Search results for: biomolecular sequence synthesis

Authors: Shiva Shakori Poshteh

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Lung cancer is a highly prevalent and devastating disease, representing a significant global health concern with profound implications for healthcare systems and society. Its high incidence, mortality rates, and late-stage diagnosis contribute to its formidable nature. To address these challenges, nanoparticle-based drug delivery has emerged as a promising therapeutic strategy. Curcumin (CUR), a natural compound derived from turmeric, has garnered attention as a potential nanomedicine for lung cancer treatment. Nanoparticle formulations of CUR offer several advantages, including improved drug delivery efficiency, enhanced stability, controlled release kinetics, and targeted delivery to lung cancer cells. CUR exhibits a diverse array of effects on cancer cells. It induces apoptosis by upregulating pro-apoptotic proteins, such as Bax and Bak, and downregulating anti-apoptotic proteins, such as Bcl-2. Additionally, CUR inhibits cell proliferation by modulating key signaling pathways involved in cancer progression. It suppresses the PI3K/Akt pathway, crucial for cell survival and growth, and attenuates the mTOR pathway, which regulates protein synthesis and cell proliferation. CUR also interferes with the MAPK pathway, which controls cell proliferation and survival, and modulates the Wnt/β-catenin pathway, which plays a role in cell proliferation and tumor development. Moreover, CUR exhibits potent antioxidant activity, reducing oxidative stress and protecting cells from DNA damage. Utilizing CUR as a standalone treatment is limited by poor bioavailability, lack of targeting, and degradation susceptibility. Nanoparticle-based delivery systems can overcome these challenges. They enhance CUR’s bioavailability, protect it from degradation, and improve absorption. Further, Nanoparticles enable targeted delivery to lung cancer cells through surface modifications or ligand-based targeting, ensuring sustained release of CUR to prolong therapeutic effects, reduce administration frequency, and facilitate penetration through the tumor microenvironment, thereby enhancing CUR’s access to cancer cells. Thus, nanoparticle-based CUR delivery systems promise to improve lung cancer treatment outcomes. This article provides an overview of lung cancer, explores CUR nanoparticles as a treatment approach, discusses the benefits and challenges of nanoparticle-based drug delivery, and highlights prospects for CUR nanoparticles in lung cancer treatment. Future research aims to optimize these delivery systems for improved efficacy and patient prognosis in lung cancer.

Keywords: lung cancer, curcumin, nanomedicine, nanoparticle-based drug delivery

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Authors: Luis F. Caballero-castro, Dirsa Feliciano, Daniela Novoa, Orlando Arroyo, Jesús D. Villalba-morales

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Colombia faces a critical challenge in seismic resilience due to the prevalence of informal housing, which constitutes approximately 70% of residential structures. More than 10 million Colombians (20% of the population), live in homes susceptible to collapse in the event of an earthquake. This, combined with the fact that 83% of the population is in intermediate and high seismic hazard areas, has brought serious consequences to the country. These consequences became evident during the 1999 Armenia earthquake, which affected nearly 100,000 properties and represented economic losses equivalent to 1.88% of that year's Gross Domestic Product (GDP). Despite previous efforts to reinforce informal housing through methods like externally reinforced masonry walls, alternatives related to seismic protection systems (SPDs), such as Buckling-Restrained Braces (BRB), have not yet been explored in the country. BRBs are reinforcement elements capable of withstanding both compression and tension, making them effective in enhancing the lateral stiffness of structures. In this study, the use of low-cost and easily installable BRBs for the retrofit of informal housing in Colombia was evaluated, considering the economic limitations of the communities. For this purpose, a case study was selected involving an informally constructed dwelling in the country, from which field information on its structural characteristics and construction materials was collected. Based on the gathered information, nonlinear models with and without BRBs were created, and their seismic performance was analyzed and compared through incremental static (pushover) and nonlinear dynamic analyses. In the first analysis, the capacity curve was identified, showcasing the sequence of failure events occurring from initial yielding to structural collapse. In the second case, the model underwent nonlinear dynamic analyses using a set of seismic records consistent with the country's seismic hazard. Based on the results, fragility curves were calculated to evaluate the probability of failure of the informal housings before and after the intervention with BRBs, providing essential information about their effectiveness in reducing seismic vulnerability. The results indicate that low-cost BRBs can significantly increase the capacity of informal housing to withstand earthquakes. The dynamic analysis revealed that retrofit structures experienced lower displacements and deformations, enhancing the safety of residents and the seismic performance of informally constructed houses. In other words, the use of low-cost BRBs in the retrofit of informal housing in Colombia is a promising strategy for improving structural safety in seismic-prone areas. This study emphasizes the importance of seeking affordable and practical solutions to address seismic risk in vulnerable communities in earthquake-prone regions in Colombia and serves as a model for addressing similar challenges of informal housing worldwide.

Keywords: buckling-restrained braces, fragility curves, informal housing, incremental dynamic analysis, seismic retrofit

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Authors: Yifan Qiao, Huanying Sun, Shimeng Hao

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Objective: This study aims to investigate the therapeutic benefits of comprehensive street renovations and their individual components from a multisensory perspective, identifying key factors that enhance the multisensory experience in urban public spaces. Method: The study employed a combination of physiological measurements and subjective ratings to investigate the healing effects of street renovations from three sensory perspectives: visual, auditory (single soundscape vs. mixed soundscape), and olfactory (single scent vs. mixed scents). Forty university students (balanced gender) were recruited and randomly assigned to three independent groups to experience different stimuli: (1) Visual only group (N=14); (2) Visual and auditory group (N=13); (3) Visual, auditory, and olfactory group (N=13). Each group experienced three VR scenarios in sequence: Visual - current scenario; Auditory - single bird song (sparrow); Olfactory - single scent (bush). Visual - pavement and seating renovation scenario (adding bike lanes, shallow grass ditches, seats, changing ground pavement); Auditory - two bird songs (sparrow, blackbird); Olfactory - two scents (bush, grass). Visual - increased plant configuration scenario (peach tree, rose); Auditory - three bird songs (sparrow, blackbird, and blue-throated); Olfactory - three scents (bush, grass, rose, and peach tree). Participants wore devices to monitor physiological data (EEG, GSR, and HRV), used the Perceived Restorativeness Scale (PRS) to assess recovery effects, and a self-made sensory evaluation scale to evaluate indicators such as sensory pleasure and richness. Results: Physiological measurements indicated that renovated scenarios (pavement and seating renovation and increased plant configuration) had better relaxation effects. In the visual-only group, emotional healing increased with renovations, but this trend weakened when auditory elements were added, especially in the visual, auditory, and olfactory groups. Subjective evaluations of all sensory combinations showed a significant increase with renovation improvements. The sensory evaluation scale revealed that positive olfactory evaluations enhanced visual and auditory sensory ratings, and floral scents effectively countered the negative impacts of adverse auditory factors. Conclusion: Overall, renovated streets demonstrated greater restorative potential than before the renovation. The multisensory combination after renovation (visual, auditory, and olfactory) showed the highest recovery scores. Participants preferred visually biodiverse environments, which induced pleasure and a sense of safety. However, overly diverse auditory and olfactory stimuli could lead to sensory overload and discomfort. This study demonstrates that the healing effects of multisensory combinations are closely related to sensory pleasure, sensory coordination, and sensory overload, providing valuable insights for future street renovation designs and multisensory urban design strategies.

