Search results for: in vitro release

Authors: Rida Pervaiz, Bushra Allah Rakha, Muhammad Sajjad Ansari, Shamim Akhter, Kainat Waseem, Sumiyyah Zuha, Tooba Javed

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Ring-necked pheasant (Phasianus colchicus) belongs to order Galliformes and family Phasianidae. It has been recognized as the most hunted bird due to its attractive colorful appearance and meat. Loss of habitat and hunting pressure has caused population fluctuations in the native range. Under these circumstances, this species can be conserved by employing ex-situ in vitro conservation techniques. Captive breeding, in combination with semen cryobanking is the most appropriate option to conserve/propagate this species without deteriorating the genetic diversity. Cryopreservation protocols of adequate efficiency are necessary to establish semen cryobanking for a species. Therefore, present study was designed to devise an efficient extender for cryopreservation of ring-necked pheasant semen. For this purpose, a range of extenders (Beltsville Poultry, red fowl, Lake, EK, Tselutin Poultry and Chicken semen extenders) were evaluated for cryopreservation of ring-necked pheasant semen. Semen collected from 10 cocks, diluted in the Beltsville Poultry (BPSE), Red Fowl (RFE), Lake (LE), EK (EKE), Tselutin Poultry (TPE) and Chicken Semen (CSE) extenders and cryopreserved. Glycerol (10%) was added to semen at 4°C, equilibrated for 10 min, filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min, cryopreserved in LN2 and stored. Sperm motility (%), viability (%), live/dead ratio (%), plasma membrane (%) and DNA Integrity (%) were evaluated at post-dilution, post-cooling, post-equilibration and post-thawing stage of cryopreservation. Sperm motility (83.8 ± 3.1; 81.3 ± 3.8; 73.8 ± 2.4; 62.5 ± 1.4), viability (79.0 ± 1.7; 75.5 ± 1.6; 69.5 ± 2.3; 65.5 ± 2.4), live/dead ratio (80.5 ± 5.7; 77.3 ± 4.9; 76.0 ± 2.7; 68.3 ± 2.3), plasma membrane (74.5 ± 2.9; 73.8 ± 3.4; 71.3 ± 2.3; 75.0 ± 3.4) and DNA integrity (78.3 ± 1.7; 73.0 ± 1.2; 68.0 ± 2.0; 63.0 ± 2.5) at all four stages of cryopreservation were recorded higher (P < 0.05) in red fowl extender compared to all experimental extenders. It is concluded that red fowl extender is the best extender for cryopreservation of ring-necked pheasant semen and can be used in establishing cryobank for ex situ conservation.

Keywords: ring-necked pheasant; extenders; cryopreservation; semen quality; DNA integrity

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Authors: Zou Xuan, Liu Hongchen

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The ideal dentin desensitizing agent should not only have good biological safety, simple clinical operation mode, the superior treatment effect, but also should have a durable effect to resist the oral environmental temperature change and oral mechanical abrasion, so as to achieve a persistent desensitization effect. Also, when using desensitizing agent to prevent the post-operative hypersensitivity, we should not only prevent it from affecting crowns’ retention, but must understand its effects on bond strength of dentin adhesives. There are various of desensitizers and dentin adhesives in clinical treatment. They have different chemical or physical properties. Whether the use of desensitizing agent would affect the bond strength of dentin adhesives still need further research. In this in vitro study, we built the hypersensitive dentin model and post-operative dentin model, to evaluate the sealing effects and durability on exposed tubule by three different dentin desensitizers and to evaluate the sealing effects and the bond strength of dentin adhesives after using three different dentin desensitizers on post-operative dentin. The result of this study could provide some important references for clinical use of dentin desensitizing agent. 1. As to the three desensitizers, the hypersensitive dentin model was built to evaluate their sealing effects on exposed tubule by SEM observation and dentin permeability analysis. All of them could significantly reduce the dentin permeability. 2. Test specimens of three groups treated by desensitizers were subjected to aging treatment with 5000 times thermal cycling and toothbrush abrasion, and then dentin permeability was measured to evaluate the sealing durability of these three desensitizers on exposed tubule. The sealing durability of three groups were different. 3. The post-operative dentin model was built to evaluate the sealing effects of the three desensitizers on post-operative dentin by SEM and methylene blue. All of three desensitizers could reduce the dentin permeability significantly. 4. The influences of three desensitizers on the bonding efficiency of total-etch and self-etch adhesives were evaluated with the micro-tensile bond strength study and bond interface morphology observation. The dentin bond strength for Green or group was significantly lower than the other two groups (P<0.05).

Keywords: dentin, desensitizer, dentin permeability, thermal cycling, micro-tensile bond strength

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Authors: Rami Faraj, Cezary Graczykowski, Błażej Popławski, Grzegorz Mikułowski, Rafał Wiszowaty

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Progress in the field of sensors, electronics and computing results in more and more often applications of adaptive techniques for dynamic response mitigation. When it comes to systems excited with mechanical impacts, the control system has to take into account the significant limitations of actuators responsible for system adaptation. The paper provides a comprehensive discussion of the problem of appropriate design and implementation of adaptation techniques and mechanisms. Two case studies are presented in order to compare completely different adaptation schemes. The first example concerns a double-chamber pneumatic shock absorber with a fast piezo-electric valve and parameters corresponding to the suspension of a small unmanned aerial vehicle, whereas the second considered system is a safety air cushion applied for evacuation of people from heights during a fire. For both systems, it is possible to ensure adaptive performance, but a realization of the system’s adaptation is completely different. The reason for this is technical limitations corresponding to specific types of shock-absorbing devices and their parameters. Impact mitigation using a pneumatic shock absorber corresponds to much higher pressures and small mass flow rates, which can be achieved with minimal change of valve opening. In turn, mass flow rates in safety air cushions relate to gas release areas counted in thousands of sq. cm. Because of these facts, both shock-absorbing systems are controlled based on completely different approaches. Pneumatic shock-absorber takes advantage of real-time control with valve opening recalculated at least every millisecond. In contrast, safety air cushion is controlled using the semi-passive technique, where adaptation is provided using prediction of the entire impact mitigation process. Similarities of both approaches, including applied models, algorithms and equipment, are discussed. The entire study is supported by numerical simulations and experimental tests, which prove the effectiveness of both adaptive impact mitigation techniques.

Keywords: adaptive control, adaptive system, impact mitigation, pneumatic system, shock-absorber

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Authors: Prasamsha Panta, Da Yeon Kim, Ja Yong Jang, Min Jae Kim, Jae Ho Kim, Moon Suk Kim

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Introduction: Among the many anticancer drugs used clinically, doxorubicin (Dox), was one of widely used drugs to treat many types of solid tumors such as liver, colon, breast, or lung. Intratumoral injection of chemotherapeutic agents is a potentially more effective alternative to systemic administration because direct delivery of the anticancer drug to the target may improve both the stability and efficacy of anticancer drugs. Injectable in situ-forming gels have attracted considerable attention because they can achieve site specific drug delivery, long term action periods, and improved patient compliance. Objective: Objective of present study is to confirm clinical benefit of intratumoral chemotherapy using injectable in situ-forming poly(ethylene glycol)-b-polycaprolactone diblock copolymer (MP) and Dox with increase in efficacy and reducing the toxicity in patients with cancer diseases. Methods and methodology: We prepared biodegradable MP hydrogel and measured viscosity for the evaluation of thermo-sensitive property. In vivo antitumor activity was performed with normal saline, MP only, single free Dox, repeat free Dox, and Dox-loaded MP gel. The remaining amount of Dox drug was measured using HPLC after the mouse was sacrified. For cytotoxicity studies WST-1 assay was performed. Histological analysis was done with H&E and TUNEL processes respectively. Results: The works in this experiment showed that Dox-loaded MP have biodegradable drug depot property. Dox-loaded MP gels showed remarkable in vitro cytotoxicity activities against cancer cells. Finally, this work indicates that injection of Dox-loaded MP allowed Dox to act effectively in the tumor and induced long-lasting supression of tumor growth. Conclusion: This work has examined the potential clinical utility of intratumorally injected Dox-loaded MP gel, which shows significant effect of higher local Dox retention compared with systemically administered Dox.

