Search results for: gene expression microarray

Authors: KuiDong Kang, Hye Bin Yim, Su Ah Kim

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Purpose: Tectorigenin is an isoflavone derived from the rhizome of Belamacanda chinensis. In this study, oxygen-induced retinopathy was used to characterize the anti-angiogenic properties of tectorigenin in mice. Methods: ICR neonatal mice were exposed to 75% oxygen from postnatal day P7 until P12 and returned to room air (21% oxygen) for five days (P12 to P17). Mice were subjected to daily intraperitoneal injection of tectorigenin (1 mg/kg, 10 mg/kg) and vehicle from P12 to P17. Retro-orbital injection of FITC-dextran was performed and retinal flat mounts were viewed by fluorescence microscopy. The Central avascular area was quantified from the digital images in a masked fashion using image analysis software (NIH ImageJ). Neovascular tufts were quantified by using SWIFT_NV and neovascular lumens were quantified from a histologic section in a masked fashion. Immunohistochemistry and Western blot analysis were also performed to demonstrate the anti-angiogenic activity of this compound in vivo. Results: In the retina of tectorigenin injected mouse (10mg/kg), the central non-perfusion area was significantly decreased compared to the vehicle injected group (1.76±0.5 mm2 vs 2.85±0.6 mm2, P<0.05). In vehicle-injected group, 33.45 ± 5.51% of the total retinal area was avascular, whereas the retinas of pups treated with high-dose (10 mg/kg) tectorigenin showed avascular retinal areas of 21.25 ±4.34% (P<0.05). High dose of tectorigenin also significantly reduced the number of vascular lumens in the histologic section. Tectorigenin (10 mg/kg) significantly reduced the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and angiotensin II compared to the vehicle injected group. Tectorigenin did not affect CD31 abundance at any tested dose. Conclusions: Our results show that tectorigenin possesses powerful anti-angiogenic properties and can attenuate new vessel formation in the retina after systemic administration. These results imply that this compound can be considered as a candidate substance for therapeutic inhibition of retinal angiogenesis.

Keywords: tectorigenin, anti-angiogenic, retinopathy, Belamacanda chinensis

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Authors: Rumana Keyani, Haleema Sadia, Asia Nosheen, Rabia Naz, Humaira Yasmin, Sidra Zahoor

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Tomato is a significant crop of the world and is one of the staple foods of Pakistan. A vast number of plant pathogens from simple viruses to complex parasites cause diseases in tomatoes but fungal infection in our country is quite high. Sometimes the symptoms are too harsh destroying the crop altogether. Countries like our own with continuously increasing massive population and limited resources cannot afford such an economic loss. There is an array of morphological, genetic, biochemical and molecular processes involved in plant resistance mechanisms to biotic stress. The study of different metabolic pathways like Jasmonic acid (JA) pathways and most importantly signaling molecules like ROS/RNS and their redoxin enzymes i.e. TRX and NRX is crucial to disease management, contributing to healthy plant growth. So, improving tolerance in crop plants against biotic stresses is a dire need of our country and world as whole. In the current study, fungal pathogenic strains Alternaria solani and Rhizoctonia solani were used to inoculate tomatoes to check the defense responses of tomato plant against these pathogens at molecular as well as phenotypic level with jasmonic acid and spermidine pretreatment. All the growth parameters (root and shoot length, dry and weight root, shoot weight measured 7 days post-inoculation, exhibited that infection drastically declined the growth of the plant whereas jasmonic acid and spermidine assisted the plants to cope up with the infection. Thus, JA and Spermidine treatments maintained comparatively better growth factors. Antioxidant assays and expression analysis through real time quantitative PCR following time course experiment at 24, 48 and 72 hours intervals also exhibited that activation of JA defense genes and a polyamine Spermidine helps in mediating tomato responses against fungal infection when used alone but the two treatments combined mask the effect of each other.

Keywords: fungal infection, jasmonic acid defence, tomato, spermidine

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Authors: Marden Silva, Danielle Guerra

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Some studies have indicated that pronunciation teaching hasn’t been paid enough attention by teachers regarding EFL contexts. In particular, segmental and suprasegmental features through genre-based approach may be an opportunity on how to integrate pronunciation into a more meaningful learning practice. Therefore, the aim of this project was to carry out a survey on some aspects related to English pronunciation that Brazilian students consider more difficult to learn, thus enabling the discussion of strategies that can facilitate the development of oral skills in English classes by integrating the teaching of phonetic-phonological aspects into the genre-based approach. Notions of intelligibility, fluency and accuracy were proposed by some authors as an ideal didactic sequence. According to their proposals, basic learners should be exposed to activities focused on the notion of intelligibility as well as intermediate students to the notion of fluency, and finally more advanced ones to accuracy practices. In order to test this hypothesis, data collection was conducted during three high school English classes at Federal Center for Technological Education of Minas Gerais (CEFET-MG), in Brazil, through questionnaires and didactic activities, which were recorded and transcribed for further analysis. The genre debate was chosen to facilitate the oral expression of the participants in a freer way, making them answering questions and giving their opinion about a previously selected topic. The findings indicated that basic students demonstrated more difficulty with aspects of English pronunciation than the others. Many of the intelligibility aspects analyzed had to be listened more than once for a better understanding. For intermediate students, the speeches recorded were considerably easier to understand, but nevertheless they found it more difficult to pronounce the words fluently, often interrupting their speech to think about what they were going to say and how they would talk. Lastly, more advanced learners seemed to express their ideas more fluently, but still subtle errors related to accuracy were perceptible in speech, thereby confirming the proposed hypothesis. It was also seen that using genre-based approach to promote oral communication in English classes might be a relevant method, considering the socio-communicative function inherent in the suggested approach.