Keywords: multisensory integration, street renovation, urban landscape, sensory healing, visual enhancement

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Authors: Yogita Patil-Sen

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Superparamagnetic Iron Oxide Nanoparticles (SPIONs) have become increasingly important materials for separation of specific bio-molecules, drug delivery vehicle, contrast agent for MRI and magnetic hyperthermia for cancer therapy. Hyperthermia is emerging as an alternative cancer treatment to the conventional radio- and chemo-therapy, which have harmful side effects. When subjected to an alternating magnetic field, the magnetic energy of SPIONs is converted into thermal energy due to movement of particles. The ability of SPIONs to generate heat and potentially kill cancerous cells, which are more susceptible than the normal cells to temperatures higher than 41 °C forms the basis of hyerpthermia treatement. The amount of heat generated depends upon the magnetic properties of SPIONs which in turn is affected by their properties such as size and shape. One of the main problems associated with SPIONs is particle aggregation which limits their employability in in vivo drug delivery applications and hyperthermia cancer treatments. Coating the iron oxide core with thermally responsive lipid based nanostructures tend to overcome the issue of aggregation as well as improve biocompatibility and can enhance drug loading efficiency. Herein we report suitability of SPIONs and silica coated core-shell SPIONs, which are further, coated with various lipids for drug delivery and magnetic hyperthermia applications. The synthesis of nanoparticles is carried out using the established methods reported in the literature with some modifications. The nanoparticles are characterised using Infrared spectroscopy (IR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM). The heating ability of nanoparticles is tested under alternating magnetic field. The efficacy of the nanoparticles as drug carrier is also investigated. The loading of an anticancer drug, Doxorubicin at 18 °C is measured up to 48 hours using UV-visible spectrophotometer. The drug release profile is obtained under thermal incubation condition at 37 °C and compared with that under the influence of alternating magnetic field. The results suggest that the nanoparticles exhibit superparamagnetic behaviour, although coating reduces the magnetic properties of the particles. Both the uncoated and coated particles show good heating ability, again it is observed that coating decreases the heating behaviour of the particles. However, coated particles show higher drug loading efficiency than the uncoated particles and the drug release is much more controlled under the alternating magnetic field. Thus, the results demonstrate that lipid coated SPIONs exhibit potential as drug delivery vehicles for magnetic hyperthermia based cancer therapy.

Keywords: drug delivery, hyperthermia, lipids, superparamagnetic iron oxide nanoparticles (SPIONS)

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Authors: Nabil Al-Zaqri

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Semiconductor photocatalysts with surface defects display incredible light absorption bandwidth, and these defects function as highly active sites for oxidation processes by interacting with the surface band structure. Accordingly, engineering the photocatalyst with surface oxygen vacancies will enhance the semiconductor nanostructure's photocatalytic efficiency. Herein, a CeO2₋ₓ nanostructure is designed under the influence of low-frequency ultrasonic waves to create surface oxygen vacancies. This approach enhances the photocatalytic efficiency compared to many heterostructures while keeping the intrinsiccrystal structure intact. Ultrasonic waves induce the acoustic cavitation effect leading to the dissemination of active elements on the surface, which results in vacancy formation in conjunction with larger surface area and smaller particle size. The structural analysis of CeO₂₋ₓ revealed higher crystallinity, as well as morphological optimization, and the presence of oxygen vacancies is verified through Raman, X-rayphotoelectron spectroscopy, temperature-programmed reduction, photoluminescence, and electron spinresonance analyses. Oxygen vacancies accelerate the redox cycle between Ce₄+ and Ce₃+ by prolongingphotogenerated charge recombination. The ultrasound-treated pristine CeO₂ sample achieved excellenthydrogen production showing a quantum efficiency of 1.125% and efficient organic degradation. Ourpromising findings demonstrated that ultrasonic treatment causes the formation of surface oxygenvacancies and improves photocatalytic hydrogen evolution and pollution degradation. Conclusion: Defect engineering of the ceria nanoparticles with oxygen vacancies was achieved for the first time using low-frequency ultrasound treatment. The U-CeO₂₋ₓsample showed high crystallinity, and morphological changes were observed. Due to the acoustic cavitation effect, a larger surface area and small particle size were observed. The ultrasound treatment causes particle aggregation and surface defects leading to oxygen vacancy formation. The XPS, Raman spectroscopy, PL spectroscopy, and ESR results confirm the presence of oxygen vacancies. The ultrasound-treated sample was also examined for pollutant degradation, where 1O₂was found to be the major active species. Hence, the ultrasound treatment influences efficient photocatalysts for superior hydrogen evolution and an excellent photocatalytic degradation of contaminants. The prepared nanostructure showed excellent stability and recyclability. This work could pave the way for a unique post-synthesis strategy intended for efficient photocatalytic nanostructures.

Keywords: surface defect, CeO₂₋ₓ, photocatalytic, water treatment, H₂ production

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Authors: Luis Enrique Escalante Ochoa

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This paper attempts to assess the impacts of Climate change (CC) on inequalities in the labour market. CC will have the most serious effects on some vulnerable economic sectors, such as agriculture, livestock or tourism, but also on the most vulnerable population groups. The objective of this research is to evaluate the impact of CC on the labour market and particularly on Mexican women. Influential documents such as the synthesis reports produced by the Intergovernmental Panel on Climate Change (IPCC) in 2007 and 2014 revived a global effort to counteract the effects of CC, called for an analysis of the impacts on vulnerable socio-economic groups and on economic activities, and for the development of decision-making tools to enable policy and other decisions based on the complexity of the world in relation to climate change, taking into account socio-economic attributes. We follow up this suggestion and determine the impact of CC on vulnerable populations in the Mexican labour market, taking into account two attributes (gender and level of qualification of workers). Most studies have focused on the effects of CC on the agricultural sector, as it is considered a highly vulnerable economic sector to the effects of climate variability. This research seeks to contribute to the existing literature taking into account, in addition to the agricultural sector, other sectors such as tourism, water availability, and energy that are of vital importance to the Mexican economy. Likewise, the effects of climate change will be extended to the labour market and specifically to women who in some cases have been left out. The studies are sceptical about the impact of CC on the female labour market because of the perverse effects on women's domestic work, which are too often omitted from analyses. This work will contribute to the literature by integrating domestic work, which in the case of Mexico is much higher among women than among men (80.9% vs. 19.1%), according to the 2009 time use survey. This study is relevant since it will allow us to analyse impacts of climate change not only in the labour market of the formal economy, but also in the non-market sphere. Likewise, we consider that including the gender dimension is valid for the Mexican economy as it is a country with high degrees of gender inequality in the labour market. In the OECD economic study for Mexico (2017), the low labour participation of Mexican women is highlighted. Although participation has increased substantially in recent years (from 36% in 1990 to 47% in 2017), it remains low compared to the OECD average where women participate around 70% of the labour market. According to Mexico's 2009 time use survey, domestic work represents about 13% of the total time available. Understanding the interdependence between the market and non-market spheres, and the gender division of labour within them is the necessary premise for any economic analysis aimed at promoting gender equality and inclusive growth.

Keywords: climate change, labour market, domestic work, rural sector

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Authors: Y. Roumila, D. Meziani, R. Bagtache, K. Abdmeziem, M. Trari

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Heterogeneous photocatalysis over semiconductors has proved its effectiveness in the treatment of wastewaters since it works under soft conditions. It has emerged as a promising technique, giving rise to less toxic effluents and offering the opportunity of using sunlight as a sustainable and renewable source of energy. Many compounds have been used as photocatalysts. Though synthesized ones are intensively used, they remain expensive, and their synthesis involves special conditions. We thus thought of implementing a natural material, a phosphate ore, due to its low cost and great availability. Our work is devoted to the removal of hazardous organic pollutants, which cause several environmental problems and health risks. Among them, dye pollutants occupy a large place. This work relates to the study of the photodegradation of methyl violet (MV) and rhodamine B (RhB), in single and binary systems, under UV light and sunlight irradiation. Methyl violet is a triarylmethane dye, while RhB is a heteropolyaromatic dye belonging to the Xanthene family. In the first part of this work, the natural compound was characterized using several physicochemical and photo-electrochemical (PEC) techniques: X-Ray diffraction, chemical, and thermal analyses scanning electron microscopy, UV-Vis diffuse reflectance measurements, and FTIR spectroscopy. The electrochemical and photoelectrochemical studies were performed with a Voltalab PGZ 301 potentiostat/galvanostat at room temperature. The structure of the phosphate material was well characterized. The photo-electrochemical (PEC) properties are crucial for drawing the energy band diagram, in order to suggest the formation of radicals and the reactions involved in the dyes photo-oxidation mechanism. The PEC characterization of the natural phosphate was investigated in neutral solution (Na₂SO₄, 0.5 M). The study revealed the semiconducting behavior of the phosphate rock. Indeed, the thermal evolution of the electrical conductivity was well fitted by an exponential type law, and the electrical conductivity increases with raising the temperature. The Mott–Schottky plot and current-potential J(V) curves recorded in the dark and under illumination clearly indicate n-type behavior. From the results of photocatalysis, in single solutions, the changes in MV and RhB absorbance in the function of time show that practically all of the MV was removed after 240 mn irradiation. For RhB, the complete degradation was achieved after 330 mn. This is due to its complex and resistant structure. In binary systems, it is only after 120 mn that RhB begins to be slowly removed, while about 60% of MV is already degraded. Once nearly all of the content of MV in the solution has disappeared (after about 250 mn), the remaining RhB is degraded rapidly. This behaviour is different from that observed in single solutions where both dyes are degraded since the first minutes of irradiation.

