on the surfaces functionalized with the REDV sequence. In conclusion, a selective coating able to promote a new endothelial cell layer on polymeric stent surface was developed. In particular, a thin AAc film was polymerised on the polymeric surface in order to expose –COOH groups, and PEGylate-REDV and PEG were successful grafted on the polymeric substrates. The REDV peptide demonstrated to encourage cell adhesion with a consequent, expected improvement of the hemocompatibility of these polymeric surfaces in vivo. Acknowledgements— This work was funded by the European Commission 7th Framework Programme under grant agreement number 604251- ReBioStent (Reinforced Bioresorbable Biomaterials for Therapeutic Drug Eluting Stents). The authors thank all the ReBioStent partners for their support in this work.

Keywords: endothelialisation, plasma treatment, stent, surface functionalisation

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Authors: Ainur Sharip, Jose E. Perez, Nouf Alsharif, Aldo I. M. Bandeas, Enzo D. Fabrizio, Timothy Ravasi, Jasmeen S. Merzaban, Jürgen Kosel

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Bone marrow-derived human mesenchymal stem cells (MSCs) are attractive candidates for tissue engineering and regenerative medicine, due to their ability to differentiate into osteoblasts, chondrocytes or adipocytes. Differentiation is influenced by biochemical and biophysical stimuli provided by the microenvironment of the cell. Thus, altering the mechanical characteristics of a cell culture scaffold can directly influence a cell’s microenvironment and lead to stem cell differentiation. Mesenchymal stem cells were cultured on densely packed, vertically aligned magnetic iron nanowires (NWs) and the effect of NWs on the cell cytoskeleton rearrangement and differentiation were studied. An electrochemical deposition method was employed to fabricate NWs into nanoporous alumina templates, followed by a partial release to reveal the NW array. This created a cell growth substrate with free-standing NWs. The Fe NWs possessed a length of 2-3 µm, with each NW having a diameter of 33 nm on average. Mechanical stimuli generated by the physical movement of these iron NWs, in response to a magnetic field, can stimulate osteogenic differentiation. Induction of osteogenesis was estimated using an osteogenic marker, osteopontin, and a reduction of stem cell markers, CD73 and CD105. MSCs were grown on the NWs, and fluorescent microscopy was employed to monitor the expression of markers. A magnetic field with an intensity of 250 mT and a frequency of 0.1 Hz was applied for 12 hours/day over a period of one week and two weeks. The magnetically activated substrate enhanced the osteogenic differentiation of the MSCs compared to the culture conditions without magnetic field. Quantification of the osteopontin signal revealed approximately a seven-fold increase in the expression of this protein after two weeks of culture. Immunostaining staining against CD73 and CD105 revealed the expression of antibodies at the earlier time point (two days) and a considerable reduction after one-week exposure to a magnetic field. Overall, these results demonstrate the application of a magnetic NW substrate in stimulating the osteogenic differentiation of MSCs. This method significantly decreases the time needed to induce osteogenic differentiation compared to commercial biochemical methods, such as osteogenic differentiation kits, that usually require more than two weeks. Contact-free stimulation of MSC differentiation using a magnetic field has potential uses in tissue engineering, regenerative medicine, and bone formation therapies.

Keywords: cell substrate, magnetic nanowire, mesenchymal stem cell, stem cell differentiation

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Authors: Wael M. Rabeh, Cesar Carrasco-Lopez, Juliana C. Ferreira, Pance Naumov

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Bioluminescence, the emission of light from a biological process, is found in various living organisms including bacteria, fireflies, beetles, fungus and different marine organisms. Luciferase is an enzyme that catalyzes a two steps oxidation of luciferin in the presence of Mg2+ and ATP to produce oxyluciferin and releases energy in the form of light. The luciferase assay is used in biological research and clinical applications for in vivo imaging, cell proliferation, and protein folding and secretion analysis. The luciferase enzyme consists of two domains, a large N-terminal domain (1-436 residues) that is connected to a small C-terminal domain (440-544) by a flexible loop that functions as a hinge for opening and closing the active site. The two domains are separated by a large cleft housing the active site that closes after binding the substrates, luciferin and ATP. Even though all insect luciferases catalyze the same chemical reaction and share 50% to 90% sequence homology and high structural similarity, they emit light of different colors from green at 560nm to red at 640 nm. Currently, the majority of the structural and biochemical studies have been conducted on green-emitting firefly luciferases. To address the color emission mechanism, we expressed and purified two luciferase enzymes with blue-shifted green and red emission from indigenous Brazilian species Amydetes fanestratus and Phrixothrix, respectively. The two enzymes naturally emit light of different colors and they are an excellent system to study the color-emission mechanism of luciferases, as the current proposed mechanisms are based on mutagenesis studies. Using a vapor-diffusion method and a high-throughput approach, we crystallized and solved the crystal structure of both enzymes, at 1.7 Å and 3.1 Å resolution respectively, using X-ray crystallography. The free enzyme adopted two open conformations in the crystallographic unit cell that are different from the previously characterized firefly luciferase. The blue-shifted green luciferase crystalized as a monomer similar to other luciferases reported in literature, while the red luciferases crystalized as an octamer and was also purified as an octomer in solution. The octomer conformation is the first of its kind for any insect’s luciferase, which might be relate to the red color emission. Structurally designed mutations confirmed the importance of the transition between the open and close conformations in the fine-tuning of the color and the characterization of other interesting mutants is underway.

Keywords: bioluminescence, enzymology, structural biology, x-ray crystallography

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Authors: Gail Louw, Helena Boshoff, Taeksun Song, Clifton Barry

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Drug resistance in Mycobacterium tuberculosis is primarily attributed to mutations in target genes. These mutations incur a fitness cost and result in bacterial generations that are less fit, which subsequently acquire compensatory mutations to restore fitness. We hypothesize that mutations in specific drug target genes influence bacterial metabolism and cellular function, which affects its ability to develop subsequent resistance to additional agents. We aim to determine whether the sequential acquisition of drug resistance and specific mutations in a well-defined clinical M. tuberculosis strain promotes or limits the development of additional resistance. In vitro mutants resistant to pretomanid, linezolid, moxifloxacin, rifampicin and kanamycin were generated from a pan-susceptible clinical strain from the Beijing lineage. The resistant phenotypes to the anti-TB agents were confirmed by the broth microdilution assay and genetic mutations were identified by targeted gene sequencing. Growth of mono-resistant mutants was done in enriched medium for 14 days to assess in vitro fitness. Double resistant mutants were generated against anti-TB drug combinations at concentrations 5x and 10x the minimum inhibitory concentration. Subsequently, mutation frequencies for these anti-TB drugs in the different mono-resistant backgrounds were determined. The initial level of resistance and the mutation frequencies observed for the mono-resistant mutants were comparable to those previously reported. Targeted gene sequencing revealed the presence of known and clinically relevant mutations in the mutants resistant to linezolid, rifampicin, kanamycin and moxifloxacin. Significant growth defects were observed for mutants grown under in vitro conditions compared to the sensitive progenitor. Mutation frequencies determination in the mono-resistant mutants revealed a significant increase in mutation frequency against rifampicin and kanamycin, but a significant decrease in mutation frequency against linezolid and sutezolid. This suggests that these mono-resistant mutants are more prone to develop resistance to rifampicin and kanamycin, but less prone to develop resistance against linezolid and sutezolid. Even though kanamycin and linezolid both inhibit protein synthesis, these compounds target different subunits of the ribosome, thereby leading to different outcomes in terms of fitness in the mutants with impaired cellular function. These observations showed that oxazolidinone treatment is instrumental in limiting the development of multi-drug resistance in M. tuberculosis in vitro.