Keywords: EFL, genre-based approach, oral skills, pronunciation

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Authors: Farahnaz Sadat Golestan Hashemi, Mohd Razi Ismail, Mohd Y. Rafii

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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system can facilitate targeted genome editing in organisms. Dual or single guide RNA (gRNA) can program the Cas9 nuclease to cut target DNA in particular areas; thus, introducing concise mutations either via error-prone non-homologous end-joining repairing or via incorporating foreign DNAs by homologous recombination between donor DNA and target area. In spite of high demand of such promising technology, developing a well-organized procedure in order for reliable mining of potential target sites for gRNAs in large genomic data is still challenging. Hence, we aimed to perform high-throughput detection of target sites by specific PAMs for not only common Streptococcus pyogenes (SpCas9) but also for Neisseria meningitides (NmCas9) CRISPR-Cas systems. Previous research confirmed the successful application of such RNA-guided Cas9 orthologs for effective gene targeting and subsequently genome manipulation. However, Cas9 orthologs need their particular PAM sequence for DNA cleavage activity. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of the target site for the two orthogonals of Cas9 protein, we created a reliable procedure to explore possible gRNA sequences. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. Finally, a complete list of all potential gRNAs along with their locations, strands, and PAMs sequence orientation can be provided for both SpCas9 as well as another potential Cas9 ortholog (NmCas9). The artificial design of potential gRNAs in a genome of interest can accelerate functional genomic studies. Consequently, the application of such novel genome editing tool (CRISPR/Cas technology) will enhance by presenting increased versatility and efficiency.

Keywords: CRISPR/Cas9 genome editing, gRNA mining, SpCas9, NmCas9

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Authors: Aoxue Yang, Weili Tian, Yonghe Wu, Haikun Liu

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Brain tumor stem cells (BTSCs) are resistant to therapy and give rise to recurrent tumors. These rare and elusive cells are likely to disseminate during cancer progression, and some may enter dormancy, remaining viable but not increasing. The identification of dormant BTSCs is thus necessary to design effective therapies for glioblastoma (GBM) patients. Little progress has been made in therapeutic treatment of glioblastoma in the last decade despite rapid progress in molecular understanding of brain tumors1. Here we show that the stress hormone glucocorticoid is essential for the maintenance of brain tumor stem cells (BTSCs), which are resistant to conventional therapy. The glucocorticoid receptor (GR) regulates metabolic plasticity and chemoresistance of the dormant BTSC via controlling expression of GPD1 (glycerol-3-phosphate dehydrogenase 1), which is an essential regulator of lipid metabolism in BTSCs. Genomic, lipidomic and cellular analysis confirm that GR/GPD1 regulation is essential for BTSCs metabolic plasticity and survival. We further demonstrate that the GR agonist dexamethasone (DEXA), which is commonly used to control edema in glioblastoma, abolishes the effect of chemotherapy drug temozolomide (TMZ) by upregulating GPD1 and thus promoting tumor cell dormancy in vivo, this provides a mechanistic explanation and thus settle the long-standing debate of usage of steroid in brain tumor patient edema control. Pharmacological inhibition of GR/GPD1 pathway disrupts metabolic plasticity of BTSCs and prolong animal survival, which is superior to standard chemotherapy. Patient case study shows that GR antagonist mifepristone blocks tumor progression and leads to symptomatic improvement. This study identifies an important mechanism regulating cancer stem cell dormancy and provides a new opportunity for glioblastoma treatment.

Keywords: cancer stem cell, dormancy, glioblastoma, glycerol-3-phosphate dehydrogenase 1, glucocorticoid receptor, dexamethasone, RNA-sequencing, phosphoglycerides.

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Authors: Eriko Ochiai, Fumiko Satoh, Keiko Miyashita, Yu Kakimoto, Motoki Osawa