Keywords: environment, organic pollutant, phosphate ore, photodegradation

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Authors: Sophio Kobauri, Temur Kantaria, Nina Kulikova, David Tugushi, Ramaz Katsarava

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The elaboration of effective drug delivery vehicles is still topical nowadays since targeted drug delivery is one of the most important challenges of the modern nanomedicine. The last decade has witnessed enormous research focused on synthetic cationic polymers (CPs) due to their flexible properties, in particular as non-viral gene delivery systems, facile synthesis, robustness, not oncogenic and proven gene delivery efficiency. However, the toxicity is still an obstacle to the application in pharmacotherapy. For overcoming the problem, creation of new cationic compounds including the polymeric nano-size particles – nano-containers (NCs) loading with different pharmaceuticals and biologicals is still relevant. In this regard, a variety of NCs-based drug delivery systems have been developed. We have found that amino acid-based biodegradable polymers called as pseudo-proteins (PPs), which can be cleared from the body after the fulfillment of their function are highly suitable for designing pharmaceutical NCs. Among them, one of the most promising are NCs made of biodegradable Cationic PPs (CPPs). For preparing new cationic NCs (CNCs), we used CPPs composed of positively charged amino acid L-arginine (R). The CNCs were fabricated by two approaches using: (1) R-based homo-CPPs; (2) Blends of R-based CPPs with regular (neutral) PPs. According to the first approach NCs we prepared from CPPs 8R3 (composed of R, sebacic acid and 1,3-propanediol) and 8R6 (composed of R, sebacic acid and 1,6-hexanediol). The NCs prepared from these CPPs were 72-101 nm in size with zeta potential within +30 ÷ +35 mV at a concentration 6 mg/mL. According to the second approach, CPPs 8R6 was blended in organic phase with neutral PPs 8L6 (composed of leucine, sebacic acid and 1,6-hexanediol). The NCs prepared from the blends were 130-140 nm in size with zeta potential within +20 ÷ +28 mV depending on 8R6/8L6 ratio. The stability studies of fabricated NCs showed that no substantial change of the particle size and distribution and no big particles’ formation is observed after three months storage. In vitro biocompatibility study of the obtained NPs with four different stable cell lines: A549 (human), U-937 (human), RAW264.7 (murine), Hepa 1-6 (murine) showed both type cathionic NCs are biocompatible. The obtained data allow concluding that the obtained CNCs are promising for the application as biodegradable drug delivery vehicles. This work was supported by the joint grant from the Science and Technology Center in Ukraine and Shota Rustaveli National Science Foundation of Georgia #6298 'New biodegradable cationic polymers composed of arginine and spermine-versatile biomaterials for various biomedical applications'.

Keywords: biodegradable polymers, cationic pseudo-proteins, nano-containers, drug delivery vehicles

Procedia PDF Downloads 132

Authors: Hung-Chih Hsu, Pey-Jium Chang, Ching-Hsein Chen, Jer-Liang Andrew Yeh

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Osteoarthritis (OA) is a common disorder in rehabilitation clinic. The main characteristics include joint pain, localized tenderness and enlargement, joint effusion, cartilage destruction, loss of adhesion of perichondrium, synovium hyperplasia. Synoviocytes inflammation might be a cause of local tenderness and effusion. Inflammation cytokines might also play an important role in joint pain, cartilage destruction, decrease adhesion of perichondrium to the bone. Treatments of osteoarthritis include non-steroid anti-inflammation drugs (NSAID), glucosamine supplementation, hyaluronic acid, arthroscopic debridement, and total joint replacement. Total joint replacement is commonly used in patients with severe OA who failed respond to pharmacological treatment. However, some patients received surgery had serious adverse events, including instability of the implants due to insufficient adhesion to the adjacent bony tissue or synovial inflammation. We tried to develop ideal nano-topographic oxidized silicon nanosponges by using with various chemicals to produce thickness difference in nanometers in order to study more about the cell-environment interactions in vitro like the alterations of cell adhesion, morphology, extracellular matrix secretions in the pathogenesis of osteoarthritis. Cytokines studies like growth factor, reactive oxygen species, reactive inflammatory materials (Like nitrous oxide and prostaglandin E2), extracellular matrix (ECM) degradation enzymes, and synthesis of collagen will also be observed and discussed. Extracellular and intracellular expression transforming growth factor beta (TGF-β) will be studied by reverse transcription-polymerase chain reaction (RT-PCR). The degradation of ECM will be observed by the bioactivity ratio of matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase by ELISA (Enzyme-linked immunosorbent assay). When rabbit synoviocytes were cultured on these nano-topographic structures, they demonstrate better cell adhesion rate, decreased expression of MMP-2,9 and PGE2, and increased expression of TGF-β when cultured in nano-topographic oxidized silicon nanosponges than in the planar oxidized silicon ones. These results show cell behavior, cytokine production can be influenced by physical characteristics from different nano-topographic structures. Our study demonstrates the possibility of manipulating cell behavior in these nano-topographic biomaterials.

Keywords: osteoarthritis, synoviocyte, oxidized silicon surfaces, reactive oxygen species

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Authors: Natalie Tyldesley-Marshall, Janette Parr, Anna Brown, Yen-Fu Chen, Amy Grove

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It is widely recognised in policy and research that the provision of services for children and young people (CYP) with Special Educational Needs and Disabilities (SEND) is enhanced when health and social care, and education services collaborate and interact effectively. In the UK, there have been significant changes to policy and provisions which support and improve collaboration. However, professionals responsible for implementing these changes face multiple challenges, including a lack of specific implementation guidance or framework to illustrate how effective multi-agency working could or should work. This systematic review will identify the key components of effective multi-agency working in services for CYP with SEND; and the most effective forms of partnership working in this setting. The review highlights interventions that lead to service improvements; and the conditions in the local area that support and encourage success. A protocol was written and registered with PROSPERO registration: CRD42022352194. Searches were conducted on several health, care, education, and applied social science databases from the year 2012 onwards. Citation chaining has been undertaken, as well as broader grey literature searching to enrich the findings. Qualitative, quantitative, mixed methods studies and systematic reviews were included, assessed independently, and critically appraised or assessed for risk of bias using appropriate tools based on study design. Data were extracted in NVivo software and checked by a more experienced researcher. A convergent segregated approach to synthesis and integration was used in which the quantitative and qualitative data were synthesised independently and then integrated using a joint display integration matrix. Findings demonstrate the key ingredients for effective partnership working for services delivering SEND. Interventions deemed effective are described, and lessons learned across interventions are summarised. Results will be of interest to educators and health and social care professionals that provide services to those with SEND. These will also be used to develop policy recommendations for how UK healthcare, social care, and education services for CYP with SEND aged 0-25 can most effectively collaborate and achieve service improvement. The review will also identify any gaps in the literature to recommend areas for future research. Funding for this review was provided by the Department for Education.

Keywords: collaboration, joint commissioning, service delivery, service improvement

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Authors: Ruwani N. Nugara, Masashi Inafuku, Kensaku Takara, Hironori Iwasaki, Hirosuke Oku