Keywords: oxazolidinones, mutations, resistance, tuberculosis

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Authors: Boutheina Ben Abdelmoumen Mardassi, Salim Chibani, Safa Boujemaa, Amaury Vaysse, Julien Guglielmini, Elhem Yacoub

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Background and aim: Mycoplasma hominis (MH) is a pathogenic bacterium belonging to the Mollicutes class. It causes a wide range of gynecological infections and infertility among adults. Recently, we have explored for the first time the phylodistribution of Tunisian M. hominis clinical strains using an expanded MLST. We have demonstrated their distinction into two pure lineages, which each corresponding to a specific pathotype: genital infections and infertility. The aim of this project is to gain further insight into the evolutionary dynamics and the specific genetic factors that distinguish MH pathotypes Methods: Whole genome sequencing of Mycoplasma hominis clinical strains was performed using illumina Miseq. Denovo assembly was performed using a publicly available in-house pipeline. We used prokka to annotate the genomes, panaroo to generate the gene presence matrix and Jolytree to establish the phylogenetic tree. We used treeWAS to identify genetic loci associated with the pathothype of interest from the presence matrix and phylogenetic tree. Results: Our results revealed a clear categorization of the 62 MH clinical strains into two distinct genetic lineages, with each corresponding to a specific pathotype.; gynecological infections and infertility[AV1] . Genome annotation showed that GC content is ranging between 26 and 27%, which is a known characteristic of Mycoplasma genome. Housekeeping genes belonging to the core genome are highly conserved among our strains. TreeWas identified 4 virulence genes associated with the pathotype gynecological infection. encoding for asparagine--tRNA ligase, restriction endonuclease subunit S, Eco47II restriction endonuclease, and transcription regulator XRE (involved in tolerance to oxidative stress). Five genes have been identified that have a statistical association with infertility, tow lipoprotein, one hypothetical protein, a glycosyl transferase involved in capsule synthesis, and pyruvate kinase involved in biofilm formation. All strains harbored an efflux pomp that belongs to the family of multidrug resistance ABC transporter, which confers resistance to a wide range of antibiotics. Indeed many adhesion factors and lipoproteins (p120, p120', p60, p80, Vaa) have been checked and confirmed in our strains with a relatively 99 % to 96 % conserved domain and hypervariable domain that represent 1 to 4 % of the reference sequence extracted from gene bank. Conclusion: In summary, this study led to the identification of specific genetic loci associated with distinct pathotypes in M hominis.

Keywords: mycoplasma hominis, infertility, gynecological infections, virulence genes, antibiotic resistance

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Authors: Banu Pradheepa Kamarajan, Muthusamy Ananthasubramanian

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Biofilm forming Pseudomonads occupy the top third position in causing hospital acquired infections. P. aeruginosa is notoriously known for its tendency to develop drug resistance. Major classes of drug such as β-lactams, aminoglycosides, quinolones, and polymyxins are found ineffective against multi-drug resistance Pseudomonas. To combat the infections, rather than administration of a single antibiotic, use of combinations (tobramycin and essential oils from plants and/or silver nanoparticles, chitosan, nitric oxide, cis-2-decenoic acid) in single formulation are suggested to control P. aeruginosa biofilms. Conventional techniques to prevent hospital-acquired implant infections such as coatings with antibiotics, controlled release of antibiotics from the implant material, contact-killing surfaces, coating the implants with functional DNase I and, coating with glycoside hydrolase are being followed. Coatings with bioactive components besides having limited shelf-life, require cold-chain and, are likely to fail when bacteria develop resistance. Recently identified nano-scale physical architectures on the insect wings are expected to have potential bactericidal property. Nanopillars are bactericidal to Staphylococcus aureus, Bacillus subtilis, K. pnuemoniae and few species of Pseudomonas. Our study aims to investigate the survival rate of biofilm forming Pseudomonas aeruginosa strain over non-biofilm forming strain on the nanopillar architecture of dragonfly (Pantala flavescens) wing. Dragonflies were collected near house-hold areas and, insect identification was carried out by the Department of Entomology, Tamilnadu Agricultural University, Coimbatore, India. Two strains of P. aeruginosa such as PAO1 (potent biofilm former) and MTCC 1688 (non-weak biofilm former) were tested against the glass coverslip (control) and wings of dragonfly (test) for 48 h. The wings/glass coverslips were incubated with bacterial suspension in 48-well plate. The plates were incubated at 37 °C under static condition. Bacterial attachment on the nanopillar architecture of the wing surface was visualized using FESEM. The survival rate of P. aeruginosa was tested using colony counting technique and flow cytometry at 0.5 h, 1 h, 2 h, 7 h, 24 h, and 48 h post-incubation. Cell death was analyzed using propidium iodide staining and DNA quantification. The results indicated that the survival rate of non-biofilm forming P. aeruginosa is 0.2 %, whilst that of biofilm former is 45 % on the dragonfly wings at the end of 48 h. The reduction in the survival rate of biofilm and non-biofilm forming P. aeruginosa was 20% and 40% respectively on the wings compared to the glass coverslip. In addition, Fourier Transformed Infrared Radiation was used to study the modification in the surface chemical composition of the wing during bacterial attachment and, post-sonication. This result indicated that the chemical moieties are not involved in the bactericidal property of nanopillars by the conserved characteristic peaks of chitin pre and post-sonication. The nanopillar architecture of the dragonfly wing efficiently deters the survival of non-biofilm forming P. aeruginosa, but not the biofilm forming strain. The study highlights the ability of biofilm formers to survive on wing architecture. Understanding this survival strategy will help in designing the architecture that combats the colonization of biofilm forming pathogens.

Keywords: biofilm, nanopillars, Pseudomonas aeruginosa, survival rate

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Authors: Jyh-Ping Chen, Yu-Tin Lai

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Wound healing is a complicated process involving overlapping hemostasis, inflammation, proliferation, and maturation phases. Ideal wound dressings can replace native skin functions in full thickness skin wounds through faster healing rate and also by reducing scar formation. Poly(lactic-co-glycolic acid) (PLGA) is an U.S. FDA approved biodegradable polymer to be used as ideal wound dressing material. Several in vitro and in vivo studies have demonstrated the effectiveness of curcumin in decreasing the release of inflammatory cytokines, inhibiting enzymes associated with inflammations, and scavenging free radicals that are the major cause of inflammation during wound healing. Heparin has binding affinities to various growth factors. With the unique and beneficial features offered by those molecules toward the complex process of wound healing, we postulate a composite wound dressing constructed from PLGA, curcumin and heparin would be a good candidate to accelerate scarless wound healing. In this work, we use electrospinning to prepare curcumin-loaded aligned PLGA nanofibrous membranes (PC NFMs). PC NFMs were further subject to oxygen plasma modification and surfaced-grafted with heparin through carbodiimide-mediated covalent bond formation to prepare curcumin-loaded PLGA-g-heparin (PCH) NFMs. The nanofibrous membranes could act as three-dimensional scaffolds to attract fibroblast migration, reduce inflammation, and increase wound-healing related growth factors concentrations at wound sites. From scanning electron microscopy analysis, the nanofibers in each NFM are with diameters ranging from 456 to 479 nm and with alignment angles within  0.5°. The NFMs show high tensile strength and good water absorptivity and provide suitable pore size for nutrients/wastes transport. Exposure of human dermal fibroblasts to the extraction medium of PC or PCH NFM showed significant protective effects against hydrogen peroxide than PLGA NFM. In vitro wound healing assays also showed that the extraction medium of PCH NFM showed significantly better migration ability toward fibroblasts than PC NFM, which is further better than PLGA NFM. The in vivo healing efficiency of the NFMs was further evaluated by a full thickness excisional wound healing diabetic rat model. After 14 days, PCH NFMs exhibits 86% wound closure rate, which is significantly different from other groups (79% for PC and 73% for PLGA NFM). Real-time PCR analysis indicated PC and PCH NFMs down regulated anti-oxidative enzymes like glutathione peroxidase (GPx) and superoxide dismutase (SOD), which are well-known transcription factors involved in cellular inflammatory responses to stimuli. From histology, the wound area treated with PCH NFMs showed more vascular lumen formation from immunohistochemistry of α-smooth muscle actin. The wound site also had more collagen type III (65.8%) expression and less collagen type I (3.5%) expression, indicating scar-less wound healing. From Western blot analysis, the PCH NFM showed good affinity toward growth factors from increased concentration of transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2) at the wound site to accelerate wound healing. From the results, we suggest PCH NFM as a promising candidate for wound dressing applications.