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Sudden and unexpected deaths from unknown causes occur in infants and youths. Recently, molecular links between a part of these deaths and several genetic diseases are examined in the postmortem. For instance, hereditary long QT syndrome and Burgada syndrome are occasionally fatal through critical ventricular tachyarrhythmia. There are a large number of target genes responsible for such diseases, the conventional analysis using the Sanger’s method has been laborious. In this report, we attempted to analyze sudden deaths comprehensively using the next generation sequencing (NGS) technique. Multiplex PCR to subject’s DNA was performed using Ion AmpliSeq Library Kits 2.0 and Ion AmpliSeq Inherited Disease Panel (Life Technologies). After the library was constructed by emulsion PCR, the amplicons were sequenced 500 flows on Ion Personal Genome Machine System (Life Technologies) according to the manufacture instruction. SNPs and indels were analyzed to the sequence reads that were mapped on hg19 of reference sequences. This project has been approved by the ethical committee of Tokai University School of Medicine. As a representative case, the molecular analysis to a 40 years old male who received a diagnosis of Brugada syndrome demonstrated a total of 584 SNPs or indels. Non-synonymous and frameshift nucleotide substitutions were selected in the coding region of heart disease related genes of ANK2, AKAP9, CACNA1C, DSC2, KCNQ1, MYLK, SCN1B, and STARD3. In particular, c.629T-C transition in exon 3 of the SCN1B gene, resulting in a leu210-to-pro (L210P) substitution is predicted “damaging” by the SIFT program. Because the mutation has not been reported, it was unclear if the substitution was pathogenic. Sudden death that failed in determining the cause of death constitutes one of the most important unsolved subjects in forensic pathology. The Ion AmpliSeq Inherited Disease Panel can amplify the exons of 328 genes at one time. We realized the difficulty in selection of the true source from a number of candidates, but postmortem genetic testing using NGS analysis deserves of a diagnostic to date. We now extend this analysis to SIDS suspected subjects and young sudden death victims.

Keywords: postmortem genetic testing, sudden death, SIDS, next generation sequencing

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Authors: Reena Aggarwal

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Wall art is a manifestation of the human mind and an absorbing form of cultural expression. Solapur wall hanging making art reflects cultural values, regional sensibilities, beliefs, and identity and helps to preserve the many different communities. The tango of warp and weft in many ways than one tells the story of civilization itself. Solapur wall hanging is a poem in multicolor, written with the warp and weft having long, rich, and complex history with indigenous design vocabularies made by the Padmasali communities. The wall-hanging weaving of Solapur has remained unaltered for years, from being very basic and monochrome having landscapes and portraits catering only to the local market, thereby becoming a potential family income generation tool. The study focuses on the need for contemporization of the Solapur wall hanging and also deliberates on the fact that wherever the culture of native people has been aided by intervention, in nearly every case, the quality of their craft has began to be enhanced. The study also found the underlying reason for diminishing sales to a declining market, low sales, lack of innovation in design, and product development. Keeping in mind that the artisans of Solapur have heroically always hold on to their ancient beliefs and practices, which give them strength and identity, and a sense of pride, an intervention program was developed with an objective of widening the market and help artisans have a sustaining income which include urban consumers and create designs suitable for the urban market. The process of defining and measuring the advantages of design intervention was achieved by using qualitative research methods. An ethnographic research methodology was adopted, which includes six months of close interface with artisans from ten families engaged in making of wall hanging in Solapur. Design solutions were proposed in terms of product diversification and design extensions of the existing product line for increased variety. A collection of contemporary wall arts (wall decor) and room dividers were designed and developed.

Keywords: wall hanging, Solapur, contemporization, traditional, sustainable

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Authors: Shalini TV, Gangadharan GG, Sriranjini S Jaideep, ASN Seshasayee, Awadhesh Pandit

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Introduction: Prakriti (body-mind constitution of an individual) is a conventional, customized and unique understanding of which is essential for the personalized medicine described in Ayurveda, Indian System of Medicine. Based on the Doshas( functional, bio humoral unit in the body), individuals are categorized into three major Prakriti- Vata, Pitta, and Kapha. The human gut microbiome hosts plenty of highly diverse and metabolically active microorganisms, mainly dominated by the bacteria, which are known to influence the physiology of an individual. Few researches have shown the correlation between the Prakriti and the biochemical parameters. In this study, an attempt was made to explore any correlation between the Prakriti (phenotype of an individual) with the Genetic makeup of the gut microbiome in healthy individuals. Materials and methods: 270 multi-ethnic, healthy volunteers of both sex with the age group between 18 to 40 years, with no history of antibiotics in the last 6 months were recruited into three groups of Vata, Pitta, and Kapha. The Prakriti of the individual was determined using Ayusoft, a software designed by CDAC, Pune, India. The volunteers were subjected to initial screening for the assessment of their height, weight, Body Mass Index, Vital signs and Blood investigations to ensure they are healthy. The stool and saliva samples of the recruited volunteers were collected as per the standard operating procedure developed, and the bacterial DNA was isolated using Qiagen kits. The extracted DNA was subjected to 16s rRNA sequencing using the Illumina kits. The sequencing libraries are targeting the variable V3 and V4 regions of the 16s rRNA gene. Paired sequencing was done on the MiSeq system and data were analyzed using the CLC Genomics workbench 11. Results: The 16s rRNA sequencing of the V3 and V4 regions showed a diverse pattern in both the oral and stool microbial DNA. The study did not reveal any specific pattern of bacterial flora amongst the Prakriti. All the p-values were more than the effective alpha values for all OTUs in both the buccal cavity and stool samples. Therefore, there was no observed significant enrichment of an OTU in the patient samples from either the buccal cavity or stool samples. Conclusion: In healthy volunteers of multi-ethnicity, due to the influence of the various factors, the correlation between the Prakriti and the gut microbiome was not seen.