Keywords: injectable in-situ forming hydrogel, anticancer, doxorubicin, intratumoral injection

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Authors: Sher Mohammed, Purushottam Lal, Pawan K. Kasera

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The Thar Desert of Rajasthan covers a wide geographical area spreading between 23.3° to 30.12°, North latitude and 69.3◦ to 76◦ Eastern latitudes; having a unique spectrum of arid zone vegetation. This desert is spreading over 12 districts having a rich source of economically important/threatened plant diversity interacting and growing with adverse climatic conditions of the area. Due to variable geological, physiographic, climatic, edaphic and biotic factors, the arid zone medicinal flora exhibit a wide collection of angiosperm families. The herbal diversity of this arid region is medicinally important in household remedies among tribal communities as well as in traditional systems. The on-going increasing disturbances in natural ecosystems are due to climatic and biological, including anthropogenic factors. The unique flora and subsequently dependent faunal diversity of the desert ecosystem is losing its biotic potential. A large number of plants have no future unless immediate steps are taken to arrest the causes, leading to their biological improvement. At present the potential loss in ecological amplitude of various genera and species is making several plant species as red listed plants of arid zone vegetation such as Commmiphora wightii, Tribulus rajasthanensis, Calligonum polygonoides, Ephedra foliata, Leptadenia reticulata, Tecomella undulata, Blepharis sindica, Peganum harmala, Sarcostoma vinimale, etc. Mostly arid zone species are under serious pressure against prevailing ecosystem factors to continuation their life cycles. Genetic, molecular, cytological, biochemical, metabolic, reproductive, germination etc. are the several points where the floral diversity of the arid zone area is facing severe ecological influences. So, there is an urgent need to conserve them. There are several opportunities in the field to carry out remarkable work at particular levels to protect the native plants in their natural habitat instead of only their in vitro multiplication.

Keywords: ecology, evaluation, xerophytes, economically, threatened plants, conservation

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Authors: Marina Arino Martin, John McEvoy, Eakalak Khan

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Endoxifen is an active metabolite responsible for the effectiveness of tamoxifen, a chemotherapeutic drug widely used for endocrine responsive breast cancer and chemo-preventive long-term treatment. Tamoxifen and endoxifen are not completely metabolized in human body and are actively excreted. As a result, they are released to the water environment via wastewater treatment plants (WWTPs). The presence of tamoxifen in the environment produces negative effects on aquatic lives due to its antiestrogenic activity. Because endoxifen is 30-100 times more potent than tamoxifen itself and also presents antiestrogenic activity, its presence in the water environment could result in even more toxic effects on aquatic lives compared to tamoxifen. Data on actual concentrations of endoxifen in the environment is limited due to recent discovery of endoxifen pharmaceutical activity. However, endoxifen has been detected in hospital and municipal wastewater effluents. The detection of endoxifen in wastewater effluents questions the treatment efficiency of WWTPs. Studies reporting information about endoxifen removal in WWTPs are also scarce. There was a study that used chlorination to eliminate endoxifen in wastewater. However, an inefficient degradation of endoxifen by chlorination and the production of hazardous disinfection by-products were observed. Therefore, there is a need to remove endoxifen from wastewater prior to chlorination in order to reduce the potential release of endoxifen into the environment and its possible effects. The aim of this research is to isolate and identify bacteria strain(s) capable of degrading endoxifen into less hazardous compound(s). For this purpose, bacteria strains from WWTPs were exposed to endoxifen as a sole carbon and nitrogen source for 40 days. Bacteria presenting positive growth were isolated and tested for endoxifen biodegradation. Endoxifen concentration and by-product formation were monitored. The Monod kinetic model was used to determine endoxifen biodegradation rate. Preliminary results of the study suggest that isolated bacteria from WWTPs are able to growth in presence of endoxifen as a sole carbon and nitrogen source. Ongoing work includes identification of these bacteria strains and by-product(s) of endoxifen biodegradation.

Keywords: biodegradation, bacterial degraders, endoxifen, wastewater

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Authors: Li Yang, Yang Song, Martin Y. M. Chiang

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Objective: To experimentally substantiate the micromechanical compatibility between cell and scaffold, in the regenerative medicine approach for restoring bone volume, is essential for phenotypic transitions Methods: Through nanotechnology and electrospinning process, nanofibrous scaffolds were fabricated to host dental follicle stem cells (DFSCs). Blends (50:50) of polycaprolactone (PCL) and silk fibroin (SF), mixed with various content of cellulose nanocrystals (CNC, up to 5% in weight), were electrospun to prepare nanofibrous scaffolds with heterogeneous microstructure in terms of fiber size. Colloidal probe atomic force microscopy (AFM) and conventional uniaxial tensile tests measured the scaffold stiffness at the micro-and macro-scale, respectively. The cell elastic modulus and cell-scaffold adhesive interaction (i.e., a chemical function) were examined through single-cell force spectroscopy using AFM. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine if the mechanotransduction signal (i.e., Yap1, Wwr2, Rac1, MAPK8, Ptk2 and Wnt5a) is upregulated by the scaffold stiffness at the micro-scale (cellular scale). Results: The presence of CNC produces fibrous scaffolds with a bimodal distribution of fiber diameter. This structural heterogeneity, which is CNC-composition dependent, remarkably modulates the mechanical functionality of scaffolds at microscale and macroscale simultaneously, but not the chemical functionality (i.e., only a single material property is varied). In in vitro tests, the osteogenic differentiation and gene expression associated with mechano-sensitive cell markers correlate to the degree of micromechanical compatibility between DFSCs and the scaffold. Conclusion: Cells require compliant scaffolds to encourage energetically favorable interactions for mechanotransduction, which are converted into changes in cellular biochemistry to direct the phenotypic evolution. The micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Keywords: phenotype transition, scaffold stiffness, electrospinning, cellulose nanocrystals, single-cell force spectroscopy

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Authors: James Britton, Vijaya Krishna, Manus Biggs, Abhay Pandit