Keywords: Curcumin, heparin, nanofibrous membrane, poly(lactic-co-glycolic acid) (PLGA), wound dressing

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Authors: Sultan Z. Mahmud, Emily C. Graff, Adil Bashir

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Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is a multifunctional adapter protein which is associated with the regulation of apoptotic cell death. Recently it has been discovered that PEA-15 is crucial in normal neurodevelopment of domestic cats, a gyrencephalic animal model, although the exact function of PEA-15 in neurodevelopment is unknown. This study investigates how PEA-15 affects the blood-brain barrier (BBB) permeability in cat brain, which can cause abnormalities in tissue metabolite and energy supplies. Severe polymicrogyria and microcephaly have been observed in cats with a loss of function PEA-15 mutation, affecting the normal neurodevelopment of the cat. This suggests that the vital role of PEA-15 in neurodevelopment is associated with gyrification. Neurodevelopment is a highly energy demanding process. The mammalian brain depends on glucose as its main energy source. PEA-15 plays a very important role in glucose uptake and utilization by interacting with phospholipase D1 (PLD1). Mitochondria also plays a critical role in bioenergetics and essential to supply adequate energy needed for neurodevelopment. Cerebral blood flow regulates adequate metabolite supply and recent findings also showed that blood plasma contains mitochondria as well. So the BBB can play a very important role in regulating metabolite and energy supply in the brain. In this study the blood-brain permeability in cat brain was measured using MRI magnetization transfer (MT) effect on the perfusion signal. Perfusion is the tissue mass normalized supply of blood to the capillary bed. Perfusion also accommodates the supply of oxygen and other metabolites to the tissue. A fraction of the arterial blood can diffuse to the tissue, which depends on the BBB permeability. This fraction is known as water extraction fraction (EF). MT is a process of saturating the macromolecules, which has an effect on the blood that has been diffused into the tissue while having minimal effect on intravascular blood water that has not been exchanged with the tissue. Measurement of perfusion signal with and without MT enables to estimate the microvascular blood flow, EF and permeability surface area product (PS) in the brain. All the experiments were performed with Siemens 7T Magnetom with 32 channel head coil. Three control cats and three PEA-15 mutant cats were used for the study. Average EF in white and gray matter was 0.9±0.1 and 0.86±0.15 respectively, perfusion in white and gray matter was 85±15 mL/100g/min and 97±20 mL/100g/min respectively, PS in white and gray matter was 201±25 mL/100g/min and 225±35 mL/100g/min respectively for control cats. For PEA-15 mutant cats, average EF in white and gray matter was 0.81±0.15 and 0.77±0.2 respectively, perfusion in white and gray matter was 140±25 mL/100g/min and 165±18 mL/100g/min respectively, PS in white and gray matter was 240±30 mL/100g/min and 259±21 mL/100g/min respectively. This results show that BBB is compromised in PEA-15 mutant cat brain, where EF is decreased and perfusion as well as PS are increased in the mutant cats compared to the control cats. This findings might further explain the function of PEA-15 in neurodevelopment.

Keywords: BBB, cat brain, magnetization transfer, PEA-15

Procedia PDF Downloads 109

Authors: Benedicta N. Nkeh-Chungag, Constance R. Sewani-Rusike, Isaac M. Malema, Daniel T. Goon, Oladele V. Adeniyi, Idowu A. Ajayi

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Background: Childhood and adolescent obesity is an important predictor of adult cardiometabolic diseases. Current data on age- and gender-specific cardiometabolic risk factors are lacking in the peri-urban Eastern Cape Province, South Africa. However, such information is important in designing innovative strategies to promote healthy living among children and adolescents. The purpose of this pilot study was to compare and determine the extent of cardiometabolic risk factors between samples of lean and overweight/obese adolescent population in a peri-urban township of South Africa. Methods: In this case-control study, age-matched, non-pregnant and non-lactating female adolescents consisting of equal number of cases (50 overweight/obese) and control (50 lean) participated in the study. Fasting venous blood samples were obtained for total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (Trig), highly sensitive C-reactive protein (hsCRP) and blood sugar. Anthropometric measurements included weight, height, waist and hip circumferences. Body mass index was calculated. Blood pressure was measured; and metabolic syndrome was assessed using appropriate diagnostic criteria for children and adolescents. Results: Of the 76 participants with complete data, 12/38 of the overweight/obese and 1/38 of the lean group met the criteria for adolescent metabolic syndrome. All cardiometabolic risk factors were elevated in the overweight/obese group compared with the lean group: low HDL-C (RR = 2.21), elevated TC (RR = 1.23), elevated LDL-C (RR = 1.42), elevated Trig (RR = 1.73), and elevated hsCRP (RR = 1.9). There were significant atherosclerotic indices among the overweight/obese group compared with the lean group: TC/HDL and LDL/HDL (2.99±0.91 vs 2.63±0.48; p=0.016 and 1.73±0.61 vs 1.41±0.46; p= 0.014, respectively). Conclusion: There are multiple cardiometabolic risk factors among the overweight/obese female adolescent group compared with lean adolescent group in the study. Female adolescent who are overweight and obese have higher relative risks of developing cardiometabolic diseases compared with their lean counterparts in the peri-urban Mthatha, South Africa. School health programme focusing on promoting physical exercise, healthy eating and keeping appropriate weight are needed in the country.

Keywords: adolescents, cardiometabolic risk factors, obesity, peri-urban South Africa

Procedia PDF Downloads 445

Authors: G. J. Fan, T. T. Lee, M. H. Chen, T. F. Shiao, B. Yu, C. F. Lee

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Rice straw with poor feed quality and spent mushroom substrate are both the most abundant agricultural residues in Taiwan. Edible mushrooms from white rot fungi possess lignocellulase activity. It was expected to improve the feeding value of rice straw for ruminant by solid-state fermentation pretreatment using spent mushroom substrate. Six varieties or subspecies of spent edible mushrooms (Pleurotus ostreatus (blue or white color), P. sajor-caju, P. citrinopileatus, P. eryngii and Ganoderma lucidium) substrate were evaluated in solid-state fermentation process with rice straw for 8 wks. Quality improvement of fermented rice straw was determined by its in vitro digestibility, lignocellulose degradability, and cell wall breakdown checked by scanning electron microscope. Results turned out that Pleurotus ostreatus (white color) and P. sajor-caju had the better lignocellulose degradation effect than the others and was chosen for advance in vivo study. Rice straw fermented with spent Pleurotus ostreatus or Pleurotus sajor-caju mushroom substrate 8 wks was prepared for growing and lactating feeding trials of dairy goat, respectively. Pangolagrass hay at 15% diet dry matter was the control diet. Fermented or original rice straw was added to substitute pangolagrass hay in both feeding trials. A total of 30 head of Alpine castrated ram were assigned into three groups for 11 weeks, 5 pens (2 head/pen) each group. A total of 21 head of Saanen and Alpine goats were assigned into three treatments and individually fed in two repeat lactating trials with 28-d each. In castrated ram study, results showed that fermented rice straw by spent Pleurotus ostreatus mushroom substrate attributed the higher daily dry matter intakes (DMI, 1.53 vs. 1.20 kg) and body weight gain (138 vs. 101 g) than goats fed original rice straw. DMI (2.25 vs. 1.81 kg) and milk yield (3.31 vs. 3.02 kg) of lactating goats fed control pangolagrass diet and fermented rice straw by spent Pleurotus sajor-caju mushroom substrate were also higher than those fed original rice straw diet (P < 0.05). Milk compositions, milk fat, protein, total solid and lactose, were similar among treatments. In conclusion, solid-state fermentation by spent Pleurotus ostreatus or Pleurotus sajor-caju mushroom substrate could effectively improve the feeding value of rice straw. Fermented rice straw is a good alternative fiber feed resource for growing and lactating dairy goats and 15% in diet dry matter is recommended.