Keywords: gut microbiome, ayurveda Prakriti, sequencing, multi-ethnic urban population

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Authors: Min-Chien Su, Tzu-Hsuan Hsu, Guan-Xuan Wu, Shyh-Ming Kuo

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Lung cancer is one of the clinically challenging malignant diseases worldwide. For efficient therapeutics in cancer, combination therapy has developed to acquire a better outcome. PLK-1 was one of the major factors affecting cell mitosis in cancer cells, its inhibitor Bi6727 was proven effective in treating several different cancers namely oral cancer, colon cancer and lung cancer. Despite its low toxicity toward normal cells compared to traditional chemotherapy, it is still yet to be evaluated in detail. Livistona Chinensis (LC) is a Chinese herb that used as a traditional prescription to treat lung cancer. Due to the uncertainty of the efficacy of LC, we utilized a water extraction method to extract the Livistona Chinensis and then lyophilized into powder for further study. In this study we investigated the antiproliferation activities of Bi6727 and LC extracts (LCE) on A549 non-small lung cancer cells. The IC50 of Bi6727 and LCE on A549 are 60 nM and 0.8 mg/mL, respectively. The fluorescent staining images shown nucleolus damage in cells treated with Bi6727 and mitochondrial damage after treated with LCE. A549 cells treated with Bi6727 and LCE showed increased expression of Bax, Caspase-3 and Caspase-9 proteins from Western blot assay. LCE also inhibited A549 cells growth keeping cells at G2-M phase from cell cycle assay. Apoptosis assay results showed that LCE induced late apoptosis of A549 cells. JC-1 assay showed that the mitochondria damaged at the LCE concentration of 0.4 mg/mL. In our preliminary anti-proliferation test of combined LCE and Bi-6727 on A549 cells, we found a dramatically decrease in proliferation after treated with LCE first for 24-h and then Bi-6727 for extra 24-h. This was an important finding regarding synergistic anti-proliferation effect of these drugs, However, the usage, the application sequence of LCE and Bi-6727 on A549 cells and their related mechanisms still need to be evaluated. In summary, the drugs exerted anti-proliferation effect on A549 cells independently. We hopefully combine the usage of these two drugs will bring a different and potential outcome in treating lung cancer.

Keywords: anti-proliferation, A549, Livistona Chinensis fruit extracts, PLK-1 inhibitor

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Authors: Oshma Chakoory, Sophie Comtet-Marre, Pierre Peyret

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Metagenomic classifiers are widely used for the taxonomic profiling of metagenomic data and estimation of taxa relative abundance. Small subunit rRNA genes are nowadays a gold standard for the phylogenetic resolution of complex microbial communities, although the power of this marker comes down to its use as full-length. We benchmarked the performance and accuracy of rRNA-specialized versus general-purpose read mappers, reference-targeted assemblers and taxonomic classifiers. We then built a pipeline called RiboTaxa to generate a highly sensitive and specific metataxonomic approach. Using metagenomics data, RiboTaxa gave the best results compared to other tools (Kraken2, Centrifuge (1), METAXA2 (2), PhyloFlash (3)) with precise taxonomic identification and relative abundance description, giving no false positive detection. Using real datasets from various environments (ocean, soil, human gut) and from different approaches (metagenomics and gene capture by hybridization), RiboTaxa revealed microbial novelties not seen by current bioinformatics analysis opening new biological perspectives in human and environmental health. In a study focused on corals’ health involving 20 metagenomic samples (4), an affiliation of prokaryotes was limited to the family level with Endozoicomonadaceae characterising healthy octocoral tissue. RiboTaxa highlighted 2 species of uncultured Endozoicomonas which were dominant in the healthy tissue. Both species belonged to a genus not yet described, opening new research perspectives on corals’ health. Applied to metagenomics data from a study on human gut and extreme longevity (5), RiboTaxa detected the presence of an uncultured archaeon in semi-supercentenarians (aged 105 to 109 years) highlighting an archaeal genus, not yet described, and 3 uncultured species belonging to the Enorma genus that could be species of interest participating in the longevity process. RiboTaxa is user-friendly, rapid, allowing microbiota structure description from any environment and the results can be easily interpreted. This software is freely available at https://github.com/oschakoory/RiboTaxa under the GNU Affero General Public License 3.0.