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Regeneration of the spinal cord after injury remains a great challenge due to the complexity of this organ. Inflammation and gliosis at the injury site hinder the outgrowth of axons and hence prevent synaptic reconnection and reinnervation. Hyaluronic acid (HA) is the main component of the spinal cord extracellular matrix and plays a vital role in cell proliferation and axonal guidance. In this study, we have synthesized and characterized a photo-cross-linkable HA-tyramine (tyr) hydrogel from a chemical, mechanical, electrical, biological and structural perspective. From our experimentation, we have found that HA-tyr can be synthesized with controllable degrees of tyramine substitution using click chemistry. The complex modulus (G*) of HA-tyr can be tuned to mimic the mechanical properties of the native spinal cord via optimization of the photo-initiator concentration and UV exposure. We have examined the degree of tyramine-tyramine covalent bonding (polymerization) as a function of UV exposure and photo-initiator use via Photo and Nuclear magnetic resonance spectroscopy. Both swelling and enzymatic degradation assays were conducted to examine the resilience of our 3D printed hydrogel constructs in-vitro. Using a femtosecond 780nm laser, the two-photon polymerization of HA-tyr hydrogel in the presence of riboflavin photoinitiator was optimized. A laser power of 50mW and scan speed of 30,000 μm/s produced high-resolution spatial patterning within the hydrogel with sustained mechanical integrity. Using dorsal root ganglion explants, the cytocompatibility of photo-crosslinked HA-tyr was assessed. Using potentiometry, the electrical conductivity of photo-crosslinked HA-tyr was assessed and compared to that of native spinal cord tissue as a function of frequency. In conclusion, we have developed a biocompatible hydrogel that can be used for photolithographic 3D printing to fabricate tissue engineered constructs for neural tissue regeneration applications.

Keywords: 3D printing, hyaluronic acid, photolithography, spinal cord injury

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Authors: Lavrentiadou S. N., Angelou V., Chatzimisios K., Papazoglou L.

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The aim of this study was the isolation and culture of keratinocytes and fibroblasts from feline skin to ultimately create an artificial engineered skin (including dermis and epidermis) useful for the effective treatment of large cutaneous deficits in cats. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies using an 8 mm biopsy punch obtained from 8 healthy cats that had undergone ovariohysterectomy. The owner’s consent was obtained. All cats had a complete blood count and a serum biochemical analysis and were screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) preoperatively. The samples were cut into small pieces and incubated with collagenase (2 mg/ml) for 5-6 hours. Following digestion, cutaneous cells were filtered through a 100 μm cell strainer, washed with DMEM, and grown in DMEM supplemented with 10% FBS. The undigested epidermis was washed with DMEM and incubated with 0.05% Trypsin/0.02% EDTA (TE) solution. Keratinocytes recovered in the TE solution were filtered through a 100 μm and a 40 μm cell strainer and, following washing, were grown on a collagen type I matrix in DMEM: F12 (3:1) medium supplemented with 10% FΒS, 1 μm hydrocortisone, 1 μm isoproterenol and 0.1 μm insulin. Both fibroblasts and keratinocytes were grown in a humidified atmosphere with 5% CO2 at 37oC. The medium was changed twice a week and cells were cultured up to passage 4. Cells were grown to 70-85% confluency, at which point they were trypsinized and subcultured in a 1:4 dilution. The majority of the cells in each passage were transferred to a freezing medium and stored at -80oC. Fibroblasts were frozen in DMEM supplemented with 30% FBS and 10% DMSO, whereas keratinocytes were frozen in a complete keratinocyte growth medium supplemented with 10% DMSO. Both cell types were thawed and successfully grown as described above. Therefore, we can create a bank of fibroblasts and keratinocytes, from which we can recover cells for further culture and use for the generation of skin equivalent in vitro. In conclusion, cutaneous cell isolation and cell culture and expansion were successfully developed. To the authors’ best knowledge, this is the first study reporting isolation and culture of keratinocytes and fibroblasts from feline skin. However, these are preliminary results and thus, the development of autologous-engineered feline skin is still in process.

Keywords: cat, fibroblasts, keratinocytes, skin equivalent, wound

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Authors: Joelle Penniston, E. B. Gueguim-Kana

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Biohydrogen has been identified as a promising alternative to the use of non-renewable fossil reserves, owing to its sustainability and non-polluting nature. pH is considered as a key parameter in fermentative biohydrogen production processes, due to its effect on the hydrogenase activity, metabolic activity as well as substrate hydrolysis. The present study assesses the influence of regulating pH on dark fermentative biohydrogen production. Four experimental hydrogen production schemes were evaluated. Two were implemented using suspended cells under regulated pH growth conditions (Sus_R) and suspended and non-regulated pH (Sus_N). The two others regimes consisted of alginate immobilized cells under pH regulated growth conditions (Imm_R) and immobilized and non-pH regulated conditions (Imm_N). All experiments were carried out at 37.5°C with glucose as sole source of carbon. Sus_R showed a lag time of 5 hours and a peak hydrogen fraction of 36% and a glucose degradation of 37%, compared to Sus_N which showed a peak hydrogen fraction of 44% and complete glucose degradation. Both suspended culture systems showed a higher peak biohydrogen fraction compared to the immobilized cell system. Imm_R experiments showed a lag phase of 8 hours, a peak biohydrogen fraction of 35%, while Imm_N showed a lag phase of 5 hours, a peak biohydrogen fraction of 22%. 100% glucose degradation was observed in both pH regulated and non-regulated processes. This study showed that biohydrogen production in batch mode with suspended cells in a non-regulated pH environment results in a partial degradation of substrate, with lower yield. This scheme has been the culture mode of choice for most reported studies in biohydrogen research. The relatively lower slope in pH trend of the non-regulated pH experiment with immobilized cells (Imm_N) compared to Sus_N revealed that that immobilized systems have a better buffering capacity compared to suspended systems, which allows for the extended production of biohydrogen even under non-regulated pH conditions. However, alginate immobilized cultures in flask systems showed some drawbacks associated to high rate of gas production that leads to increased buoyancy of the immobilization beads. This ultimately impedes the release of gas out of the flask.

Keywords: biohydrogen, sustainability, suspended, immobilized

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Authors: Surbhi Surbhi, Andrea Erni, Gunter Meister, Harold Cremer, Christophe Beclin

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MicroRNAs (miRNAs) are short 20-23 nucleotide long non-coding RNAs; there are 2605 miRNA in humans and 1936 miRNA in mouse in total (miRBase). The nervous system expresses the most abundant miRNA and most diverse. MiRNAs play a role in many steps during neurogenesis, like cell proliferation, differentiation, neural patterning, axon pathfinding, etc. Moreover, in vitro studies suggested a role in the regulation of local translation at the synapse, thus controlling neuronal plasticity. However, due to the specific structure of miRNA molecules, an in-vivo confirmation of the general role of miRNAs in the control of neuronal plasticity is still pending. For example, their small size and their high level of sequence homology make difficult the analysis of their cellular and sub-cellular localization in-vivo by in-situ hybridization. Moreover, it was found that only 40% of the expressed miRNA molecules in a cell are included in RNA-Induced Silencing Complexes (RISC) and, therefore, involved in inhibitory interactions while the rest is silent. Definitively, the development of new tools is needed to have a better understanding of the cellular function of miRNAs, in particular their role in neuronal plasticity. Here we describe a new technique called in-vivo AGO-APP designed to investigate miRNA expression and function in-vivo. This technique is based on the expression of a small peptide derived from the human RISC-complex protein TNRC6B, called T6B, which binds all known Argonaute (Ago) proteins with high affinity allowing the efficient immunoprecipitation of AGO-bound miRNAs. We have generated two transgenic mouse lines conditionally expressing T6B either ubiquitously in the cell or targeted at the synapse. A comparison of the repertoire of miRNAs immuno-precipitated from mature neurons of both mouse lines will provide us with a list of miRNAs showing a specific activity at the synapse. The physiological role of these miRNAs will be subsequently addressed through gain and loss of function experiments.