Keywords: feeding value, fermented rice straw, growing and lactating dairy goat, spent Pleurotus ostreatus and Pleurotus sajor-caju mushroom substrate

Procedia PDF Downloads 143

Authors: Piotr Jablonski, Krzysztof Mars, Wiktor Niemiec, Agnieszka Kyziol, Marek Hebda, Halina Krawiec, Karol Kyziol

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NiTi alloys, possessing unique mechanical properties such as pseudoelasticity and shape memory effect (SME), are suitable for many applications, including implanthology and biomedical devices. Additionally, these alloys have similar values of elastic modulus to those of human bones, what is very important in orthopedics. Unfortunately, the environment of physiological fluids in vivo causes unfavorable release of Ni ions, which in turn may lead to metalosis as well as allergic reactions and toxic effects in the body. For these reasons, the surface properties of NiTi alloys should be improved to increase corrosion resistance, taking into account biological properties, i.e. excellent biocompatibility. The prospective in this respect are layers based on DLC (Diamond-Like Carbon) structures, which are an attractive solution for many applications in implanthology. These coatings (DLC), usually obtained by PVD (Physical Vapour Deposition) and PA CVD (Plasma Activated Chemical Vapour Deposition) methods, can be also modified by doping with other elements like silicon, nitrogen, oxygen, fluorine, titanium and silver. These methods, in combination with a suitably designed structure of the layers, allow the possibility co-decide about physicochemical and biological properties of modified surfaces. Mentioned techniques provide specific physicochemical properties of substrates surface in a single technological process. In this work, the following types of layers based on DLC structures (incl. Si-DLC or Si/N-DLC) were proposed as prospective and attractive approach in surface functionalization of shape memory alloy. Nitinol substrates were modified in plasma conditions, using RF CVD (Radio Frequency Chemical Vapour Deposition). The influence of plasma treatment on the useful properties of modified substrates after deposition DLC layers doped with silica and/or nitrogen atoms, as well as only pre-treated in O2 NH3 plasma atmosphere in a RF reactor was determined. The microstructure and topography of the modified surfaces were characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Furthermore, the atomic structure of coatings was characterized by IR and Raman spectroscopy. The research also included the evaluation of surface wettability, surface energy as well as the characteristics of selected mechanical and biological properties of the layers. In addition, the corrosion properties of alloys after and before modification in the physiological saline were also investigated. In order to determine the corrosion resistance of NiTi in the Ringer solution, the potentiodynamic polarization curves (LSV – Linear Sweep Voltamperometry) were plotted. Furthermore, the evolution of corrosion potential versus immersion time of TiNi alloy in Ringer solution was performed. Based on all carried out research, the usefullness of proposed modifications of nitinol for medical applications was assessed. It was shown, inter alia, that the obtained Si-DLC layers on the surface of NiTi alloy exhibit a characteristic complex microstructure, increased surface development, which is an important aspect in improving the osteointegration of an implant. Furthermore, the modified alloy exhibits biocompatibility, the transfer of the metal (Ni, Ti) to Ringer’s solution is clearly limited.

Keywords: bioactive coatings, corrosion resistance, doped DLC structure, NiTi alloy, RF CVD

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Authors: Shraddha Rane, Richard Warren, Stephen Eyre

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L-23 is a pro-inflammatory molecule that signals T cells to release cytokines such as IL-17A and IL-22. Psoriasis is driven by a dysregulated immune response, within which IL-23 is now thought to play a key role. Genome-wide association studies (GWAS) have identified a number of genetic risk loci that support the involvement of IL-23 signalling in psoriasis; in particular a robust susceptibility locus at a gene encoding a subunit of the IL-23 receptor (IL23R) (Stuart et al., 2015; Tsoi et al., 2012). The lead psoriasis-associated SNP rs9988642 is located approximately 500 bp downstream of IL23R but is in tight linkage disequilibrium (LD) with a missense SNP rs11209026 (R381Q) within IL23R (r2 = 0.85). The minor (G) allele of rs11209026 is present in approximately 7% of the population and is protective for psoriasis and several other autoimmune diseases including IBD, ankylosing spondylitis, RA and asthma. The psoriasis-associated missense SNP R381Q causes an arginine to glutamine substitution in a region of the IL23R protein between the transmembrane domain and the putative JAK2 binding site in the cytoplasmic portion. This substitution is expected to affect the receptor’s surface localisation or signalling ability, rather than IL23R expression. Recent studies have also identified a psoriatic arthritis (PsA)-specific signal at IL23R; thought to be independent from the psoriasis association (Bowes et al., 2015; Budu-Aggrey et al., 2016). The lead PsA-associated SNP rs12044149 is intronic to IL23R and is in LD with likely causal SNPs intersecting promoter and enhancer marks in memory CD8+ T cells (Budu-Aggrey et al., 2016). It is therefore likely that the PsA-specific SNPs affect IL23R function via a different mechanism compared with the psoriasis-specific SNPs. It could be hypothesised that the risk allele for PsA located within the IL23R promoter causes an increase IL23R expression, relative to the protective allele. An increased expression of IL23R might then lead to an exaggerated immune response. The independent genetic signals identified for psoriasis and PsA in this locus indicate that different mechanisms underlie these two conditions; although likely both affecting the function of IL23R. It is very important to further characterise these mechanisms in order to better understand how the IL-23 receptor and its downstream signalling is affected in both diseases. This will help to determine how psoriasis and PsA patients might differentially respond to therapies, particularly IL-23 biologics. To investigate this further we have developed an in vitro model using CD4 T cells which express either wild type IL23R and IL12Rβ1 or mutant IL23R (R381Q) and IL12Rβ1. Model expressing different isotypes of IL23R is also underway to investigate the effects on IL23R expression. We propose to further investigate the variants for Ps and PsA and characterise key intracellular processes related to the variants.

Keywords: IL23R, psoriasis, psoriatic arthritis, SNP

Procedia PDF Downloads 144

Authors: Mohammad Afzal Khan, Fatimah Alanazi, Hala Abdalrahman Ahmed, Talal Shamma, Kilian Kelly, Mohammed A. Hammad, Abdullah O. Alawad, Abdullah Mohammed Assiri, Dieter Clemens Broering

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Lung transplantation is a life-saving surgical replacement of diseased lungs in patients with end-stage respiratory malfunctions. Despite the remarkable short-term recovery, long-term lung survival continues to face several significant challenges, including chronic rejection and severe toxic side-effects due to global immunosuppression. Stem cell-based immunotherapy has been recognized as a crucial immunoregulatory regimen in various preclinical and clinical studies. Despite initial therapeutic outcomes, conventional stem cells face key limitations. The Cymerus™ manufacturing facilitates the production of a virtually limitless supply of consistent human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells, which could play a key role in selective immunosuppression and graft repair during rejection. Here, we demonstrated the impact of iPSC-derived human MSCs on the development of immune-tolerance and long-term graft survival in mouse orthotopic airway allografts. BALB/c→C57BL/6 allografts were reconstituted with iPSC-derived MSCs (2 million/transplant/ at d0), and allografts were examined for regulatory T cells (Tregs), oxygenation, microvascular blood flow, airway epithelium and collagen deposition during rejection. We demonstrated that iPSC-derived MSC treatment leads to significant increase in tissue expression of hTSG-6 protein, followed by an upregulation of mouse Tregs and IL-5, IL-10, IL-15 cytokines, which augments graft microvascular blood flow and oxygenation, and thereby maintained a healthy airway epithelium and prevented the subepithelial deposition of collagen at d90 post-transplantation. Collectively, these data confirmed that iPSC-derived MSC-mediated immunosuppression has potential to establish immune-tolerance and rescue allograft from sustained hypoxic/ischemic phase and subsequently limits long-term airway epithelial injury and collagen progression, which therapeutically warrant a study of Cymerus iPSC-derived MSCs as a potential management option for immunosuppression in transplant recipients.