Keywords: metagenomics profiling, microbial diversity, SSU rRNA genes, full-length phylogenetic marker

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Authors: Israel B. Bitton

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“Memory” is associated with various phenomena, from physical to mental, personal to collective and historical to cultural. As part of a broader exploration of memory studies in philosophy and science (slated for academic publication October 2021), this specific study employs analytical methods of cognitive psychology and philosophy of memory to theorize that A) the primary human will (drive) is to significance, in that every human action and expression can be rooted in a most primal desire to be cosmically significant (however that is individually perceived); and B) that the will to significance manifests as the will to memorability, an innate desire to be remembered by others after death. In support of these broad claims, a review of various popular culture “touchpoints”—historic and contemporary records spanning literature, film and television, traditional news media, and social media—is presented to demonstrate how this very theory is repeatedly and commonly expressed (and has been for a long time) by many popular public figures as well as “everyday people.” Though developed before COVID, the crisis only increased the theory’s relevance: so many people were forced to die alone, leaving them and their loved ones to face even greater existential angst than what ordinarily accompanies death since the usual expectations for one’s “final moments” were shattered. To underscore this issue of, and response to, what can be considered a sociocultural “memory gap,” this study concludes with a summary of several projects launched by journalists at the height of the pandemic to document the memorable human stories behind COVID’s tragic warped speed death toll that, when analyzed through the lens of Viktor E. Frankl’s psychoanalytical perspective on “existential meaning,” shows how countless individuals were robbed of the last wills and testaments to their self-significance and memorability typically afforded to the dying and the aggrieved. The resulting insight ought to inform how government and public health officials determine what is truly “non-essential” to human health, physical and mental, at times of crisis.

Keywords: cognitive psychology, covid, neuroscience, philosophy of memory

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Authors: Hamed Ahari, Behrouz Akbari Adreghani, Vadood Razavilar, Amirali Anvar, Sima Moradi, Hourieh Shalchi

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Considering the ever increasing population and industrialization of the developmental trend of humankind's life, we are no longer able to detect the toxins produced in food products using the traditional techniques. This is due to the fact that the isolation time for food products is not cost-effective and even in most of the cases, the precision in the practical techniques like the bacterial cultivation and other techniques suffer from operator errors or the errors of the mixtures used. Hence with the advent of nanotechnology, the design of selective and smart sensors is one of the greatest industrial revelations of the quality control of food products that in few minutes time, and with a very high precision can identify the volume and toxicity of the bacteria. Methods and Materials: In this technique, based on the bacterial antibody connection to nanoparticle, a sensor was used. In this part of the research, as the basis for absorption for the recognition of bacterial toxin, medium sized silica nanoparticles of 10 nanometer in form of solid powder were utilized with Notrino brand. Then the suspension produced from agent-linked nanosilica which was connected to bacterial antibody was positioned near the samples of distilled water, which were contaminated with Staphylococcus aureus bacterial toxin with the density of 10-3, so that in case any toxin exists in the sample, a connection between toxin antigen and antibody would be formed. Finally, the light absorption related to the connection of antigen to the particle attached antibody was measured using spectrophotometry. The gene of 23S rRNA that is conserved in all Staphylococcus spp., also used as control. The accuracy of the test was monitored by using serial dilution (l0-6) of overnight cell culture of Staphylococcus spp., bacteria (OD600: 0.02 = 107 cell). It showed that the sensitivity of PCR is 10 bacteria per ml of cells within few hours. Result: The results indicate that the sensor detects up to 10-4 density. Additionally, the sensitivity of the sensors was examined after 60 days, the sensor by the 56 days had confirmatory results and started to decrease after those time periods. Conclusions: Comparing practical nano biosensory to conventional methods like that culture and biotechnology methods(such as polymerase chain reaction) is accuracy, sensitiveness and being unique. In the other way, they reduce the time from the hours to the 30 minutes.

Keywords: exotoxin, nanobiosensor, recognition, Staphylococcus aureus

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Authors: Kamran Jawed, Anu Jose Mattam, Zia Fatma, Saima Wajid, Malik Z. Abdin, Syed Shams Yazdani

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Worldwide demand of natural and sustainable fuels and chemicals have encouraged researchers to develop microbial platform for synthesis of short chain fatty acids as they are useful precursors to replace petroleum-based fuels and chemicals. In this study, we evaluated the role of fatty acid synthesis and β-oxidation cycle of Escherichia coli to produce butyric acid, a 4-carbon short chain fatty acid, with the help of three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron. We found that E. coli strain transformed with gene for TesBT and grown in presence of 8 g/L glucose produced maximum butyric acid titer at 1.46 g/L, followed by that of TesBF at 0.85 g/L and TesAT at 0.12 g/L, indicating that these thioesterases were efficiently converting short chain fatty acyl-ACP intermediate of fatty acid synthesis pathway into the corresponding acid. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. Deletion of genes for fatty acyl-CoA synthetase and acyl-CoA dehydrogenase, which are involved in initiating the fatty acid degradation cycle, and overexpression of FadR, which is a dual transcriptional regulator and exerts negative control over fatty acid degradation pathway, reduced up to 30% of butyric acid titer. This observation suggested that β-oxidation pathway is working synergistically with fatty acid synthesis pathway in production of butyric acid. Moreover, accelerating the fatty acid elongation cycle by overexpressing acetyl-CoA carboxyltransferase (Acc) and 3-hydroxy-acyl-ACP dehydratase (FabZ) or by deleting FabR, the transcription suppressor of elongation, did not improve the butyric acid titer, rather favored the long chain fatty acid production. Finally, a balance between cell growth and butyric acid production was achieved with the use of phosphorous limited growth medium and 14.3 g/L butyric acid, and 17.5 g/L total free fatty acids (FFAs) titer was achieved during fed-batch cultivation. We have engineered an E. coli strain which utilizes the intermediate of both fatty acid synthesis and degradation pathway, i.e. butyryl-ACP and -CoA, to produce butyric acid from glucose. The strategy used in this study resulted in highest reported titers of butyric acid and FFAs in engineered E. coli.