Keywords: RNA-induced silencing complexes, TNRC6B, miRNA, argonaute, synapse, neuronal plasticity, neurogenesis

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Authors: Mohammad Mosaddik Rahman

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This study embarks on an exploratory journey to assess the pervasive issue of heavy metal contamination in the water bodies along Chittagong Coast, Bangladesh. Situated along the mesmerizing Bay of Bengal, known for its potential as an emerging tourist haven, economic zone, ship breaking yard, confronts significant environmental hurdles. The core of these challenges lies in the contamination from heavy metals such as lead, cadmium, chromium, and mercury, which detrimentally impact both the ecological integrity and public health of the region. This contamination primarily stems from industrial activities, particularly those involving metallurgical and chemical processes, which release these metals into the environment, leading to their accumulation in soil and water bodies. The study's primary aim is to conduct a thorough assessment of heavy metal pollution levels, alongside an analysis of nutrient variations, focusing on nitrates and nitrites. Methodologically, the study leverages systematic sampling and advanced analytical tools like the Hach 3900 spectrophotometer to ensure precise and reliable data collection. The implications of heavy metal presence are multifaceted, affecting microbial and aquatic life, and posing severe health risks to the local population, including respiratory problems, neurological disorders, and an increased risk of cancer. The results of this study highlight the urgent need for effective mitigation strategies and regulatory measures to address this critical issue. By providing a comprehensive understanding of the environmental and public health implications of heavy metal contamination in Chittagong Coast, this research endeavours to serve as a catalyst for change, emphasising the need for pollution control and advancements in water management policies. It is envisioned that the outcomes of this study will guide stakeholders in collaborating to develop and implement sustainable solutions, ultimately safeguarding the region’s environment and public health.

Keywords: heavy metal, environmental health, pollution control policies, shipbreaking yard

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Authors: Amina Aloui, Jalila Ben Salah-Abbès, Abdellah Zinedine, Amar Riba, Noel Durand, Catherine Brabet, Didier Montet, Samir Abbès

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Mycotoxin contamination of food and feed-isa globaconcern, both economically and for public health. Aflatoxin M1 (AFM1) is the principal hydroxylated metabolite of aflatoxin B1. It is frequently found in milk and other dairy products. It is responsible for the development of hepatocellular carcinoma and immunotoxic in humans and animals. The reduction of its bioavailabilitybecomesa great demand in order to protect human and animal health. The use of probiotic bacteria and clay are demonstrated to be able to bind AFM1 in vitro. This study aimed to investigate, in vivo, the activity of two-component mixture: L. rhamnosusGAF06 (LR) and bentonite for reducing the oxidative stress and the histological alterationsinduced by AFM1 in the liver andkidneys. For the experiment, male mice were divided into 7 groups (6 mice/group) and treated, orally, by AFM1, alone or in combination with LR and/or bentonite, for 10 days as follows: group 1 control, group 2 treated with LR alone (2.108 CFU/ml), group 3 treated with bentonite alone (1g/kg), group 4 treated with AFM1 alone (100μg/kg), group 5 co-treated with LR+AFM1, group 6 co-treated with bentonite+AFM1, group 7 co-treated with bentonite+LR+AFM1. At the end of the treatment, the mice were sacrificed, and the livers and kidneys were collected for histological assays. Intracellular antioxidant activities and lipid peroxidation were also studied. The results showed that AFM1causeddamage in liver and kidney tissues, being evidence of hepatotoxicity and nephrotoxicity marked by necrotic cells. It increased the MDA level and decreased the antioxidant enzyme activities (SOD) in both organs. In contrast, the co-treatment with AFM1 plus LR and/or bentonitesignificantly improved the hepatic and renal tissues, regulated kidney, and liver antioxidant enzyme activities. This improvement was more remarkable with the administration of LR-bentonite mixture with AFM1.LR and bentonite alone showed to be safe during the treatment. This mixture can be a promising candidate for future applications in biotechnological processes that aimed to detoxify AFM1in food and feed.

Keywords: aflatoxin M1, bentonite, L. rhamnosus GAF06, oxidative stress, prevention

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Authors: Xiaoquan Huang, Congcong Li, Tingting Wei, Changcun Bai, Na Liu, Meng Tang

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Silver is often doped on nano-titanium dioxide photocatalysts (Ag-TiO₂) by photodeposition method to improve their utilization of visible-light while increasing the toxicity of TiO₂。 However, it is not known what factors influence this toxicity and how to reduce toxicity while maintaining the maximum catalytic activity. In this study, Ag-TiO₂ photocatalysts were synthesized by the photodeposition method with different silver content (AgC) and photodeposition time (PDT). Characterization and catalytic experiments demonstrated that silver was well assembled on TiO₂ with excellent visible-light catalytic activity, and the size of silver increased with PDT. In vitro, the cell viability of lung epithelial cells A549 and BEAS-2B showed that the higher content and smaller size of silver doping caused higher toxicity. In vivo, Ag-TiO₂ catalysts with lower AgC or larger silver particle size obviously caused less pulmonary pro-inflammatory and pro-fibrosis responses. However, the visible light catalytic activity decreased with the increase in silver size. Therefore, in order to optimize the Ag-TiO₂ photocatalyst with the lowest pulmonary toxicity and highest catalytic performance, response surface methodology (RSM) was further performed to optimize the two independent variables of AgC and PDT. Visible-light catalytic activity was evaluated by the degradation rate of Rhodamine B, the antibacterial property was evaluated by killing log value for Escherichia coli, and cytotoxicity was evaluated by IC50 to BEAS-2B cells. As a result, the RSM model showed that AgC and PDT exhibited an interaction effect on catalytic activity in the quadratic model. AgC was positively correlated with antibacterial activity. Cytotoxicity was proportional to AgC while inversely proportional to PDT. Finally, the optimization values were AgC 3.08 w/w% and PDT 28 min. Under this optimal condition, the relatively high silver proportion ensured the visible-light catalytic and antibacterial activity, while the longer PDT effectively reduced the cytotoxicity. This study is of significance for the safe and efficient application of silver-doped TiO₂ photocatalysts.

Keywords: Ag-doped TiO₂, cytotoxicity, inflammtion, fibrosis, response surface methodology

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Authors: Mohammed Y. Elsherbeny, Mohamed Salama, Ahmed Lotfy, Hossam Fareed, Nora Mohammed

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Background: Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on brain during the early childhood when human brains are vulnerable during this period. DNT study in vivo cannot determine the effect of the neurotoxins, as it is not applicable, so using the neurosphere cells of lab animals as an alternative is applicable and time saving. Methods: Cell culture: Rat neural progenitor cells were isolated from rat embryos’ brain. The cortices were aseptically dissected out and the tissues were triturated. The dispersed tissues were allowed to settle. The supernatant was then transferred to a fresh tube and centrifuged. The pellet was placed in Hank’s balanced salt solution and cultured as free-floating neurospheres in proliferation medium. Differentiation was initiated by growth factor withdrawal in differentiation medium and plating onto a poly-d-lysine/ laminin matrix. Chemical Exposure: Neurospheres were treated for 2 weeks with papaverine in proliferation medium. Proliferation analyses: Spheres were cultured. After 0, 4, 5, 11 and 14 days, sphere size was determined by software analyses (CellProfiler, version 2.1; Broad Institute). Diameter of each neurosphere was measured and exported to excel file further to statistical analysis. Viability test: Trypsin-EDTA solution was added to neurospheres to dissociate neurospheres into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Result: As regards proliferation analysis and percentage of viable cells of papaverin treated groups: There was no significant change in cells proliferation compared to control at 0, 4, 5, 11 and 14 days with concentrations 1, 5 and 10 µM of papaverine, but there is a significant change in cell viability compared to control after 1 week and 2 weeks with the same concentrations of papaverine. Conclusion: Papaverine has toxic effect on viability of neural cell, not on their proliferation, so it may produce focal neural lesions not growth morphological changes.