Keywords: stem cell therapy, immunotolerance, regulatory T cells, hypoxia and ischemia, microvasculature

Procedia PDF Downloads 133

Authors: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

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The alternative oxidase (AOX) mediates cyanide-resistant respiration, which bypasses proton-pumping complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In Chlamydomonas reinhardtii, AOX is a monomeric protein that is encoded by two genes of discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In C. reinhardtii, AOX2 was initially identified as one of constitutively low expressed genes. Like other photosynthetic organisms C. reinhardtii cells frequently experience periods of hypoxia. To examine AOX2 transcriptional regulation and role of AOX2 in hypoxia adaptation, real-time PCR analysis and artificial microRNA method were employed. Two experimental approaches have been used to induce the anoxic conditions: dark-anaerobic and light-anaerobic conditions. C. reinhardtii cells exposed to the oxygen deprivation have shown increased AOX2 mRNA levels. By contrast, AOX1 was not an anoxia-responsive gene. In C. reinhardtii, a subset of genes is regulated by transcription factor CRR1 in anaerobic conditions. Notable, the AOX2 promoter region contains the potential motif for CRR1 binding. Therefore, the role of CRR1 in the control of AOX2 transcription was tested. The CRR1-underexpressing strains, that were generated and characterized in this work, exhibited low levels of AOX2 transcripts under anoxic conditions. However, the transformants still slightly induced AOX2 gene expression in the darkness. These confirmed our suggestions that darkness is a regulatory stimulus for AOX genes in C. reinhardtii. Thus, other factors must contribute to AOX2 promoter activity under dark-anoxic conditions. Moreover, knock-down of CRR1 caused a complete reduction of AOX2 expression under light-anoxic conditions. These results indicate that (1) CRR1 is required for AOX2 expression during hypoxia, and (2) AOX2 gene is regulated by CRR1 together with yet-unknown regulatory factor(s). In addition, the AOX2-underexpressing strains were generated. The analysis of amiRNA-AOX2 strains suggested a role of this alternative oxidase in hypoxia adaptation of the alga. In conclusion, the results reported here show that C. reinhardtii AOX2 gene is stress inducible. CRR1 transcriptional factor is involved in the regulation of the AOX2 gene expression in the absence of oxygen. Moreover, AOX2 but not AOX1 functions under oxygen deprivation. This work was supported by Russian Science Foundation (research grant № 16-14-10004).

Keywords: alternative oxidase 2, artificial microRNA approach, chlamydomonas reinhardtii, hypoxia

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Authors: Igor N. Kosenko

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The present contribution is an analysis of more than 300 specimens of Upper Jurassic to Lower Cretaceous oysters collected by V.A. Zakharov during the 1960s and currently stored in the Trofimuk Institute of Geology and Geophysics SB RAS (Novosibirsk, Russia). They were sampled in the northwestern bounder of Western Siberia (Yatriya, Maurynia, Tol’ya and Lopsiya rivers) and the north of Eastern Siberia (Boyarka, Bolshaya Romanikha and Dyabaka-Tari rivers). During the last five years, they were examined with taxonomical and palaeoecological purposes. Based on carbonate material of oyster’s shells were performed isotopic analyses and associated palaeotemperatures. Taxonomical study consists on classical morphofunctional and biometrical analyses. It is completed by another large amount of Cretaceous oysters from Crimea as well as modern Pacific oyster - Crassostrea gigas. Those were studied to understand the range of modification variability between different species. Oysters previously identified as Liostrea are attributed now to four genera: Praeexogyra and Helvetostrea (Flemingostreidae), Pernostrea (Gryphaeidae) and one new genus (Gryphaeidae), including one species “Liostrea” roemeri (Quenstedt). This last is characterized by peculiar ethology, being attached to floating ammonites and morphology, outlined by a beak-shaped umbo on the right (!) valve. Endemic Siberian species from the Pernostrea genus have been included into the subgenus Boreiodeltoideum subgen. nov. Pernostrea and Deltoideum genera have been included into the tribe Pernostreini n. trib. from the Gryphaeinae subfamily. Model of phylogenetic relationships between species of this tribe has been proposed. Siberian oyster complexes were compared with complexes from Western Europe, Poland and East European Platform. In western Boreal and Subboreal Realm (England, northern France and Poland) two stages of oyster’s development were recognized: Jurassic-type and Cretaceous-type. In Siberia, Jurassic and Lower Cretaceous oysters formed a unique complex. It may be due to the isolation of the Siberian Basin toward the West during the Early Cretaceous. Seven oyster’s shells of Pernostrea (Pernostrea) uralensis (Zakharov) from the Jurassic/Cretaceous Boundary Interval (Upper Volgian – Lower Ryazanian) of Maurynia river were used to perform δ13C and δ18O isotopic analyses. The preservation of the carbonate material was controlled by: cathodoluminescence analyses; content of Fe, Mn, Sr; absence of correlation between δ13C and δ18O and content of Fe and Mn. The obtained δ13C and δ18O data were compared with isotopic data based on belemnites from the same stratigraphical interval of the same section and were used to trace palaeotemperatures. A general trend towards negative δ18O values is recorded in the Maurynia section, from the lower part of the Upper Volgian to the middle part of the Ryazanian Chetaites sibiricus ammonite zone. This trend was previously recorded in the Nordvik section. The higher palaeotemperatures (2°C in average) determined from oyster’s shells indicate that belemnites likely migrated laterally and lived part of their lives in cooler waters. This work financially supported by the Russian Foundation for Basic Researches (grant no. 16-35-00003).

Keywords: isotopes, oysters, Siberia, taxonomy

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Authors: Mustafa M. Donma, Orkide Donma

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Obesity is a clinical state associated with low-grade inflammation. It is also a major risk factor for insulin resistance (IR). In its advanced stages, metabolic syndrome (MetS), a much more complicated disease which may lead to life-threatening problems, may develop. Obesity-mediated IR seems to correlate with the inflammation. Human studies performed particularly on pediatric population are scarce. The aim of this study is to detect possible associations between inflammation and IR in terms of some related ratios. 549 children were grouped according to their age- and sex-based body mass index (BMI) percentile tables of WHO. MetS components were determined. Informed consent and approval from the Ethics Committee for Clinical Investigations were obtained. The principles of the Declaration of Helsinki were followed. The exclusion criteria were infection, inflammation, chronic diseases and those under drug treatment. Anthropometric measurements were obtained. Complete blood cell, fasting blood glucose, insulin, and C-reactive protein (CRP) analyses were performed. Homeostasis model assessment of insulin resistance (HOMA-IR), systemic immune inflammation (SII) index, tense index, alanine aminotransferase to aspartate aminotransferase ratio (ALT/AST), neutrophils to lymphocyte (NLR), platelet to lymphocyte, and lymphocyte to monocyte ratios were calculated. Data were evaluated by statistical analyses. The degree for statistical significance was 0.05. Statistically significant differences were found among the BMI values of the groups (p < 0.001). Strong correlations were detected between the BMI and waist circumference (WC) values in all groups. Tense index values were also correlated with both BMI and WC values in all groups except overweight (OW) children. SII index values of children with normal BMI were significantly different from the values obtained in OW, obese, morbid obese and MetS groups. Among all the other lymphocyte ratios, NLR exhibited a similar profile. Both HOMA-IR and ALT/AST values displayed an increasing profile from N towards MetS3 group. BMI and WC values were correlated with HOMA-IR and ALT/AST. Both in morbid obese and MetS groups, significant correlations between CRP versus SII index as well as HOMA-IR versus ALT/AST were found. ALT/AST and HOMA-IR values were correlated with NLR in morbid obese group and with SII index in MetS group, (p < 0.05), respectively. In conclusion, these findings showed that some parameters may exhibit informative differences between the early and late stages of obesity. Important associations among HOMA-IR, ALT/AST, NLR and SII index have come to light in the morbid obese and MetS groups. This study introduced the SII index and NLR as important inflammatory markers for the discrimination of normal and obese children. Interesting links were observed between inflammation and IR in morbid obese children and those with MetS, both being late stages of obesity.