Keywords: butenoic acid, butyric acid, Escherichia coli, fed-batch fermentation, short chain fatty acids, thioesterase

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Authors: Mohammad Hossein Habibi, Atena Sadat Hosseini

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Colorectal cancer is the third leading cause of cancer-related death in the world. Colorectal cancer screening, early detection, and treatment programs could benefit from the most up-to-date information on the disease's burden, given the present worldwide trend of increasing colorectal cancer incidence. Tumor recurrence and resistance are exacerbated by the presence of chemotherapy-resistant cancer stem cells that can generate rapidly proliferating tumor cells. In addition, tumor cells can evolve chemoresistance through adaptation mechanisms. In this work, we used in silico analysis to select suitable GEO datasets. In this study, we compared slow-growing cancer stem cells with high-growth colorectal cancer-derived cancer stem cells. We then evaluated the signal pathways, transcription factors, and kinases associated with these two types of cancer stem cells. A total of 980 upregulated genes and 870 downregulated genes were clustered. MAPK signaling pathway, AGE-RAGE signaling pathway in diabetic complications, Fc gamma R-mediated phagocytosis, and Steroid biosynthesis signaling pathways were observed in upregulated genes. Also, caffeine metabolism, amino sugar and nucleotide sugar metabolism, TNF signaling pathway, and cytosolic DNA-sensing pathway were involved in downregulated genes. In the next step, we evaluated the best transcription factors and kinases in two types of cancer stem cells. In this regard, NR2F2, ZEB2, HEY1, and HDGF as transcription factors and PRDM5, SMAD, CBP, and KDM2B as critical kinases in upregulated genes. On the other hand, IRF1, SPDEF, NCOA1, and STAT1 transcription factors and CTNNB1 and CDH7 kinases were regulated low expression genes. Using bioinformatics analysis in the present study, we conducted an in-depth study of colorectal cancer stem cells at low and high growth rates so that we could take further steps to detect and even target these cells. Naturally, more additional tests are needed in this direction.

Keywords: colorectal cancer, bioinformatics analysis, transcription factor, kinases, cancer stem cells

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Authors: Kamsiah Jaarin, Yusof Kamisah, Faizah Othman Nurul Akmal Muhammad, Zakiah Jubri, Qodriyah Mohd Saad, Srijit Das

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Forty (40) normotensive adult male Sprague-Dawley rats aged three months weighing 180-200 g were divided into 4 groups with 10 rats per group: (1) normotensive control; (2) hypertensive rats; (3) hypertensive rats treated with Nigella sativa (2.5 ml/kg/day); and (4) hypertensive rats treated with nicardipine (5 mg/kg/day). After acclimatization, the hypertensive rats of the group 2, 3 and 4 were induced to be hypertensive by giving NW–nitro-L-arginine methyl ester (L-NAME; 30 mg/kg/day) in their drinking water for consecutive 7 days. After one week, rats in the group 3 were given a daily oral dose of 2.5 ml/kg/day of Nigella sativa (NS) by oral gavage. Rats in the group 4 were given nicardipine (5 mg/kg/day) via oral gavages. All rats in this study received L-NAME continuously throughout the treatment duration. The blood pressure will be measured pre-treatment and weekly for 8 weeks using power lab. Blood was taken before and at the end of study for measurement of nitric oxide. At the end of 8 weeks, the rats are sacrificed and descending thoracic aorta was disserted for measurement of vascular reactivity, and intracellular adhesion molecules (ICAM-1) and vascular cell adhesion molecules (VCAM-1). Nigella sativa reduced both systolic and diastolic BP compared to control and L-name group. The BP lowering effect of NS was comparable to nicardipine a calcium antagonist. The blood pressure lowering effect of NS was accompanied with an increasing relaxation response to nitroprusside and acetylcholine and reducing vasoconstriction response to epinephrine. L-NAME and nicardipine on the other hand, reduced plasma nitric oxide concentration. In contrast, NS increased NO concentration. However, Nigella sativa had no significant effect on aortic VCAM- 1 and ICAM-1 expression. In conclusion; Nigella sativa oil reduces both systolic and diastolic blood pressure in L-NAME treated rats. The antihypertensive effect of NS was comparable to nicardipine. The BP lowering effect may be mediated via stimulating nitric oxide release from vascular endothelium.