Keywords: developmental neurotoxicity, neurotoxin, papaverine, neuroshperes

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Authors: Grace McCambridge, Alanna Keady, Madhur Agrawal, Dequina Nicholas Alvarado, Barbara Nikolajczyk, Leena Panneerseelan-Bharath

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Age is a non-modifiable risk factor for the inflammation that underlies pathologies such as type 2 diabetes mellitus (T2DM). Inflammation, as indicated by circulating cytokines, rises in aging, but mechanisms that promote this ‘inflammaging’ remain poorly defined. Furthermore, downstream consequences of inflammaging, including the development of an inflammatory profile that predicts comorbidities like T2DM, remain speculative. We tested the possibility that natural aging-associated changes in autophagy, a process that is compromised in both aging and T2DM, regulates inflammatory profiles in older subjects. Our data showed that circulating CD4⁺ T cells from older compared to younger subjects have (i) defects in autophagy; (ii) higher mitochondria accumulation; (iii) a failure to metabolically shift from oxidative phosphorylation to anaerobic glycolysis upon αCD3/CD28 activation; (iv) more reactive oxygen species (ROS) accumulation; and (v) a cytokine profile that recapitulates the Th17 profile that predicts T2DM. ROS scavenging in cells from older subjects restored mitochondrial mass and membrane potential (indicators of improved autophagy) and reduced Th17 cytokines to amounts made by T cells from younger subjects. Knock-down of the autophagy protein Atg3 in T cells from younger subjects increased mitochondrial accumulation and Th17 cytokines. To begin translating these findings to clinical practice, we showed that physiological concentrations of the diabetes drug metformin (100 µM) added in vitro enhanced autophagy, prevented mitochondria and ROS accumulation, increased anaerobic glycolysis, and decreased Th17 cytokines in activated CD4⁺ T cells from older subjects. Metformin therefore improves autophagy and multiple downstream pro-inflammatory mechanisms CD4⁺ T cells from older subjects. We conclude that autophagy improvement ameliorates the development of a T2DM-predictive Th17 profile in aging, and thus holds promise for delay or prevention of aging-associated metabolic decline.

Keywords: autophagy, mitochondrial turnover, ROS, glycolysis

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

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

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

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Authors: Raquel Galante, Marina Braga, Daniela Ghisleni, Terezinha J. A. Pinto, Rogério Colaço, Ana Paula Serro

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The sensitive nature of soft biomaterials, such as hydrogels, renders their sterilization a particularly challenging task for the biomedical industry. Widely used contact lenses are now studied as promising platforms for topical corneal drug delivery. However, to the best of the authors knowledge, the influence of sterilization methods on these systems has yet to be evaluated. The main goal of this study was to understand how different pairs drug-hydrogel would interact under an ozone-based sterilization method in comparison with two conventional processes (steam heat and gamma irradiation). For that, Si-Hy containing hydroxylethyl methacrylate (HEMA) and [tris(trimethylsiloxy)silyl]propyl methacrylate (TRIS) was produced and soaked in different drug solutions, commonly used for the treatment of ocular diseases (levofloxacin, chlorhexidine, diclofenac and timolol maleate). The drug release profiles and main material properties were evaluated before and after the sterilization. Namely, swelling capacity was determined by water uptake studies, transparency was accessed by UV-Vis spectroscopy, surface topography/morphology by scanning electron microscopy (SEM) and mechanical properties by performing tensile tests. The drug released was quantified by high performance liquid chromatography (HPLC). The effectiveness of the sterilization procedures was assured by performing sterility tests. Ozone gas method led to a significant reduction of drug released and to the formation of degradation products specially for diclofenac and levofloxacin. Gamma irradiation led to darkening of the loaded Si-Hys and to the complete degradation of levofloxacin. Steam heat led to smoother surfaces and to a decrease of the amount of drug released, however, with no formation of degradation products. This difference in the total drug released could be the related to drug/polymer interactions promoted by the sterilization conditions in presence of the drug. Our findings offer important insights that, in turn, could be a useful contribution to the safe development of actual products.

Keywords: drug delivery, silicone hydrogels, sterilization, gamma irradiation, steam heat, ozone gas

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Authors: Wadha Alqahtani

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In recent times, polymers have seen a great surge in interest in the field of medicine, particularly chemotherapeutics. One recent innovation is the conversion of polymeric materials into “polymeric nanoparticles”. These nanoparticles can be designed and modified to encapsulate and transport drugs selectively to cancer cells, minimizing collateral damage to surrounding healthy tissues, and improve patient quality of life. In this study, we have synthesized pseudo-branched polyester polymers from bio-based small molecules, including sorbitol, glutaric acid and a propargylic acid derivative to further modify the polymer to make it “click-able" with an azide-modified target ligand. Melt polymerization technique was used for this polymerization reaction, using lipase enzyme catalyst NOVO 435. This reaction was conducted between 90- 95 °C for 72 hours. The polymer samples were collected in 24-hour increments for characterization and to monitor reaction progress. The resulting polymer was purified with the help of methanol dissolving and filtering with filter paper then characterized via NMR, GPC, FTIR, DSC, TGA and MALDI-TOF. Following characterization, these polymers were converted to a polymeric nanoparticle drug delivery system using solvent diffusion method, wherein DiI optical dye and chemotherapeutic drug Taxol can be encapsulated simultaneously. The efficacy of the nanoparticle’s apoptotic effects were analyzed in-vitro by incubation with prostate cancer (LNCaP) and healthy (CHO) cells. MTT assays and fluorescence microscopy were used to assess the cellular uptake and viability of the cells after 24 hours at 37 °C and 5% CO2 atmosphere. Results of the assays and fluorescence imaging confirmed that the nanoparticles were successful in both selectively targeting and inducing apoptosis in 80% of the LNCaP cells within 24 hours without affecting the viability of the CHO cells. These results show the potential of using biodegradable polymers as a vehicle for receptor-specific drug delivery and a potential alternative for traditional systemic chemotherapy. Detailed experimental results will be discussed in the e-poster.