Keywords: children, inflammation, insulin resistance, metabolic syndrome, obesity

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Authors: Sulalit Bandyopadhyay, Muhammad Awais Ashfaq Alvi, Anuvansh Sharma, Wilhelm R. Glomm

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Release of drugs from nanogels and nanogel-based systems can occur under the influence of external stimuli like temperature, pH, magnetic fields and so on. pNIPAm-AAc nanogels respond to the combined action of both temperature and pH, the former being mostly determined by hydrophilic-to-hydrophobic transitions above the volume phase transition temperature (VPTT), while the latter is controlled by the degree of protonation of the carboxylic acid groups. These nanogels based systems are promising candidates in the field of drug delivery. Combining nanogels with magneto-plasmonic nanoparticles (NPs) introduce imaging and targeting modalities along with stimuli-response in one hybrid system, thereby incorporating multifunctionality. Fe@Au core-shell NPs possess optical signature in the visible spectrum owing to localized surface plasmon resonance (LSPR) of the Au shell, and superparamagnetic properties stemming from the Fe core. Although there exist several synthesis methods to control the size and physico-chemical properties of pNIPAm-AAc nanogels, yet, there is no comprehensive study that highlights the dependence of incorporation of one or more layers of NPs to these nanogels. In addition, effective determination of volume phase transition temperature (VPTT) of the nanogels is a challenge which complicates their uses in biological applications. Here, we have modified the swelling-collapse properties of pNIPAm-AAc nanogels, by combining with Fe@Au NPs using different solution based methods. The hydrophilic-hydrophobic transition of the nanogels above the VPTT has been confirmed to be reversible. Further, an analytical method has been developed to deduce the average VPTT which is found to be 37.3°C for the nanogels and 39.3°C for nanogel coated Fe@Au NPs. An opposite swelling –collapse behaviour is observed for the latter where the Fe@Au NPs act as bridge molecules pulling together the gelling units. Thereafter, Cyt C, a model protein drug and L-Dopa, a drug used in the clinical treatment of Parkinson’s disease were loaded separately into the nanogels and nanogel coated Fe@Au NPs, using a modified breathing-in mechanism. This gave high loading and encapsulation efficiencies (L Dopa: ~9% and 70µg/mg of nanogels, Cyt C: ~30% and 10µg/mg of nanogels respectively for both the drugs. The release kinetics of L-Dopa, monitored using UV-vis spectrophotometry was observed to be rather slow (over several hours) with highest release happening under a combination of high temperature (above VPTT) and acidic conditions. However, the release of L-Dopa from nanogel coated Fe@Au NPs was the fastest, accounting for release of almost 87% of the initially loaded drug in ~30 hours. The chemical structure of the drug, drug incorporation method, location of the drug and presence of Fe@Au NPs largely alter the drug release mechanism and the kinetics of these nanogels and Fe@Au NPs coated with nanogels.

Keywords: controlled release, nanogels, volume phase transition temperature, l-dopa

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Authors: Despoina Chochtoula, Aristidis Ilias, Yannis Stamatiou

Abstract:

Modbus is a protocol that enables the communication among devices which are connected to the same network. This protocol is, often, deployed in connecting sensor and monitoring units to central supervisory servers in Supervisory Control and Data Acquisition, or SCADA, systems. These systems monitor critical infrastructures, such as factories, power generation stations, nuclear power reactors etc. in order to detect malfunctions and ignite alerts and corrective actions. However, due to their criticality, SCADA systems are vulnerable to attacks that range from simple eavesdropping on operation parameters, exchanged messages, and valuable infrastructure information to malicious modification of vital infrastructure data towards infliction of damage. Thus, the SCADA research community has been active over strengthening SCADA systems with suitable data protection mechanisms based, to a large extend, on cryptographic methods for data encryption, device authentication, and message integrity protection. However, due to the limited computation power of many SCADA sensor and embedded devices, the usual public key cryptographic methods are not appropriate due to their high computational requirements. As an alternative, Elliptic Curve Cryptography has been proposed, which requires smaller key sizes and, thus, less demanding cryptographic operations. Until now, however, no such implementation has been proposed in the SCADA literature, to the best of our knowledge. In order to fill this gap, our methodology was focused on integrating Modbus, a frequently used SCADA communication protocol, with Elliptic Curve based cryptography and develop a server/client application to demonstrate the proof of concept. For the implementation we deployed two C language libraries, which were suitably modify in order to be successfully integrated: libmodbus (https://github.com/stephane/libmodbus) and ecc-lib https://www.ceid.upatras.gr/webpages/faculty/zaro/software/ecc-lib/). The first library provides a C implementation of the Modbus/TCP protocol while the second one offers the functionality to develop cryptographic protocols based on Elliptic Curve Cryptography. These two libraries were combined, after suitable modifications and enhancements, in order to give a modified version of the Modbus/TCP protocol focusing on the security of the data exchanged among the devices and the supervisory servers. The mechanisms we implemented include key generation, key exchange/sharing, message authentication, data integrity check, and encryption/decryption of data. The key generation and key exchange protocols were implemented with the use of Elliptic Curve Cryptography primitives. The keys established by each device are saved in their local memory and are retained during the whole communication session and are used in encrypting and decrypting exchanged messages as well as certifying entities and the integrity of the messages. Finally, the modified library was compiled for the Android environment in order to run the server application as an Android app. The client program runs on a regular computer. The communication between these two entities is an example of the successful establishment of an Elliptic Curve Cryptography based, secure Modbus wireless communication session between a portable device acting as a supervisor station and a monitoring computer. Our first performance measurements are, also, very promising and demonstrate the feasibility of embedding Elliptic Curve Cryptography into SCADA systems, filling in a gap in the relevant scientific literature.

Keywords: elliptic curve cryptography, ICT security, modbus protocol, SCADA, TCP/IP protocol

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Authors: Alsu F. Saifitdinova, Alexey S. Komissarov, Svetlana A. Galkina, Elena I. Koshel, Maria M. Kulak, Stephen J. O'Brien, Elena R. Gaginskaya

Abstract:

The mystery of sex determination is one of the most ancient and still not solved until the end so far. In many species, sex determination is genetic and often accompanied by the presence of dimorphic sex chromosomes in the karyotype. Genomic sequencing gave the information about the gene content of sex chromosomes which allowed to reveal their origin from ordinary autosomes and to trace their evolutionary history. Female-specific W chromosome in birds as well as mammalian male-specific Y chromosome is characterized by the degeneration of gene content and the accumulation of repetitive DNA. Tandem repeats complicate the analysis of genomic data. Despite the best efforts chicken W chromosome assembly includes only 1.2 Mb from expected 55 Mb. Supplementing the information on the sex chromosome composition not only helps to complete the assembly of genomes but also moves us in the direction of understanding of the sex-determination systems evolution. A whole-genome survey to the assembly Gallus_gallus WASHUC 2.60 was applied for repeats search in assembled genome and performed search and assembly of high copy number repeats in unassembled reads of SRR867748 short reads datasets. For cytogenetic analysis conventional methods of fluorescent in situ hybridization was used for previously cloned W specific satellites and specifically designed directly labeled synthetic oligonucleotide DNA probe was used for bioinformatically identified repetitive sequence. Hybridization was performed with mitotic chicken chromosomes and manually isolated giant meiotic lampbrush chromosomes from growing oocytes. A novel chicken W specific satellite (GGAAA)n which is not co-localizes with any previously described classes of W specific repeats was identified and mapped with high resolution. In the composition of autosomes this repeat units was found as a part of upstream regions of gonad specific protein coding sequences. These findings may contribute to the understanding of the role of tandem repeats in sex specific differentiation regulation in birds and sex chromosome evolution. This work was supported by the postdoctoral fellowships from St. Petersburg State University (#1.50.1623.2013 and #1.50.1043.2014), the grant for Leading Scientific Schools (#3553.2014.4) and the grant from Russian foundation for basic researches (#15-04-05684). The equipment and software of Research Resource Center “Chromas” and Theodosius Dobzhansky Center for Genome Bioinformatics of Saint Petersburg State University were used.

Keywords: birds, lampbrush chromosomes, sex chromosomes, tandem repeats

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Authors: Cai J. T. Ladd, Mollie F. Duggan-Edwards, Tjeerd J. Bouma, Jordi F. Pages, Martin W. Skov

Abstract:

Salt marshes are valued for their role in coastal flood protection, carbon storage, and for supporting biodiverse ecosystems. As a biogeomorphic landscape, marshes evolve through the complex interactions between sea level rise, sediment supply and wave/current forcing, as well as and socio-economic factors. Climate change and direct human modification could lead to a global decline marsh extent if left unchecked. Whilst the processes of saltmarsh erosion and expansion are well understood, empirical evidence on the key drivers of long-term lateral marsh dynamics is lacking. In a GIS, saltmarsh areal extent in 25 estuaries across Great Britain was calculated from historical maps and aerial photographs, at intervals of approximately 30 years between 1846 and 2016. Data on the key perceived drivers of lateral marsh change (namely sea level rise rates, suspended sediment concentration, bedload sediment flux rates, and frequency of both river flood and storm events) were collated from national monitoring centres. Continuous datasets did not extend beyond 1970, therefore predictor variables that best explained rate change of marsh extent between 1970 and 2016 was calculated using a Partial Least Squares Regression model. Information about the spread of Spartina anglica (an invasive marsh plant responsible for marsh expansion around the globe) and coastal engineering works that may have impacted on marsh extent, were also recorded from historical documents and their impacts assessed on long-term, large-scale marsh extent change. Results showed that salt marshes in the northern regions of Great Britain expanded an average of 2.0 ha/yr, whilst marshes in the south eroded an average of -5.3 ha/yr. Spartina invasion and coastal engineering works could not explain these trends since a trend of either expansion or erosion preceded these events. Results from the Partial Least Squares Regression model indicated that the rate of relative sea level rise (RSLR) and availability of suspended sediment concentration (SSC) best explained the patterns of marsh change. RSLR increased from 1.6 to 2.8 mm/yr, as SSC decreased from 404.2 to 78.56 mg/l along the north-to-south gradient of Great Britain, resulting in the shift from marsh expansion to erosion. Regional differences in RSLR and SSC are due to isostatic rebound since deglaciation, and tidal amplitudes respectively. Marshes exposed to low RSLR and high SSC likely leads to sediment accumulation at the coast suitable for colonisation by marsh plants and thus lateral expansion. In contrast, high RSLR with are likely not offset deposition under low SSC, thus average water depth at the marsh edge increases, allowing larger wind-waves to trigger marsh erosion. Current global declines in sediment flux to the coast are likely to diminish the resilience of salt marshes to RSLR. Monitoring and managing suspended sediment supply is not common-place, but may be critical to mitigating coastal impacts from climate change.

Keywords: lateral saltmarsh dynamics, sea level rise, sediment supply, wave forcing

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Authors: Paola Modesto, Floriana Fruscione, Isabella Martini, Simona Perga, Federica Riccardo, Mariateresa Camerino, Davide Giacobino, Cecilia Gola, Luca Licenziato, Elisabetta Razzuoli, Katia Varello, Lorella Maniscalco, Elena Bozzetta, Angelo Ferrari

Abstract:

Canine and human melanoma, osteosarcoma (OSA), and mammary carcinomas are aggressive tumors with common characteristics making dogs a good model for comparative oncology. Novel therapeutic strategies against these tumors could be useful to both species. In humans, chondroitin sulphate proteoglycan 4 (CSPG4) is a marker involved in tumor progression and could be a candidate target for immunotherapy. The anti-CSPG4 DNA electrovaccination has shown to be an effective approach for canine malignant melanoma (CMM) [1]. An immunohistochemistry evaluation of CSPG4 expression in tumour tissue is generally performed prior to electrovaccination. To assess the possibility to perform a rapid molecular evaluation and in order to validate these spontaneous canine tumors as the model for human studies, we investigate the CSPG4 gene expression by RT qPCR in CMM, OSA, and canine mammary tumors (CMT). The total RNA was extracted from RNAlater stored tissue samples (CMM n=16; OSA n=13; CMT n=6; five paired normal tissues for CMM, five paired normal tissues for OSA and one paired normal tissue for CMT), retro-transcribed and then analyzed by duplex RT-qPCR using two different TaqMan assays for the target gene CSPG4 and the internal reference gene (RG) Ribosomal Protein S19 (RPS19). RPS19 was selected from a panel of 9 candidate RGs, according to NormFinder analysis following the protocol already described [2]. Relative expression was analyzed by CFX Maestro™ Software. Student t-test and ANOVA were performed (significance set at P<0.05). Results showed that gene expression of CSPG4 in OSA tissues is significantly increased by 3-4 folds when compared to controls. In CMT, gene expression of the target was increased from 1.5 to 19.9 folds. In melanoma, although an increasing trend was observed, no significant differences between the two groups were highlighted. Immunohistochemistry analysis of the two cancer types showed that the expression of CSPG4 within CMM is concentrated in isles of cells compared to OSA, where the distribution of positive cells is homogeneous. This evidence could explain the differences in gene expression results.CSPG4 immunohistochemistry evaluation in mammary carcinoma is in progress. The evidence of CSPG4 expression in a different type of canine tumors opens the way to the possibility of extending the CSPG4 immunotherapy marker in CMM, OSA, and CMT and may have an impact to translate this strategy modality to human oncology.

Keywords: canine melanoma, canine mammary carcinomas, canine osteosarcoma, CSPG4, gene expression, immunotherapy

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Authors: Ching-Yi Yiu, Hui-Chen Hsu

Abstract:

Objectives: Head and neck cancer (HNC) is a big global health problem in the world. Despite the development of diagnosis and treatment, the overall survival of HNC is still low. The well recognition of the interaction of the host immune system and cancer cells has led to realizing the processes of tumor initiation, progression and metastasis. Many systemic inflammatory responses have been shown to play a crucial role in cancer progression. The pre and post-treatment nutritional and immunological status of HNC patients is a reliable prognostic indicator of tumor outcomes and survivors. Methods: Between July 2020 to June 2022, We have enrolled 60 HNC patients, including 59 males and 1 female, in Chi Mei Medical Center, Liouying, Taiwan. The age distribution was from 37 to 81 years old (y/o), with a mean age of 57.6 y/o. We evaluated the pre-and post-treatment nutritional and immunological status of these HNC patients with body weight, body weight loss, body mass index (BMI), whole blood count including hemoglobin (Hb), lymphocyte, neutrophil and platelet counts, biochemistry including prealbumin, albumin, c-reactive protein (CRP), with the time period of before treatment, post-treatment 3 and 6 months. We calculated the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) to assess how these biomarkers influence the outcomes of HNC patients. Results: We have carcinoma of the hypopharynx in 21 cases with 35％, carcinoma of the larynx in 9 cases, carcinoma of the tonsil and tongue every 6 cases, carcinoma soft palate and tongue base every 5 cases, carcinoma of buccal mucosa, retromolar trigone and mouth floor every 2 cases, carcinoma of the hard palate and low lip each 1 case. There were stage I 15 cases, stage II 13 cases, stage III 6 cases, stage IVA 10 cases, and stage IVB 16 cases. All patients have received surgery, chemoradiation therapy or combined therapy. We have wound infection in 6 cases, 2 cases of pharyngocutaneous fistula, flap necrosis in 2 cases, and mortality in 6 cases. In the wound infection group, the average BMI is 20.4 kg/m2; the average Hb is 12.9 g/dL, the average albumin is 3.5 g/dL, the average NLR is 6.78, and the average PLR is 243.5. In the PC fistula and flap necrosis group, the average BMI is 21.65 kg/m2; the average Hb is 11.7 g/dL, the average albumin is 3.15 g/dL, average NLR is 13.28, average PLR is 418.84. In the mortality group, the average BMI is 22.3 kg/m2; the average Hb is 13.58 g/dL, the average albumin is 3.77 g/dL, the average NLR is 6.06, and the average PLR is 275.5. Conclusion: HNC is a big challenging public health problem worldwide, especially in the high prevalence of betel nut consumption area Taiwan. Besides the definite risk factors of smoking, drinking and betel nut related, the other biomarkers may play significant prognosticators in the HNC outcomes. We concluded that the average BMI is less than 22 kg/m2, the average Hb is low than 12.0 g/dL, the average albumin is low than 3.3 g/dL, the average NLR is low than 3, and the average PLR is more than 170, the surgical complications and mortality will be increased, and the prognosis is poor in HNC patients.

Keywords: nutritional, immunological, neutrophil-to-lymphocyte ratio, paltelet-to-lymphocyte ratio.

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Authors: Chooi Fong Lee

Abstract:

Background: Maternal psychological well-being has been investigated from various aspects, such as social support, employment status. However, a perspective from self-discrepancy theory has not been employed. Moreover, most were focused on young mothers. Less is understanding the middle-aged mother’s psychological well-being. Objective: To examine the influence of maternal self-discrepancy between actual and ideal self on maternal role achievement, state anxiety, trait anxiety, and subjective well-being among Japanese middle-aged mothers across their employment status. Method: A pilot study was conducted with 20 mother participants (aged 40-55, 9 regular-employed, 8 non-regular-employed, and 3 homemaker mothers) to assess the viability of survey questionnaires (Maternal Role Achievement Scale, State-Trait Anxiety Inventory, Subjective Well-being Scale, and a self-report). Participants were randomly selected voluntarily from the college students’ mothers. Participants accessed the survey via a designated URL. The self-report questionnaire prompted participants to list up to 3 ideal selves they aspired to be and rate the extent to which their actual selves deviated from their ideal selves on a 7-point scale (1= not at all; 4 = medium; 7 = extremely). The findings confirmed the validity of the survey questionnaires, indicating their appropriateness for use in subsequent research. Self-discrepancy scores were calculated by subtracting participants’ degree ratings from a 7-point scale, summing them up, and then dividing the total by 3. Setting: We ensured participants were randomly selected from the research firm to mitigate bias. The self-report questionnaire was adapted from a validated instrument and underwent rigorous modification and testing in the pilot study. The final sample consisted of 241 participants, 97 regular-employed, 87 non-regular employed, and 57 homemaker mothers. Result: The reliability coefficient for the discrepancy score is α=.75. The findings indicate that regular-employed mothers tend to exhibit lower self-discrepancy scores compared to non-regular employed and homemaker mothers. This discrepancy negatively impacts maternal role, state anxiety, and subjective well-being while positively affecting trait anxiety. Trait anxiety arises when one feels they did not meet their ideal self, as evidenced by higher levels in homemaker mothers, who experience lower state anxiety. Conversely, regular-employed mothers exhibit higher state anxiety but lower trait anxiety, suggesting satisfaction in their professional pursuits despite balancing work and family responsibilities. Full-time maternal roles contribute to lower state anxiety but higher trait anxiety among homemaker mothers due to a lack of personal identity achievement. Non-regular employed mothers show similarities to homemaker mothers. In self-reports, regular-employed mothers highlight support and devotion to their children’s development, while non-regular-employed mothers seek life fulfillment through part-time work alongside child-rearing duties. Homemaker mothers emphasize qualities like sociability, and communication skills, potentially influencing their self-discrepancy scores. Furthermore, the hierarchical multiple regression analysis revealed that the discrepancy scores significantly predict subjective well-being. Conclusion: There may be the need for broader generalizability beyond our sample of Japanese mothers; however, the findings offer valuable insights into the impact of maternal self-discrepancy on psychological well-being among middle-aged mothers across different employment statuses. Understanding these dynamics becomes crucial as contemporary women increasingly pursue higher education and depart from traditional motherhood norms.

Keywords: maternal employment, maternal role, self-discrepancy, state-trait anxiety, subjective well-being

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Authors: Ankit Khurana

Abstract:

The sufficient heat storage capacity or ability to dissipate heat is the most decisive parameter to have an effective and efficient functioning of Friction-based Brake Disc systems. The primary aim of the research was to analyse the effect of multiple coatings on lightweight disk rotors surface which not only alleviates the mass of vehicle & also, augments heat transfer. This research is projected to aid the automobile fraternity with an enunciated view over the thermal aspects in a braking system. The results of the project indicate that with the advent of modern coating technologies a brake system’s thermal curtailments can be removed and together with forced convection, heat transfer processes can see a drastic improvement leading to increased lifetime of the brake rotor. Other advantages of modifying the surface of a lightweight rotor substrate will be to reduce the overall weight of the vehicle, decrease the risk of thermal brake failure (brake fade and fluid vaporization), longer component life, as well as lower noise and vibration characteristics. A mathematical model was constructed in MATLAB which encompassing the various thermal characteristics of the proposed coatings and substrate materials required to approximate the heat flux values in a free and forced convection environment; resembling to a real-time braking phenomenon which could easily be modelled into a full cum scaled version of the alloy brake rotor part in ABAQUS. The finite element of a brake rotor was modelled in a constrained environment such that the nodal temperature between the contact surfaces of the coatings and substrate (Wrought Aluminum alloy) resemble an amalgamated solid brake rotor element. The initial results obtained were for a Plasma Electrolytic Oxidized (PEO) substrate wherein the Aluminum alloy gets a hard ceramic oxide layer grown on its transitional phase. The rotor was modelled and then evaluated in real-time for a constant ‘g’ braking event (based upon the mathematical heat flux input and convective surroundings), which reflected the necessity to deposit a conducting coat (sacrificial) above the PEO layer in order to inhibit thermal degradation of the barrier coating prematurely. Taguchi study was then used to bring out certain critical factors which may influence the maximum operating temperature of a multi-coated brake disc by simulating brake tests: a) an Alpine descent lasting 50 seconds; b) an Autobahn stop lasting 3.53 seconds; c) a Six–high speed repeated stop in accordance to FMVSS 135 lasting 46.25 seconds. Thermal Barrier coating thickness and Vane heat transfer coefficient were the two most influential factors and owing to their design and manufacturing constraints a final optimized model was obtained which survived the 6-high speed stop test as per the FMVSS -135 specifications. The simulation data highlighted the merits for preferring Wrought Aluminum alloy 7068 over Grey Cast Iron and Aluminum Metal Matrix Composite in coherence with the multiple coating depositions.

Keywords: lightweight brakes, surface modification, simulated braking, PEO, aluminum

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