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Pteryxin from the partially purified hexane phase (HP) of Peucedanum japonicum Thunb (PJT) was identified as the active compound related to anti-obesity. Thus, in this study we investigated the mechanisms related to anti-obesity activity in-vitro. The HP was fractionated, and effect on the triglyceride (TG) content was evaluated in 3T3-L1 and HepG2 cells. Comprehensive spectroscopic analyses were used to identify the structure of the active compound. The dose dependent effect of active constituent on the TG content, and the gene expressions related to adipogenesis, fatty acid catabolism, energy expenditure, lipolysis and lipogenesis (20 μg/mL) were examined in-vitro. Furthermore, higher dosage of pteryxin (50μg/mL) was tested against 20μg/mL in 3T3-L1 adipocytes. The mRNA were subjected to SOLiD next generation sequencer and the obtained data were analyzed by Ingenuity Pathway Analysis (IPA). The active constituent was identified as pteryxin, a known compound in PJT. However, its biological activities against obesity have not been reported previously. Pteryxin dose dependently suppressed TG content in both 3T3-L1 adipocytes and HepG2 hepatocytes (P < 0.05). Sterol regulatory element-binding protein-1 (SREBP1 c), Fatty acid synthase (FASN), and acetyl-CoA carboxylase-1 (ACC1) were downregulated in pteryxin-treated adipocytes (by 18.0, 36.1 and 38.2%; P < 0.05, respectively) and hepatocytes (by 72.3, 62.9 and 38.8%, respectively; P < 0.05) indicating its suppressive effects on fatty acid synthesis. The hormone-sensitive lipase (HSL), a lipid catabolising gene was upregulated (by 15.1%; P < 0.05) in pteryxin-treated adipocytes suggesting improved lipolysis. Concordantly, the adipocyte size marker gene, paternally expressed gene1/mesoderm specific transcript (MEST) was downregulated (by 42.8%; P < 0.05), further accelerating the lipolytic activity. The upregulated trend of uncoupling protein 2 (UCP2; by 77.5%; P < 0.05) reflected the improved energy expenditure due to pteryxin. The 50μg/mL dosage of pteryxin completely suppressed PPARγ, MEST, SREBP 1C, HSL, Adiponectin, Fatty Acid Binding Protein (FABP) 4, and UCP’s in 3T3-L1 adipocytes. The IPA suggested that pteryxin at 20μg/mL and 50μg/mL suppress obesity in two different pathways, whereas the WNT signaling pathway play a key role in the higher dose of pteryxin in preadipocyte stage. Pteryxin in PJT play the key role in regulating lipid metabolism related gene network and improving energy production in vitro. Thus, the results suggests pteryxin as a new natural compound to be used as an anti-obesity drug in pharmaceutical industry.

Keywords: obesity, peucedanum japonicum thunb, pteryxin, food science

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Authors: Santhani M. Selveindran

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Background: Natural disasters are traumatic global events that are becoming increasing more common, with significant psychosocial impact on survivors. This impact results not only in psychosocial distress but, for many, can lead to psychosocial disorders and chronic psychopathology. While there are currently available interventions that seek to prevent and treat these psychosocial sequelae, their effectiveness is uncertain. The evidence-base is emerging with more primary studies evaluating the effectiveness of various psychosocial interventions for survivors of natural disasters, which remains to be synthesized. Aim of Review: To identify, critically appraise and synthesize the current evidence-base on the effectiveness of psychosocial interventions in preventing or treating Post-Traumatic Stress Disorder (PTSD), Major Depressive Disorder (MDD) and/or Generalized Anxiety Disorder (GAD) in adults and children who are survivors of natural disasters. Methods: A protocol was developed as a guide to carry out this review. A systematic search was conducted in eight international electronic databases, three grey literature databases, one dissertation and thesis repository, websites of six humanitarian and non-governmental organizations renowned for their work on natural disasters, as well as bibliographic and citation searching for eligible articles. Papers meeting the specific inclusion criteria underwent quality assessment using the Downs and Black checklist. Data were extracted from the included papers and analysed by way of narrative synthesis. Results: Database and website searching returned 3777 papers where 31 met the criteria for inclusion. Additional 2 papers were obtained through bibliographic and citation searching. Methodological quality of most papers was fair. Twenty-five studies evaluated psychological interventions, five, social interventions whereas three studies evaluated ‘mixed’ psychological and social interventions. All studies, irrespective of methodological quality, reported post-intervention reductions in symptom scores for PTSD, depression and/or anxiety and where assessed, reduced diagnosis of PTSD and MDD, and produced improvements in self-efficacy and quality of life. Statistically significant results were seen in 27 studies. However, three studies demonstrated that the evaluated interventions may not have been very beneficial. Conclusions: The overall positive results suggest that any psychosocial interventions are favourable and should be delivered to all natural disaster survivors, irrespective of age, country, and phase of disaster. Yet, heterogeneity and methodological shortcomings of the current evidence-base makes it difficult to draw definite conclusions needed to formulate categorical guidance or frameworks. Further, rigorously conducted research is needed in this area, although the feasibility of such, given the context and nature of the problem, is also recognized.

Keywords: psychosocial interventions, natural disasters, survivors, effectiveness

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Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais

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The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.

Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology

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Authors: Kirsten Techmer, Jan-Erik Rybak, Simon Rudolph

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Natural formed pyrites (FeS2) are frequent sulfides in sedimentary and metamorphic rocks. Growth textures of idiomorphic pyrite assemblages reflect the conditions during their formation in the geologic sequence, furtheron the local texture analyses of the growth patterns of pyrite assemblages by EBSD reveal the possibility to resolve the growth conditions during the formation of pyrite at the micron scale. The spatial resolution of local texture measurements in the Scanning Electron Microscope used can be in the nanomete scale. Orientation contrasts resulting from domains of smaller misorientations within larger pyrite crystals can be resolved as well. The electron optical studies have been carried out in a Field-Emission Scanning Electron Microscope (FEI Quanta 200) equipped with a CCD camera to study the orientation contrasts along the surfaces of pyrite. Idiomorphic cubic single crystals of pyrite, polycrystalline assemblages of pyrite, spherically grown spheres of pyrite as well as pyrite-bearing ammonites have been studied by EBSD in the Scanning Electron Microscope. Samples were chosen to show no or minor secondary deformation and an idiomorphic 3D crystal habit, so the local textures of pyrite result mainly from growth and minor from deformation. The samples studied derived from Navajun (Spain), Chalchidiki (Greece), Thüringen (Germany) and Unterkliem (Austria). Chemical analyses by EDAX show pyrite with minor inhomogeneities e.g., single crystals of galena and chalcopyrite along the grain boundaries of larger pyrite crystals. Intergrowth between marcasite and pyrite can be detected in one sample. Pyrite may form intense growth twinning lamellae on {011}. Twinning, e.g., contact twinning is abundant within the crystals studied and the individual twinning lamellaes can be resolved by EBSD. The ammonites studied show a replacement of the shale by newly formed pyrite resulting in an intense intergrowth of calcite and pyrite. EBSD measurements indicate a polycrystalline microfabric of both minerals, still reflecting primary surface structures of the ammonites e.g, the Septen. Discs of pyrite (“pyrite dollar”) as well as pyrite framboids show growth patterns comprising a typical microfabric. EBSD studies reveal an equigranular matrix in the inner part of the discs of pyrite and a fiber growth with larger misorientations in the outer regions between the individual segments. This typical microfabric derived from a formation of pyrite crystals starting at a higher nucleation rate and followed by directional crystal growth. EBSD studies show, that the growth texture of pyrite in the samples studied reveals a correlation between nucleation rate and following growth rate of the pyrites, thus leading to the characteristic crystal habits. Preferential directional growth at lower nucleation rates may lead to the formation of 3D framboids of pyrite. Crystallographic misorientations between the individual fibers are similar. In ammonites studied, primary anisotropies of the substrates like e.g., ammonitic sutures, influence the nucleation, crystal growth and habit of the newly formed pyrites along the surfaces.

Keywords: Electron Back Scatter Diffraction (EBSD), growth pattern, Fe-sulfides (pyrite), texture analyses

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Authors: Raha Bahreini

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This paper discusses the roll-back of women’s sexual and reproductive rights in the Islamic Republic of Iran, which has come in the wake of a striking shift in the country’s official population policies. Since the late 1980s, Iran has won worldwide praise for its sexual and reproductive health and services, which have contributed to a steady decline in the country’s fertility rate–from 7.0 births per women in 1980 to 5.5 in 1988, 2.8 in 1996 and 1.85 in 2014. This is owed to a significant increase in the voluntary use of modern contraception in both rural and urban areas. In 1976, only 37 per cent of women were using at least one method of contraception; by 2014 this figure had reportedly risen to a high of nearly 79 per cent for married girls and women living in urban areas and 73.78 per cent for those living in rural areas. Such progress may soon be halted. In July 2012, Iran’s Supreme Leader Ayatollah Sayed Ali Khamenei denounced Iran’s family planning policies as an imitation of Western lifestyle. He exhorted the authorities to increase Iran’s population to 150 to 200 million (from around 78.5 million), including by cutting subsidies for contraceptive methods and dismantling the state’s Family and Population Planning Programme. Shortly thereafter, Iran’s Minister of Health and Medical Education announced the scrapping of the budget for the state-funded Family and Population Planning Programme. Iran’s Parliament subsequently introduced two bills; the Comprehensive Population and Exaltation of Family Bill (Bill 315), and the Bill to Increase Fertility Rates and Prevent Population Decline (Bill 446). Bill 446 outlaws voluntary tubectomies, which are believed to be the second most common method of modern contraception in Iran, and blocks access to information about contraception, denying women the opportunity to make informed decisions about the number and spacing of their children. Coupled with the elimination of state funding for Iran’s Family and Population Programme, the move would undoubtedly result in greater numbers of unwanted pregnancies, forcing more women to seek illegal and unsafe abortions. Bill 315 proposes various discriminatory measures in the areas of employment, divorce, and protection from domestic violence in order to promote a culture wherein wifedom and child-bearing is seen as women’s primary duty. The Bill, for example, instructs private and public entities to prioritize, in sequence, men with children, married men without children and married women with children when hiring for certain jobs. It also bans the recruitment of single individuals as family law lawyers, public and private school teachers and members of the academic boards of universities and higher education institutes. The paper discusses the consequences of these initiatives which would, if continued, set the human rights of women and girls in Iran back by decades, leaving them with a future shaped by increased inequality, discrimination, poor health, limited choices and restricted freedoms, in breach of Iran’s international human rights obligations.