Keywords: chemotherapeutic drug, click chemistry, nanoparticle, prostat cancer

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Authors: Beth Taylor, Kojima Mituaki, Atsushi Senda, Koji Morishita, Yasuhiro Otomo

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Intestinal ischaemia-reperfusion injury drives systemic inflammation and organ failure following trauma/haemorrhagic shock (T/HS), through the release of pro-inflammatory mediators into the mesenteric lymph (ML). However, changes in the biological function of ML are not fully understood, and therefore, a specific model of intestinal ischaemia-reperfusion injury is required to obtain ML for the study of its biological function upon inflammatory cells. ML obtained from a model of intestinal ischaemia-reperfusion injury was used to assess biological function upon inflammatory cells and investigate changes in the biological function of individual ML components. An additional model was used to determine the effect of vagal nerve stimulation (VNS) upon biological function. Rat ML was obtained by mesenteric lymphatic duct cannulation before and after occlusion of the superior mesenteric artery (SMAO). ML was incubated with human polymorphonuclear neutrophils (PMNs), monocytes and lymphocytes, and the biological function of these cells was assessed. ML was then separated into supernatant, exosome and micro-vesicle components, and biological activity was compared in monocytes. A model with an additional VNS phase was developed, in which the right cervical vagal nerve was exposed and stimulated, and ML collected for comparison of biological function with the conventional model. The biological function of ML was altered by intestinal ischaemia-reperfusion injury, increasing PMN activation, monocyte activation, and lymphocyte apoptosis. Increased monocyte activation was only induced by the exosome component of ML, with no significant changes induced by the supernatant or micro-vesicle components. VNS partially attenuated monocyte activation, but no attenuation of PMN activation was observed. Intestinal ischaemia-reperfusion injury induces changes in the biological function of ML upon both innate and adaptive inflammatory cells, supporting the role of intestinal ischaemia-reperfusion injury in driving systemic inflammation following T/HS. The exosome component of ML appears to be critical to the transport of pro-inflammatory mediators in ML. VNS partially attenuates changes in innate inflammatory cell biological activity observed, presenting possibilities for future novel treatment development in multiple organ failure patients.

Keywords: exosomes, inflammation, intestinal ischaemia, mesenteric lymph, vagal stimulation

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Authors: N. A. P. Camaforte, P. M. P. Vareda, L. L. Saldanha, A. L. Dokkedal, J. M. Rezende-Neto, M. R. Senger, F. P. Silva-Jr, J. R. Bosqueiro

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Diabetes mellitus is a disease characterized by deficiency of insulin secretion and/or action which results in hyperglycemia. Nowadays, acarbose is a medicine used by diabetic people to inhibit alpha-glucosidases leading to the decreasing of post-feeding glycaemia, but with low effectiveness and many side effects. Medicinal plants have been used for the treatment of many diseases including diabetes and their action occurs through the modulation of insulin-depending processes, pancreas regeneration or inhibiting glucose absorption by the intestine. Previous studies in our laboratory showed that the treatment using two crude extracts of plants from Brazilian cerrado was able to decrease fasting blood glucose and improve glucose tolerance in streptozotocin-diabetic mice. Because of this and the importance of the search for new alternatives to decrease the hyperglycemia, we decided to evaluate the inhibitory action of two plants from Brazilian cerrado - B.H. and Myrcia bella. The enzymatic assay was performed in 50 µL of final volume using pancreatic α-amylase and maltase together with theirs commercial substrates. The inhibition potency (IC50) was determined by the incubation of eight different concentrations of both extracts and the enzymes for 5 minutes at 37ºC. After, the substrate was added to start the reaction. Glucosidases assay was evaluated measuring the quantity of p-nitrophenol in 405 nmin 384 wells automatic reader. The in vitro assay with the extracts of B.H. and M. bella showed an IC50 of 28,04µg/mL and 16,93 µg/mL for α-amilase, and 43,01µg/mL and 17 µg/mL for maltase, respectively. M. bella extract showed a higher inhibitory activity for those enzymes than B.H. extract. The crude extracts tested showed a higher inhibition rate to α-amylase, but were less effective against maltase in comparison to acarbose (IC50 36µg/mL and 9 µg/mL, respectively). In conclusion, the crude extract of B.H. and M. bella showed a potent inhibitory effect against α-amylase and showed promising results to the possible development of new medicines to treat diabetes with less or even without side effects.

Keywords: alfa-glucosidases, diabetes mellitus, glycaemia, medicinal plants

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Authors: Govindan Sudha, Mathumitha Subramaniam, Alamelu Govindasamy, Sasikala Gunasekaran

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The aim of this study was to investigate the protective effect of Hypsizygus ulmarius polysaccharides (HUP) on reducing oxidative stress, cognitive impairment and neurotoxicity in D-galactose induced aging mice. Mice were subcutaneously injected with D-galactose (150 mg/kg per day) for 6 weeks and were administered HUP simultaneously. Aged mice receiving vitamin E (100 mg/kg) served as positive control. Chronic administration of D-galactose significantly impaired cognitive performance oxidative defence and mitochondrial enzymes activities as compared to control group. The results showed that HUP (200 and 400 mg/kg) treatment significantly improved the learning and memory ability in Morris water maze test. Biochemical examination revealed that HUP significantly increased the decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), mitochondrial enzymes-NADH dehydrogenase, malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), Na+K+, Ca2+, Mg2+ATPase activities, elevated the lowered total anti-oxidation capability (TAOC), glutathione (GSH), vitamin C and decreased the raised acetylcholinesterase (AChE) activities, malondialdehyde (MDA), hydroperoxide (HPO), protein carbonyls (PCO), advanced oxidation protein products (AOPP) levels in brain of aging mice induced by D-gal in a dose-dependent manner. In conclusion, present study highlights the potential role of HUP against D-galactose induced cognitive impairment, biochemical and mitochondrial dysfunction in mice. In vitro studies on the effect of HUP on scavenging DPPH, ABTS, DMPD, OH radicals, reducing power, B-carotene bleaching and lipid peroxidation inhibition confirmed the free radical scavenging and antioxidant activity of HUP. The results suggest that HUP possesses anti-aging efficacy and may have potential in treatment of neurodegenerative diseases.

Keywords: aging, antioxidants, mushroom, neurotoxicity

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Authors: Dimakatso B. Gumede, Nicolette N. Houreld

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Diabetic foot ulcers (DFUs) are a complication of diabetes mellitus (DM), a metabolic disease caused by insulin resistance or insufficiency, resulting in hyperglycaemia and low-grade chronic inflammation. Current therapies for treating DFUs include wound debridement, glycaemic control, and wound dressing. However, these therapies are moderately effective as there is a recurrence of these ulcers and an increased risk of lower limb amputations. Photobiomodulation (PBM), which is the application of non-invasive low-level light for wound healing at the spectrum of 660-1000 nm, has shown great promise in accelerating the healing of chronic wounds. However, its underlying mechanisms are not clearly defined. Studies have indicated that PBM induces wound healing via the activation of signaling pathways that are involved in tissue repair, such as the transforming growth factor-β (TGF-β). However, other signaling pathways, such as the WNT/β-catenin pathway, which is also critical for wound repair, have not been investigated. This study aimed to elucidate if PBM at 660 nm and a fluence of 5 J/cm² activates the WNT/β-catenin signaling pathway for wound healing in a diabetic cellular model. Human dermal fibroblasts (WS1) were continuously cultured high-glucose (26.5 mM D-glucose) environment to create a diabetic cellular model. A central scratch was created in the diabetic model to ‘wound’ the cells. The diabetic wounded (DW) cells were thereafter irradiated at 660 nm and a fluence of 5 J/cm². Cell migration, gene expression and protein assays were conducted at 24- and 48-h post-PBM. The results showed that PBM at 660 nm and a fluence of 5 J/cm² significantly increased cell migration in diabetic wounded cells at 24-h post-PBM. The expression of CTNNB1, ACTA2, COL1A1 and COL3A1 genes was also increased in DW cells post-PBM. Furthermore, there was increased cytoplasmic accumulation and nuclear localization of β-catenin at 24 h post-PBM. The findings in this study demonstrate that PBM activates the WNT/β-catenin signaling pathway by inducing the accumulation of β-catenin in diabetic wounded cells, leading to increased cell migration and expression of wound repair markers. These results thus indicate that PBM has the potential to improve wound healing in diabetic ulcers via activation of the WNT/β-catenin signaling pathway.