Keywords: Nigella sativa, ICAM, VCAM, blood pressure, vascular reactivity

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Authors: Zeynep Yazgan, Gökhan Aygün, Reyhan Çalışkan

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Objective: Invasive fungal infections are a serious threat in terms of morbidity and mortality, especially in immunocompromised patients. The most frequently isolated agents are Aspergillus genus fungi, and sensitivity to azoles, which are the first choice in treatment, decreases. In our study, we aimed to investigate the use of the agar plate screening method as a fast, easy, and practical method in determining azole resistance in Aspergillus spp. species. Methods: Our study was conducted with 125 Aspergillus spp. isolates produced from various clinical samples. Aspergillus spp. isolates were identified by conventional methods and azole resistance was determined by gradient test and agar plate screening method. Broth microdilution method was applied to resistant isolates, and CypA-L98H and CypA-M220 mutations in the cyp51A gene were investigated. Results: In our study, 55 A. fumigatus complex (44%), 42 A. flavus (33.6%), 6 A. terreus (5%), 4 A. niger (3%) and 18 Aspergillus spp. (14%) were identified. With the gradient test method, resistance to VOR and POS was detected in 1 (1.8%) of A.fumigatus isolates, and resistance to ITR was detected in 3 (5.45%). With the agar plate method, 1 of the A.fumigatus isolates (1.8%) had VOR, ITR, POS, 1 of the A.terreus isolates (16.7%) had VOR, 1 of the A.niger isolates (25%) had ITR. Resistance to VOR and POS was detected in 2 Aspergillus spp. isolates (11%), and resistance to ITR was detected in 1 (5.6%). Sensitivity and specificity were determined as 100% for VOR and POS in A. fumigatus species, 33.3% and 100% for ITR, respectively, 100% for ITR in A. flavus species, and 100% for ITR and POS in A. terreus species. By broth microdilution method in 7 isolates in which resistance was detected by gradient test and/or agar plate screening method; 1 A.fumigatus resistant to ITR, VOR, POS, 2 A.fumigatus resistant to ITR, 2 Aspergillus spp. ITR, VOR, POS MICs were determined as 2µg/ml and 8µg/ml, 8µg/ml and >32µg/ml, 0.5µg/ml and 4µg/ml, respectively. CypA-L98H mutations were detected in 5 of these isolates, CypA-M220 mutations were detected in 6, and no mutation was detected in 1. CypA-L98H and CypA-M220 mutations were detected in 1 isolate for which resistance was not detected. Conclusion: The need for rapid antifungal susceptibility screening tests is increasing in the treatment of aspergillosis. Although the sensitivity of the agar plate method was determined to be 33.3% for A.fumigatus ITR in our study, its sensitivity and specificity were determined to be 100% for ITR, VOR, and POS in other species. The low sensitivity value detected for A.fumigatus showed that agar plate drug concentrations should be updated in accordance with the latest regulations of EUCAST guidelines. The CypA-L98H and CypA-M220 mutations detected in our study suggested that the distribution of azole resistance-related mutations in different regions in our country should be investigated. In conclusion, it is thought that the agar plate method, which can be easily applied to detect azole resistance, is a fast and practical method in routine use and can contribute to both the determination of effective treatment strategies and the generation of epidemiological data.

Keywords: Aspergillus, agar plate, azole resistance, cyp51A, cypA-L98H, cypA-M220

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Authors: Salma A. El-Marasy, Rehab F. Abdel-Rahman, Reham M. Abd-Elsalam

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The burden of stroke is intensely increasing worldwide. Brain injury following transient or permanent focal cerebral ischemia develops ischemic stroke as a consequence of a complex series of pathophysiological events. The aim of this study is to evaluate the possible neuroprotective effect of a dipeptidyl peptidase-4 inhibitor, vildagliptin, independent on its insulinotropic properties in non-diabetic rats subjected to cerebral ischemia. Anaesthetized Wistar rats were subjected to either left middle cerebral artery occlusion (MCAO) or sham operation followed by reperfusion after 30 min of MCAO. The other three groups were orally administered vildagliptin at 3 dose levels (2.5, 5, 10 mg/kg) for 3 successive weeks before subjected to left focal cerebral ischemia/reperfusion and till the end of the study. Neurological deficit scores and motor activity were assessed 24h following reperfusion. 48h following reperfusion, rats were euthanized and their left brain hemispheres were harvested and used in the biochemical, histopathological, and immunohistochemical investigations. Vildagliptin pretreatment improved neurological score deficit, locomotor activity and motor coordination in MCAO rats. Moreover, vildagliptin reduced malondialdehyde (MDA), elevated reduced glutathione (GSH), phosphotylinosital 3 kinase (PI3K), phosphorylated of protein kinase B (p-AKT), and mechanistic target of rapamycin (mTOR) brain contents in addition to reducing protein expression of caspase-3. Also, vildagliptin showed a dose-dependent attenuation in neuronal cell loss and histopathological alterations in MCAO rats. This study proves that vildagliptin exerted the neuroprotective effect in a dose-dependent manner as shown in amelioration of neuronal cell loss and histopathological damage in MCAO rats, which may be mediated by attenuating neuronal and motor deficits, it’s anti-oxidant property, activation of PI3K/AKT/mTOR pathway and its anti-apoptotic effect.