Keywords: family planning and reproductive health, gender equality and empowerment of women, human rights, population growth

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

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Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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

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

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Authors: Rama Munajat

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Discourse information levels may be viewed from either a background-foreground distinction or a multi-level perspective, and cross-linguistic studies on this area suggest that each information level is marked by a specific linguistic device. In this sense, Sundanese, spoken in Indonesia’s West Javanese Province, further differentiates the background and foreground information into ordinary and significant types. This paper will report an ongoing shift from particle-to-active voice marking in the way Sundanese signals foregrounding events. The shift relates to decades of contact with Bahasa Indonesia (Indonesia’s official language) and linguistic compatibility between the two surface marking strategies. Representative data analyzed include three groups of short stories in both Sundanese and Bahasa Indonesia (Indonesian) published in three periods: before 1945, 1965-2006, and 2016-2019. In the first group of Sundanese data, forward-moving events dominantly appear in particle KA (Kecap Anteuran, word-accompanying) constructions, where the KA represents different particles that co-occur with a special group of verbs. The second group, however, shows that the foregrounded events are more frequently described in active-voice forms with a subject-predicate (SP) order. Subsequently, the third offers stronger evidence for the use of the SP structure. As for the Indonesian data, the foregrounding events in the first group occur in verb-initial and passive-voice constructions, while in the second and third, the events more frequently appear in active-voice structures (subject-predicate sequence). The marking shift above suggests a structural influence from Indonesian, stemmed from generational differences among authors of the Sundanese short stories, particularly related to their education and language backgrounds. The first group of short stories – published before 1945 or before Indonesia's independence from Dutch – were written by native speakers of Sundanese who spoke Indonesian as a foreign language and went through the Dutch education system. The second group of authors, on the other hand, represents a generation of Sundanese native speakers who spoke Indonesian as a second language. Finally, the third group consists of authors who are bilingual speakers of both Sundanese and Indonesian. The data suggest that the last two groups of authors completed the Indonesian education system. With these, the use of subject-predicate sequences to denote foregrounding events began to appear more frequently in the second group and then became more dominant in those of the third. The coded data also signify that cohesion, coherence, and pragmatic purposes in Particle KA constructions are intact in their respective active-voice structure counterparts. For instance, the foregrounding events in Particle KA constructions occur in Sentence-initial KA and Pre-verbal KA forms, whereas those in the active-voice are described in Subject-Predicate (SP) and Zero-Subject active-voice patterns. Cross-language data further demonstrate that the Sentence-initial KA and the SP active-voice structures each contain an overt noun phrase (NP) co-referential with one of the entities introduced in a preceding context. Similarly, the pre-verbal KA and Zero-Subject active-voice patterns have a deleted noun phrase unambiguously referable to the only one entity previously mentioned. The presence and absence of an NP inform a pragmatic strategy to place prominence on topic/given and comment/new information, respectively.

Keywords: discourse analysis, foregrounding marking, pragmatics, language contact

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Authors: Dong Yue, Hua Chen

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The study region Xihu Harbor in Xiangshan County, Ningbo City is located in the central coast of Zhejiang Province. Concerning the wave dispating issue, Ningbo government firstly introduced Spartina alterniflora in 1980s. In the 1990s, S. alterniflora spread so rapidly thus a ‘grassland’ in the sea has been created nowadays. It has become the most important invasive plant of China’s coastal tidal flats. Although S. alterniflora had some ecological and economic functions, it has also brought series of hazards. It has ecological hazards on many aspects, including biomass and biodiversity, hydrodynamic force and sedimentation process, nutrient cycling of tidal flat, succession sequence of soil and plants and so on. On engineering, it courses problems of poor drainage and channel blocking. On economy, the hazard mainly reflected in the threat on aquaculture industry. The purpose of this study is to explore an ecological, feasible and economical way to manage Spartina alterniflora and use the land formed by it, taking Xihu Harbor in Xiangshan County as a case. Comparison method, mathematical modeling, qualitative and quantitative analysis are utilized to proceed the study. Main outcomes are as follows. By comparing a series of S. alterniflora managing methods which include the combination of mechanical cutting and hydraulic reclamation, waterlogging, herbicide and biological substitution from three standpoints – ecology, engineering and economy. It is inferred that the combination of mechanical cutting and hydraulic reclamation is among the top rank of S. alternifora managing methods. The combination of mechanical cutting and hydraulic reclamation means using large-scale mechanical equipment like large screw seagoing dredger to excavate the S. alterniflora with root and mud together. Then the mix of mud and grass was blown off nearby coastal tidal zone transported by pipelines, which can cushion the silt of tidal zone to form a land. However, as man-made land by coast, the reclamation area’s ecological sensitivity is quite high and will face high possibility of flood threat. Therefore, the reclamation area has many reasonability requirements, including ones on location, specific scope, water surface rate, direction of main watercourse, site of water-gate, the ratio of ecological land to urban construction land. These requirements all became important basis when the planning was being made. The water system planning, green space system planning, road structure and land use all need to accommodate the ecological requests. Besides, the profits from the formed land is the managing project’s source of funding, so how to utilize land efficiently is another considered point in the planning. It is concluded that by aiming at managing a large area of S. alterniflora, the combination of mechanical cutting and hydraulic reclamation is an ecological, feasible and economical method. The planning of reclamation area should fully respect the natural environment and possible disasters. Then the planning which makes land use efficient, reasonable, ecological will promote the development of the area’s city construction.

Keywords: ecological management, ecological planning method, reclamation area, Spartina alternifora, Xihu harbor

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Authors: Soma Ghosh, Balaram Mohapatra, Pinaki Sar, Abhijeet Mukherjee

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Microbial role in arsenic (As) mobilization in the groundwater aquifers of Brahmaputra river basin (BRB) in India, severely threatened by high concentrations of As, remains largely unknown. The present study, therefore, is a molecular and ecophysiological characterization of an indigenous bacterium strain IIIJ3-1 isolated from As contaminated groundwater of BRB and application of this strain in several microcosm set ups differing in their organic carbon (OC) source and terminal electron acceptors (TEA), to understand its role in As dissolution under aerobic and anaerobic conditions. Strain IIIJ3-1 was found to be a new facultative anaerobic, gram-positive, endospore-forming strain capable of arsenite (As3+) oxidation and dissimilatory arsenate (As5+) reduction. The bacterium exhibited low genomic (G+C)% content (45 mol%). Although, its 16S rRNA gene sequence revealed a maximum similarity of 99% with Bacillus cereus ATCC 14579(T) but the DNA-DNA relatedness of their genomic DNAs was only 49.9%, which remains well below the value recommended to delimit different species. Abundance of fatty acids iC17:0, iC15:0 and menaquinone (MK) 7 though corroborates its taxonomic affiliation with B. cereus sensu-lato group, presence of hydroxy fatty acids (HFAs), C18:2, MK5 and MK6 marked its uniqueness. Besides being highly As resistant (MTC=10mM As3+, 350mM As5+), metabolically diverse, efficient aerobic As3+ oxidizer; it exhibited near complete dissimilatory reduction of As5+ (1 mM). Utilization of various carbon sources with As5+ as TEA revealed lactate to serve as the best electron donor. Aerobic biotransformation assay yielded a lower Km for As3+ oxidation than As5+ reduction. Arsenic homeostasis was found to be conferred by the presence of arr, arsB, aioB, and acr3(1) genes. Scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) analysis of this bacterium revealed reduction in cell size upon exposure to As and formation of As-rich electron opaque dots following growth with As3+. Incubation of this strain with sediment (sterilised) collected from BRB aquifers under varying OC, TEA and redox conditions revealed that the strain caused highest As mobilization from solid to aqueous phase under anaerobic condition with lactate and nitrate as electron donor and acceptor, respectively. Co-release of highest concentrations of oxalic acid, a well known bioweathering agent, considerable fold increase in viable cell counts and SEM-EDX and X-ray diffraction analysis of the sediment after incubation under this condition indicated that As release is consequent to microbial bioweathering of the minerals. Co-release of other elements statistically proves decoupled release of As with Fe and Zn. Principle component analysis also revealed prominent role of nitrate under aerobic and/or anaerobic condition in As release by strain IIIJ3-1. This study, therefore, is the first to isolate, characterize and reveal As mobilization property of a strain belonging to the Bacillus cereus sensu lato group isolated from highly As contaminated aquifers of Brahmaputra River Basin.