Keywords: wound healing, diabetic ulcers, photobiomodulation, WNT/β-catenin, signalling pathway

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Authors: Jeremy Bost, Matthew Brett, Jacob Flynn, Weihui Li

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One response during anaphylaxis is reduced blood pressure due to blood vessels relaxing and dilating. Epinephrine causes the blood vessels to constrict, which raises blood pressure to counteract the symptoms. When going through an allergic reaction, an Epinephrine injector is used to administer a shot of epinephrine intramuscularly. Epinephrine injectors have become an integral part of day-to-day life for people with allergies. Current Epinephrine injectors (EpiPen) are completely mechanical and have no sensors to monitor the vital signs of patients or give suggestions the optimal time for the shot. The EpiPens are also large and inconvenient to carry daily. The current price of an EpiPen is roughly 600$ for a pack of two. This makes carrying an EpiPen very expensive, especially when they need to be switched out when the epinephrine expires. This new design is in the form of a bracelet, which has the ability to inject epinephrine. The bracelet will be equipped with vital signs monitors that can aid the patient to sense the allergic reaction. The vital signs that would be of interest are blood pressure, heart rate and Electrodermal activity (EDA). The heart rate of the patient will be tracked by a photoplethysmograph (PPG) that is incorporated into the sensors. The heart rate is expected to increase during anaphylaxis. Blood pressure will be monitored through a radar sensor, which monitors the phase changes in electromagnetic waves as they reflect off of the blood vessel. EDA is under autonomic control. Allergen-induced anaphylaxis is caused by a release of chemical mediators from mast cells and basophils, thus changes the autonomic activity of the patient. So by measuring EDA, it will give the wearer an alert on how their autonomic nervous system is reacting. After the vital signs are collected, they will be sent to an application on a smartphone to be analyzed, which can then alert an emergency contact if the epinephrine injector on the bracelet is activated. Overall, this design creates a safer system by aiding the user in keeping track of their epinephrine injector, while making it easier to track their vital signs. Also, our design will be more affordable and more convenient to replace. Rather than replacing the entire product, only the needle and drug will be switched out and not the entire design.

Keywords: allergy, anaphylaxis, epinephrine, injector, vital signs monitor

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Authors: Shivankar Agrawal, Sunil Kumar Deshmukh, Colin Barrow, Alok Adholeya

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A variety of genetic and environmental factors cause various cosmetics and dermatological problems. There are already claimed drugs available in market for treating these problems. However, the challenge remains in finding more potent, environmental friendly, causing minimal side effects and economical cosmeceuticals. This leads to an increased demand for natural cosmeceutical products in the last few decades. Plant derived ingredients are limited because plants either contain toxic metabolites, grow too slow or seasonal harvesting is a problem. To identify new bioactive cosmetics ingredients of marine microbial bioresource, we screened 35 marine fungi isolated from marine samples collected from Andaman Island and west coast of India. Fungal crude extracts were investigated for their antityrosinase, antioxidant and antibacterial activities for the purpose of identifying anti-aging, skin-whitening and anti-acne biomolecule with the potential in cosmetics. In the tyrosinase inhibition and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays, two fungal extracts, including “P2”, Talaromyces stipitatus and “D4”, Aspergillus terreus showed high inhibitory activity at 1mg/mL for tyrosinase inhibition and 0.5mg/mL for DPPH scavenging. The in vitro antimicrobial activity was investigated by the agar well diffusion method. In the tyrosinase inhibition assay, 8 extracts showed significant antibacterial activity against bacteria causing skin and wound infection in humans. In the course of systematic screening program for bioactive marine fungi, strain “D5” was found to be most potent strain with MIC value of 1mg/mL, which was morphologically identified as Simplicillium lamellicola. The effects of the most active crude extracts against their susceptible test microorganisms were also investigated by SEM analysis. Further investigations will focus on purification and characterization major active components responsible for these activities.

Keywords: antioxidant, antimicrobial activity, tyrosinase, cosmeceuticals, marine fungi

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Authors: Maria Giovanna Lupo, Marina Camera, Marcello Rattazzi, Nicola Ferri

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A complex interplay among chronic kidney disease, lipid metabolism and aortic calcification has been recognized starting from results of many clinical and experimental studies. Here we investigated the influence of kidney function on PCSK9 levels, both in uremic rats and in clinical observation study, and its potential direct action on cultured smooth muscle cells (SMCs) calcification. In a cohort of 594 subjects enrolled in a single centre, observational, cross-sectional and longitudinal study, a negative association between GFR and plasma PCSK9 was found. Atherosclerotic cardiovascular disease (ASCVD), as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats, induced aortic calcification and increased total cholesterol and PCSK9 levels in plasma, livers and kidneys. Immunohistochemical analysis confirmed PCSK9 expression in aortic SMCs. SMCs overexpressing PCSK9 (SMCsPCSK9), cultured for 7-days in a pro-calcification environment (2.0mM or 2.4mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to mocked SMCs. Under the same experimental conditions, the addition of exogenous recombinant PCSK9 did not increase the extracellular calcification of SMCs. By flow cytometry analysis we showed that SMCsPCSK9, in response to 2.4mM Pi, released higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALPL), compared to EVs from mocks SMCs. In conclusion, our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated to CKD condition. This effect appears to be mediated by a positive effect of endogenous PCSK9 on the release of EVs containing Ca2+ and ALP, which facilitate the deposition inorganic calcium phosphate crystals.

Keywords: PCSK9, calcification, extracellular vesicles, chronic kidney disease

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Authors: Fadia Gafri, Kathryn Mckintosh, Louise Young, Alan Harvey, Simon Mackay, Andrew Paul, Robin Plevin

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Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor found originally to play a key role in regulating inflammation. However considerable evidence links this pathway to the suppression of apoptosis, cellular transformation, proliferation and invasion (Aggarwal et al., 2006). Moreover, recent studies have also linked inflammation to cancer progression making NF-κB overall a promising therapeutic target for drug discovery (Dobrovolskaia & Kozlov, 2005). In this study we examined the effect of the natural product canthin-6-one (SU182) as part of a CRUK small molecule drug discovery programme for effects upon the NF-κB pathway. Initial studies demonstrated that SU182 was found to have good potency against the inhibitory kappa B kinases (IKKs) at 30M in vitro. However, at concentrations up to 30M, SU182 had no effect upon TNFα stimulated loss in cellular IκBα or p65 phosphorylation in the keratinocyte cell line NCTC2544. Nevertheless, 30M SU182 reduced TNF-α / PMA-induced NF-κB-linked luciferase reporter activity to (22.9 ± 5%) and (34.6± 3 %, P<0.001) respectively, suggesting an action downstream of IKK signalling. Indeed, SU182 neither decreased NF-κB-DNA binding as assayed by EMSA nor prevented the translocation of p65 (NF-κB) to the nucleus assessed by immunofluorescence and subcellular fractionation. In addition to the inhibition of transcriptional activity of TNFα-induced NF-κB reporter activity SU182 significantly reduced PMA-induced AP-1-linked luciferase reporter activity to about (48± 9% at 30M, P<0.001) . This mode of inhibition was not sufficient to prevent the activation of NF-κB dependent induction of other proteins such as COX-2 and iNOS, or activated MAP kinases (p38, JNK and ERK1/2) in LPS stimulated RAW 264.7 macrophages. Taken together these data indicate the potential for SU182 to interfere with the transcription factors NF-κB and AP-1 at transcriptional level. However, no potential anti-inflammatory effect was indicated, further investigation for other NF-κB dependent proteins linked to survival are also required to identify the exact mechanism of action.