Keywords: anaerobic microcosm, arsenic rich electron opaque dots, Arsenic release, Bacillus strain IIIJ3-1

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Authors: Olga Gomez De Miranda, Jose R. Ochoa-Gomez, Stefaan De Wildeman, Luciano Monsegue, Soraya Prieto, Leire Lorenzo, Cristina Dineiro

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The chemical industry is facing a new revolution. As long as processes based on the exploitation of fossil resources emerged with force in the XIX century, Society currently demands a new radical change that will lead to the complete and irreversible implementation of a circular sustainable economic model. The implementation of biorefineries will be essential for this. There, renewable raw materials as sugars and other biomass resources are exploited for the development of new materials that will partially replace their petroleum-derived homologs in a safer, and environmentally more benign approach. Isosorbide, (1,4:3,6-dianhydro-d-glucidol) is a primary bio-based derivative obtained from the plant (poly) saccharides and a very interesting example of a useful chemical produced in biorefineries. It can, in turn, be converted to other secondary monomers as isosorbide bis-methyl carbonate (IBMC), whose main field of application can be as a key biodegradable intermediary substitute of bisphenol-A in the manufacture of polycarbonates, or as an alternative to the toxic isocyanates in the synthesis of new polyurethanes (non-isocyanate polyurethanes) both with a huge application market. New products will present advantageous mechanical or optical properties, as well as improved behavior in non-toxicity and biodegradability aspects in comparison to their petro-derived alternatives. A robust production process of IBMC, a biomass-derived chemical, is here presented. It can be used with different raw material qualities using dimethyl carbonate (DMC) as both co-reactant and solvent. It consists of the transesterification of isosorbide with DMC under soft operational conditions, using different basic catalysts, always active with the isosorbide characteristics and purity. Appropriate isolation processes have been also developed to obtain crude IBMC yields higher than 90%, with oligomers production lower than 10%, independently of the quality of the isosorbide considered. All of them are suitable to be used in polycondensation reactions for polymers obtaining. If higher qualities of IBMC are needed, a purification treatment based on nanofiltration membranes has been also developed. The IBMC reaction-isolation conditions established in the laboratory have been successfully modeled using appropriate software programs and moved to a pilot-scale (production of 100 kg of IBMC). It has been demonstrated that a highly efficient IBMC production process able to be up-scaled under suitable market conditions has been obtained. Operational conditions involved the production of IBMC involve soft temperature and energy needs, no additional solvents, and high operational efficiency. All of them are according to green manufacturing rules.

Keywords: biomass, catalyst, isosorbide bis-methyl carbonate, polycarbonate, polyurethane, transesterification

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Authors: Mehran Nozari-Asbemarz, Italo Pisano, Simin Arshi, Edmond Magner, James J. Leahy

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Integrating electrolytic synthesis with renewable energy makes it feasible to address urgent environmental and energy challenges. Conventional water electrolyzers concurrently produce H₂ and O₂, demanding additional procedures in gas separation to prevent contamination of H₂ with O₂. Moreover, the oxygen evolution reaction (OER), which is sluggish and has a low overall energy conversion efficiency, does not deliver a significant value product on the electrode surface. Compared to conventional water electrolysis, integrating electrolytic hydrogen generation from water with thermodynamically more advantageous aqueous organic oxidation processes can increase energy conversion efficiency and create value-added compounds instead of oxygen at the anode. One strategy is to use renewable and sustainable carbon sources from biomass, which has a large annual production capacity and presents a significant opportunity to supplement carbon sourced from fossil fuels. Numerous catalytic techniques have been researched in order to utilize biomass economically. Because of its safe operating conditions, excellent energy efficiency, and reasonable control over production rate and selectivity using electrochemical parameters, electrocatalytic upgrading stands out as an appealing choice among the numerous biomass refinery technologies. Therefore, we propose a broad framework for coupling H2 generation from water splitting with oxidative biomass upgrading processes. Four representative biomass targets were considered for oxidative upgrading that used a hierarchically porous CoFe-MOF/LDH @ Graphite Paper bifunctional electrocatalyst, including glucose, ethanol, benzyl, furfural, and 5-hydroxymethylfurfural (HMF). The potential required to support 50 mA cm-2 is considerably lower than (~ 380 mV) the potential for OER. All four compounds can be oxidized to yield liquid byproducts with economic benefit. The electrocatalytic oxidation of glucose to the value-added products, gluconic acid, glucuronic acid, and glucaric acid, was examined in detail. The cell potential for combined H₂ production and glucose oxidation was substantially lower than for water splitting (1.44 V(RHE) vs. 1.82 V(RHE) for 50 mA cm-2). In contrast, the oxidation byproduct at the anode was significantly more valuable than O₂, taking advantage of the more favorable glucose oxidation in comparison to the OER. Overall, such a combination of HER and oxidative biomass valorization using electrocatalysts prevents the production of potentially explosive H₂/O₂mixtures and produces high-value products at both electrodes with lower voltage input, thereby increasing the efficiency and activity of electrocatalytic conversion.

Keywords: biomass, electrocatalytic, glucose oxidation, hydrogen evolution

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Authors: Thilini M. Yatanwala

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CCTV (Closed-Circuit-Television) Surveillance System is being used in public places over decades and a large variety of data is being produced every moment. However, most of the CCTV data is stored in isolation without having integrity. As a result, identification of the behavior of suspicious people along with their location has become strenuous. This research was conducted to acquire more accurate and reliable timely information from the CCTV video records. The implemented system can identify human objects in public places based on outfit colors. Inter-process communication technologies were used to implement the CCTV camera network to track people in the premises. The research was conducted in three stages and in the first stage human objects were filtered from other movable objects available in public places. In the second stage people were uniquely identified based on their outfit colors and in the third stage an individual was continuously tracked in the CCTV network. A face detection algorithm was implemented using cascade classifier based on the training model to detect human objects. HAAR feature based two-dimensional convolution operator was introduced to identify features of the human face such as region of eyes, region of nose and bridge of the nose based on darkness and lightness of facial area. In the second stage outfit colors of human objects were analyzed by dividing the area into upper left, upper right, lower left, lower right of the body. Mean color, mod color and standard deviation of each area were extracted as crucial factors to uniquely identify human object using histogram based approach. Color based measurements were written in to XML files and separate directories were maintained to store XML files related to each camera according to time stamp. As the third stage of the approach, inter-process communication techniques were used to implement an acknowledgement based CCTV camera network to continuously track individuals in a network of cameras. Real time analysis of XML files generated in each camera can determine the path of individual to monitor full activity sequence. Higher efficiency was achieved by sending and receiving acknowledgments only among adjacent cameras. Suspicious incidents such as a person staying in a sensitive area for a longer period or a person disappeared from the camera coverage can be detected in this approach. The system was tested for 150 people with the accuracy level of 82%. However, this approach was unable to produce expected results in the presence of group of people wearing similar type of outfits. This approach can be applied to any existing camera network without changing the physical arrangement of CCTV cameras. The study of human identification and suspicious incident detection using outfit color analysis can achieve higher level of accuracy and the project will be continued by integrating motion and gait feature analysis techniques to derive more information from CCTV videos.