Keywords: Canthin-6-one, NF-κB, AP-1, phosphorylation, Nuclear translocation, DNA-binding activity, inflammatory proteins.

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Authors: Cenza Rhoda, Falone Sunda, Elvis Kidzeru, Nonhlanhla P. Khumalo, Afolake Arowolo

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Introduction: The human FAM111B gene mutations are associated with POIKTMP, a rare multi-organ fibrosing disease. Recent studies also reported the overexpression of FAM111B in specific cancers. However, the role of FAM111B in these pathologies, particularly fibrosarcoma, remains unknown. Materials and Methods: FAM111B RNA expression in some cancer cell lines was assessed in silico and validated in vitro in these cell lines and skin fibroblasts derived from the South African family member affected by POIKTMP with the heterozygous FAM111B gene mutation: NM_198947.4: c.1861T>G (p. Tyr621Asp or Y621D) by qPCR and western blot. The cellular function of FAM111B was also studied in HT1080 using various cell-based functional assays and a simple and cost-effective PCR-RFLP method for genotyping/screening FAM111B gene mutations described. Results: Expression studies showed upregulated FAM111B mRNA and protein in the cancer cells. High FAM111B expression with robust nuclear localization occurred in HT1080. Additionally, expression data and cell-based assays indicated that FAM111B led to the upregulation of cell migration and decreased cell apoptosis and cell proliferation modulation. FAM111B Y621D mutation showed similar effects on cell migration but minimal impact on cell apoptosis. FAM111B mRNA and protein expression were markedly downregulated (p ≤ 0.05) in the patient's skin-derived fibroblasts. Lastly, the PCR-RFLP method successfully genotyped FAM111B Y621D gene mutation. Discussion: FAM111B is a cancer-associated nuclear protein: Its modulation by mutations may enhance cell migration and proliferation and decrease apoptosis, as seen in cancers and POIKTMP/fibrosis, thus representing a viable therapeutic target in these disorders. Furthermore, the PCR-RFLP method could prove a valuable tool for FAM111B mutation validation or screening in resource-constrained laboratories.

Keywords: FAM111B, POIKTMP, cancer, fibrosis, PCR-RFLP

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Authors: Christin Koch, Andreas Winkel, Martin Kahlmeyer, Stefan Böhm

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Due to environmental regulations on greenhouse gas emissions and the depletion of fossil fuels, there is an increasing interest in electric vehicles.To maximize their driving range, batteries with high storage capacities are needed. In most electric cars, rechargeable lithium-ion batteries are used because of their high energy density. However, it has to be taken into account that these batteries generate a large amount of heat during the charge and discharge processes. This leads to a decrease in a lifetime and damage to the battery cells when the temperature exceeds the defined operating range. To ensure an efficient performance of the battery cells, reliable thermal management is required. Currently, the cooling is achieved by heat sinks (e.g., cooling plates) bonded to the battery cells with a thermally conductive adhesive (TCA) that directs the heat away from the components. Especially when large amounts of heat have to be dissipated spontaneously due to peak loads, the principle of heat conduction is not sufficient, so attention must be paid to the mechanism of heat storage. An efficient method to store thermal energy is the use of phase change materials (PCM). Through an isothermal phase change, PCM can briefly absorb or release thermal energy at a constant temperature. If the phase change takes place in the transition from solid to liquid, heat is stored during melting and is released to the ambient during the freezing process upon cooling. The presented work displays the great potential of thermally conductive adhesives filled with microencapsulated PCM to limit peak temperatures in battery systems. The encapsulation of the PCM avoids the effects of aging (e.g., migration) and chemical reactions between the PCM and the adhesive matrix components. In this study, microencapsulation has been carried out by in situ polymerization. The microencapsulated PCM was characterized by FT-IR spectroscopy, and the thermal properties were measured by DSC and laser flash method. The mechanical properties, electrical and thermal conductivity, and adhesive toughness of the TCA/PCM composite were also investigated.

Keywords: phase change material, microencapsulation, adhesive bonding, thermal management

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Authors: Hongtao Zhu, Dezhi Sun

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Nitrogen and phosphorus are essential nutrients for biomass growth. But excessive nitrogen and phosphorus can contribute to accelerated eutrophication of lakes and rivers. Constructed wetland is an efficient and eco-friendly wastewater treatment technology with low operating cost and low-energy consumption. Because of high affinity with ammonium ion, zeolite, as a common substrate, is applied in constructed wetlands worldwide. Another substrate seen commonly for constructed wetlands is steel slag, which has high contents of Ca, Al, or Fe, and possesses a strong affinity with phosphate. Due to the excellent ammonium removal ability of zeolite and phosphate removal ability of steel slag, they were considered to be combined in the substrate bed of a constructed wetland in order to enhance the simultaneous removal efficiencies of nitrogen and phosphorus. In our early tests, zeolite and steel slag were combined with each other in order to simultaneously achieve a high removal efficiency of ammonium-nitrogen and phosphate-phosphorus. However, compared with the results when only zeolite was used, the removal efficiency of ammonia was sharply decreased when zeolite and steel slag were used together. The main objective of this study was to establish an overview of the interaction of steel slag and zeolite on ammonium nitrogen removal. The CaO dissolution from slag, as well as the effects of influencing parameters (i.e. pH and Ca2+ concentration) on the ammonium adsorption onto zeolite, was systematically studied. Modeling results of Ca2+ and OH- release from slag indicated that pseudo-second order reaction had a better fitness than pseudo-first order reaction. Changing pH value from 7 to 12 would result in a drastic reduction of the ammonium adsorption capacity on zeolite, from the peak at pH7. High Ca2+ concentration in solution could also inhibit the adsorption of ammonium onto zeolite. The mechanism for steel slag inhibiting the ammonium adsorption capacity of zeolite includes: on one hand, OH- released from steel slag can react with ammonium ions to produce molecular form ammonia (NH3∙H2O), which would cause the dissociation of NH4+ from zeolite. On the other hand, Ca2+ could replace the NH4+ ions to adhere onto the surface of zeolite. An innovative substrate filling configuration that zeolite and steel slag are placed sequentially was proposed to eliminate the disadvantageous effects of steel slag. Experimental results showed that the novel filling configuration was superior to the other two contrast filling configurations in terms of ammonium removal.

Keywords: ammonium nitrogen, constructed wetlands, steel slag, zeolite

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