Keywords: CCTV surveillance, human detection and identification, image processing, inter-process communication, security, suspicious detection

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Authors: Ana M. Antolín, Sandra Contreras, Francesc Medina, Didier Tichit

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Introduction: High levels of nitrates (> 50 ppm NO3-) in drinking water are potentially risky to human health. In the recent years, the trend of nitrate concentration in groundwater is rising in the EU and other countries. Conventional catalytic nitrate reduction processes into N2 and H2O lead to some toxic intermediates and by-products, such as NO2-, NH4+, and NOx gases. Alternatively, photocatalytic nitrate removal using solar irradiation and heterogeneous catalysts is a very promising and ecofriendly technique. It has been scarcely performed and more research on highly efficient catalysts is still needed. In this work, different nanocatalysts supported on Aeroxide Titania P25 (P25) have been prepared varying: 0.5-4 % wt. Ag); Pt (2, 4 % wt.); Pt precursor (H2PtCl6/K2PtCl6); and impregnation order of both metals. Pt was chosen in order to increase the selectivity to N2 and decrease that to NO2-. Catalysts were characterized by nitrogen physisorption, X-Ray diffraction, UV-visible spectroscopy, TEM and X Ray-Photoelectron Spectroscopy. The aim was to determine the influence of the composition and the preparation method of the catalysts on the conversion and selectivity in the nitrate reduction, as well as going through an overall and better understanding of the process. Nanocatalysts synthesis: For the mono and bimetallic catalysts preparation, wise-drop wetness impregnation of the precursors (AgNO3, H2PtCl6, K2PtCl6) followed by a reduction step (NaBH4) was used to obtain the metal colloids. Results and conclusions: Denitration experiments were performed in a 350 mL PTFE batch reactor under inert standard operational conditions, ultraviolet irradiations (λ=254 nm (UV-C); λ=365 nm (UV-A)), and presence/absence of hydrogen gas as a reducing agent, contrary to most studies using oxalic or formic acid. Samples were analyzed by Ionic Chromatography. Blank experiments using respectively P25 (dark conditions), hydrogen only and UV irradiations without hydrogen demonstrated a clear influence of the presence of hydrogen on nitrate reduction. Also, they demonstrated that UV irradiation increased the selectivity to N2. Interestingly, the best activity was obtained under ultraviolet lamps, especially at a closer wavelength to visible light irradiation (λ = 365 nm) and H2. 2% Ag/P25 leaded to the highest NO3- conversion among the monometallic catalysts. However, nitrite quantities have to be diminished. On the other hand, practically no nitrate conversion was observed with the monometallics based on Pt/P25. Therefore, the amount of 2% Ag was chosen for the bimetallic catalysts. Regarding the bimetallic catalysts, it is observed that the metal impregnation order, amount and Pt precursor highly affects the results. Higher selectivity to the desirable N2 gas is obtained when Pt was firstly added, especially with K2PtCl6 as Pt precursor. This suggests that when Pt is secondly added, it covers the Ag particles, which are the most active in this reaction. It could be concluded that Ag allows the nitrate reduction step to nitrite, and Pt the nitrite reduction step toward the desirable N2 gas.

Keywords: heterogeneous catalysis, hydrogenation, nanocatalyst, nitrate removal, photocatalysis

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Authors: Noury Bakrim

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When dealing with motivation/arbitrariness, forming form (Forma Formans) and morphodynamics are to be grasped as relevant implications of enunciation/enactment, schematization within the specificity of language as sound/meaning articulation. Thus, the fact that a language is a form does not contradict stasis/dynamic enunciation (reflexivity vs double articulation). Moreover, some languages exemplify the role of the forming form, uttering, and schematization (roots in Semitic languages, the Chinese case). Beyond the evolutionary biosemiotic process (form/substance bifurcation, the split between realization/representation), non-isomorphism/asymmetry between linguistic form/norm and linguistic realization (phonetics for instance) opens up a new horizon problematizing the role of Brain – sensorimotor contribution in the continuous forming form. Therefore, we hypothesize biotization as both process/trace co-constructing motivation/forming form. Henceforth, referring to our findings concerning distribution and motivation patterns within Berber written texts (pulse based obstruents and nasal-lateral levels in poetry) and oral storytelling (consonant intensity clustering in quantitative and semantic/prosodic motivation), we understand consonant clustering, motivation and schematization as a complex phenomenon partaking in patterns of oral/written iconic prosody and reflexive metalinguistic representation opening the stable form. We focus our inquiry on both Amazigh and English clusters (/spl/, /spr/) and iconic consonant iteration in [gnunnuy] (to roll/tumble), [smummuy] (to moan sadly or crankily). For instance, the syllabic structures of /splaeʃ/ and /splaet/ imply an anamorphic representation of the state of the world: splash, impact on aquatic surfaces/splat impact on the ground. The pair has stridency and distribution as distinctive features which specify its phonetic realization (and a part of its meaning) /ʃ/ is [+ strident] and /t/ is [+ distributed] on the vocal tract. Schematization is then a process relating both physiology/code as an arthron vocal/bodily, vocal/practical shaping of the motor-articulatory system, leading to syntactic/semantic thematization (agent/patient roles in /spl/, /sm/ and other clusters or the tense uvular /qq/ at the initial position in Berber). Furthermore, the productivity of serial syllable sequencing in Berber points out different expressivity forms. We postulate two Components of motivated formalization: i) the process of memory paradigmatization relating to sequence modeling under sensorimotor/verbal specific categories (production/perception), ii) the process of phonotactic selection - prosodic unconscious/subconscious distribution by virtue of iconicity. Basing on multiple tests including a questionnaire, phonotactic/visual recognition and oral/written reproduction, we aim at patterning/conceptualizing consonant schematization and motivation among EFL and Amazigh (Berber) learners and speakers integrating biolinguistic hypotheses.

Keywords: consonant motivation and prosody, language and order of life, anamorphic representation, represented representation, biotization, sensori-motor and brain representation, form, formalization and schematization

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Authors: Boriana Karamanova, Svetlana Veleva, Luybomir Soserov, Ana Arenillas, Francesco Lufrano, Antonia Stoyanova

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Supercapacitors have received a lot of research attention and are promising energy storage devices due to their high power and long cycle life. In order to developed an advanced device with significant capacity for storing charge and cheap carbon materials, efforts must focus not only on improving synthesis by controlling the morphology and pore size but also on improving electrode-electrolyte compatibility of the resulting systems. The present study examines the relationship between the surface chemistry of two activated carbon xerogels, the electrolyte type, and the electrochemical properties of supercapacitors. Activated carbon xerogels were prepared by varying the initial pH of the resorcinol-formaldehyde aqueous solution. The materials produced are physicochemical characterized by DTA/TGA, porous characterization, and SEM analysis. The carbon xerogel based electrodes were prepared by spreading over glass plate a slurry containing the carbon gel, graphite, and poly vinylidene difluoride (PVDF) binder. The layer formed was dried consecutively at different temperatures and then detached by water. After, the layer was dried again to improve its mechanical stability. The developed electrode materials and the Aquivion® E87-05S membrane (Solvay Specialty Polymers), socked in Na2SO4 as a polymer electrolyte, were used to assembly the solid-state supercapacitor. Symmetric supercapacitor cells composed by same electrodes and 1 M KOH electrolytes are also assembled and tested for comparison. The supercapacitor performances are verified by different electrochemical methods - cyclic voltammetry, galvanostatic charge/discharge measurements, electrochemical impedance spectroscopy, and long-term durability tests in neutral and alkaline electrolytes. Specific capacitances, energy, and power density, energy efficiencies, and durability were compared into studied supercapacitors. Ex-situ physicochemical analyses on the synthesized materials have also been performed, which provide information about chemical and structural changes in the electrode morphology during charge / discharge durability tests. They are discussed on the basis of electrode-electrolyte interaction. The obtained correlations could be of significance in order to design sustainable solid-state supercapacitors with high power and energy density. Acknowledgement: This research is funded by the Ministry of Education and Science of Bulgaria under the National Program "European Scientific Networks" (Agreement D01-286 / 07.10.2020, D01-78/30.03.2021). Authors gratefully acknowledge.

Keywords: carbon xerogel, electrochemical tests, neutral and alkaline electrolytes, supercapacitors

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Authors: Xiaoling Ren, Guidong Yang

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As one of the largest industrial synthetic chemicals in the world, ammonia has the advantages of high energy density, easy liquefaction, and easy transportation, which is widely used in agriculture, chemical industry, energy storage, and other fields. The industrial Haber-Bosch method process for ammonia synthesis is generally conducted under severe conditions. It is essential to develop a green, sustainable strategy for ammonia production to meet the growing demand. In this direction, photocatalytic nitrogen reduction has huge advantages over the traditional, well-established Haber-Bosch process, such as the utilization of natural sun light as the energy source and significantly lower pressure and temperature to affect the reaction process. However, the high activation energy of nitrogen and the low efficiency of photo-generated electron-hole separation in the photocatalyst result in low ammonia production yield. Many researchers focus on improving the catalyst. In addition to modifying the catalyst, improving the dispersion of the catalyst and making full use of active sites are also means to improve the overall catalytic activity. Few studies have been carried out on this, which is the aim of this work. In this work, by making full use of the nitrogen activation ability of WO3-x with defective sites, small size WO3-x photocatalyst with high dispersibility was constructed, while the growth of WO3-x was restricted by using a high specific surface area mesoporous SBA-15 molecular sieve with the regular pore structure as a template. The morphology of pure SBA-15 and WO3-x/SBA-15 was characterized byscanning electron microscopy (SEM). Compared with pure SBA-15, some small particles can be found in the WO3-x/SBA-15 material, which means that WO3-x grows into small particles under the limitation of SBA-15, which is conducive to the exposure of catalytically active sites. To elucidate the chemical nature of the material, the X-ray diffraction (XRD) analysis was conducted. The observed diffraction pattern inWO3-xis in good agreement with that of the JCPDS file no.71-2450. Compared with WO3-x, no new peaks appeared in WO3-x/SBA-15.It can be concluded that WO3-x/SBA-15 was synthesized successfully. In order to provide more active sites, the mass content of WO3-x was optimized. Then the photocatalytic nitrogen reduction performances of above samples were performed with methanol as a hole scavenger. The results show that the overall ammonia production performance of WO3-x/SBA-15 is improved than pure bulk WO3-x. The above results prove that making full use of active sites is also a means to improve overall catalytic activity.This work provides material basis for the design of high-efficiency photocatalytic nitrogen reduction catalysts.

Keywords: ammonia, photocatalytic, nitrogen reduction, WO3-x, high dispersibility

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Authors: Bastian Waduge Naveen Harindu Hemasiri, Jae-Kwan Kim, Ji-Myon Lee

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Indium tin oxide (ITO) remains the industrial standard transparent conducting oxides with better performances. Recently, graphene becomes as a strong material with unique properties to replace the ITO. However, graphene/ITO hybrid composite material is a newly born field in the electronic world. In this study, the graphene/ITO composite bi-film was synthesized by a two steps process. 10 wt.% tin-doped, ITO thin films were produced by an environmentally friendly aqueous sol-gel spin coating technique with economical salts of In(NO3)3.H2O and SnCl4 without using organic additives. The wettability and surface free energy (97.6986 mJ/m2) enhanced oxygen plasma treated glass substrates were used to form voids free continuous ITO film. The spin-coated samples were annealed at 600 0C for 1 hour under low vacuum conditions to obtained crystallized, ITO film. The crystal structure and crystalline phases of ITO thin films were analyzed by X-ray diffraction (XRD) technique. The Scherrer equation was used to determine the crystallite size. Detailed information about chemical composition and elemental composition of the ITO film were determined by X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDX) coupled with FE-SEM respectively. Graphene synthesis was done under chemical vapor deposition (CVD) method by using Cu foil at 1000 0C for 1 min. The quality of the synthesized graphene was characterized by Raman spectroscopy (532nm excitation laser beam) and data was collected at room temperature and normal atmosphere. The surface and cross-sectional observation were done by using FE-SEM. The optical transmission and sheet resistance were measured by UV-Vis spectroscopy and four point probe head at room temperature respectively. Electrical properties were also measured by using V-I characteristics. XRD patterns reveal that the films contain the In2O3 phase only and exhibit the polycrystalline nature of the cubic structure with the main peak of (222) plane. The peak positions of In3d5/2 (444.28 eV) and Sn3d5/2 (486.7 eV) in XPS results indicated that indium and tin are in the oxide form only. The UV-visible transmittance shows 91.35 % at 550 nm with 5.88 x 10-3 Ωcm specific resistance. The G and 2D band in Raman spectroscopy of graphene appear at 1582.52 cm-1 and 2690.54 cm-1 respectively when the synthesized CVD graphene on SiO2/Si. The determined intensity ratios of 2D to G (I2D/IG) and D to G (ID/IG) were 1.531 and 0.108 respectively. However, the above-mentioned G and 2D peaks appear at 1573.57 cm-1 and 2668.14 cm-1 respectively when the CVD graphene on the ITO coated glass, the positions of G and 2D peaks were red shifted by 8.948 cm-1 and 22.396 cm-1 respectively. This graphene/ITO bi-film shows modified electrical properties when compares with sol-gel derived ITO film. The reduction of sheet resistance in the bi-film was 12.03 % from the ITO film. Further, the fabricated graphene/ITO bi-film shows 88.66 % transmittance at 550 nm wavelength.

Keywords: chemical vapor deposition, graphene, ITO, Raman Spectroscopy, sol-gel

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Authors: Adenike Adesanya, Nonthaphat Wong, Xiang-Yun Lan, Shea Ping Yip, Chien-Ling Huang

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Background: The most challenging mutation of the oncokinase BCR-ABL protein T315I, which is commonly known as the “gatekeeper” mutation and is notorious for its strong resistance to almost all tyrosine kinase inhibitors (TKIs), especially imatinib. Therefore, this study aims to identify T315I-dependent downstream microRNA (miRNA) pathways associated with drug resistance in chronic myeloid leukemia (CML) for prognostic and therapeutic purposes. Methods: T315I-carrying K562 cell clones (K562-T315I) were generated by the CRISPR-Cas9 system. Imatinib-treated K562-T315I cells were subjected to small RNA library preparation and next-generation sequencing. Putative lncRNA-miRNA-mRNA networks were analyzed with (i) DESeq2 to extract differentially expressed miRNAs, using Padj value of 0.05 as cut-off, (ii) STarMir to obtain potential miRNA response element (MRE) binding sites of selected miRNAs on lncRNA H19, (iii) miRDB, miRTarbase, and TargetScan to predict mRNA targets of selected miRNAs, (iv) IntaRNA to obtain putative interactions between H19 and the predicted mRNAs, (v) Cytoscape to visualize putative networks, and (vi) several pathway analysis platforms – Enrichr, PANTHER and ShinyGO for pathway enrichment analysis. Moreover, mitochondria isolation and transcript quantification were adopted to determine the new mechanism involved in T315I-mediated resistance of CML treatment. Results: Verification of the CRISPR-mediated mutagenesis with digital droplet PCR detected the mutation abundance of ≥80%. Further validation showed the viability of ≥90% by cell viability assay, and intense phosphorylated CRKL protein band being detected with no observable change for BCR-ABL and c-ABL protein expressions by Western blot. As reported by several investigations into hematological malignancies, we determined a 7-fold increase of H19 expression in K562-T315I cells. After imatinib treatment, a 9-fold increment was observed. DESeq2 revealed 171 miRNAs were differentially expressed K562-T315I, 112 out of these miRNAs were identified to have MRE binding regions on H19, and 26 out of the 112 miRNAs were significantly downregulated. Adopting the seed-sequence analysis of these identified miRNAs, we obtained 167 mRNAs. 6 hub miRNAs (hsa-let-7b-5p, hsa-let-7e-5p, hsa-miR-125a-5p, hsa-miR-129-5p, and hsa-miR-372-3p) and 25 predicted genes were identified after constructing hub miRNA-target gene network. These targets demonstrated putative interactions with H19 lncRNA and were mostly enriched in pathways related to cell proliferation, senescence, gene silencing, and pluripotency of stem cells. Further experimental findings have also shown the up-regulation of mitochondrial transcript and lncRNA MALAT1 contributing to the lncRNA-miRNA-mRNA networks induced by BCR-ABL T315I mutation. Conclusions: Our results have indicated that lncRNA-miRNA regulators play a crucial role not only in leukemogenesis but also in drug resistance, considering the significant dysregulation and interactions in the K562-T315I cell model generated by CRISPR-Cas9. In silico analysis has further shown that lncRNAs H19 and MALAT1 bear several complementary miRNA sites. This implies that they could serve as a sponge, hence sequestering the activity of the target miRNAs.

Keywords: chronic myeloid leukemia, imatinib resistance, lncRNA-miRNA-mRNA, T315I mutation

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