Keywords: caspase-3, cerebral ischemia, dipeptidyl peptidase-4 inhibitor, oxidative stress, PI3K/AKT/mTOR pathway, rats, vildagliptin

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Authors: Tugce Duygu Koksal

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In recent years, given the fact that the acts of terrorism and the threat of the latter are taking place without any border and distinction, it has led the states to deal with the terrorism as a priority issue. More recently, as seen in different countries during state of emergency, the adoption of anti-terrorism measures motivated by the sole need of the prevention of terrorism targets directly the fundamental rights of individuals. Therefore, a contribution to the understanding of the value of the principle of legality is becoming more and more important nowadays. This paper aims to reflect the probable effects of the adoption of anti-terrorism measures regardless of the principle of legality, on the fundamental rights. In this respect, this paper will first discuss the margin of appreciation of the national authorities by countering terrorism, and then, the importance of the respect of the legality of the anti-terrorism measures will be examined in the light of actual examples. Indeed, one of the major findings of this study is the fact that the anti-terrorism laws and measures were taken in this framework must be subject to close scrutiny in democracies, which adopted the principle of the rule of law and respect human rights. Although the state's margin of appreciation in the field of counter-terrorism is broad, these measures which are based on the legitimate aim of a democracies’ legitimate right to protect itself against the activities of terrorist organizations should have the legal basis and be strictly required by the exigencies of the fight against terrorism. While combating terrorism, the legal basis shall only be achieved if the legal consequences of an individuals’ actions related to terrorism shall be clear and foreseeable by the individuals of a society. On the other hand, particularly during the state of emergency, the ambiguity of the law might be used to include a wide range of actions under acts of terrorism. This is becoming more dangerous where freedom of expression, freedom of the press, freedom of association and the right to information is in the substance of these actions. Disregarding the principle of legality is susceptible to create a chilling effect on the exercise of human rights, and therefore, the fight against terrorism can be transformed into a repressive regime on opponents. As a result, the efforts to counter terrorism of the national authorities irrespective of the principle of legality are susceptible to cause a transformation of the rule of law to a state of law which cannot be appreciated in a democratic society.

Keywords: anti-terrorism measures, chilling effect, predictability, the principle of legality, state of emergency

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Authors: Patricia Portugal Marques de Carvalho Lourenco

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A three-phase methodological multi-lingual path was designed, constructed and carried out using the 2020 Chinese Internet Drama Series We Are All Alone as a case study. Phase one, the backbone of the research, comprised of secondary data analysis, providing the structure on which the next two phases would be built on. Phase one incorporated a Google Scholar and a Baidu Index analysis, Star Network Influence Index and Mydramalist.com top two drama reviews, along with an article written about the drama and scrutiny of Chinese related blogs and websites. Phase two was field research elaborated across Latin Europe, and phase three was social media focused, having into account that perceptions are going to be memory conditioned based on past ideas recall. Overall, research has shown the poor cultural expression of Chinese entertainment in Latin Europe and demonstrated the inexistence of Chinese content in French, Italian, Portuguese and Spanish Business to Consumer retailers; a reflection of their low significance in Latin European markets and the short-life cycle of entertainment products in general, bubble-gum, disposable goods without a mid to long-term effect in consumers lives. The process of conducting comprehensive international research was complex and time-consuming, with data not always available in Mandarin, the researcher’s linguistic deficiency, limited Chinese Cultural Knowledge and cultural equivalence. Despite steps being taken to minimize the international proposed research, theoretical limitations concurrent to Latin Europe and China still occurred. Data accuracy was disputable; sampling, data collection/analysis methods are heterogeneous; ascertaining data requirements and the method of analysis to achieve a construct equivalence was challenging and morose to operationalize. Secondary data was also not often readily available in Mandarin; yet, in spite of the array of limitations, research was done, and results were produced.

Keywords: research methodologies, international research, primary data, secondary data, research limitations, online dramas, china, latin europe

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Authors: Muhammad Naeem, Juho Lee, Jin-Wook Yoo

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In this study, we hypothesized that prolonged gastrointestinal transit at the inflamed colon conferred by a pH-triggered mucoadhesive smart nanoparticulate drug delivery system aids in achieving selective and sustained levels of the drug within the inflamed colon for the treatment of ulcerative colitis. We developed budesonide-loaded pH-sensitive charge-reversal solid lipid nanoparticles (SLNs) using a hot homogenization method. Polyetylenimine (PEI) was used to render SLNs cationic (PEI-SLNs). Eudragit S100 (ES) was coated on PEI-SLNs for pH-trigger charge-reversal SLNs (ES-PEI-SLNs). Therapeutic potential of the prepared SNLs formulation was evaluated in ulcerative colitis in mice. The transmission electron microscopy, zeta size and zeta potential data showed the successful formation of SLNs formulations. SLNs and PEI-SLNs showed burst drug release in acidic pH condition mimicking stomach and early small intestine environment which limiting their application as oral delivery systems. However, ES-PEI-SLNs prevented a burst drug release in acidic pH conditions and showed sustained release at a colonic pH. Most importantly, the surface charge of ES-PEI-SLNs switched from negative to positive in colonic conditions by pH-triggered removal of ES coating and accumulated selectively in inflamed colon. Furthermore, a charge reversal ES-PEI-SLNs showed a superior mitigation of dextran sulfate sodium (DSS)-induced acute colitis in mice as compared to SLNs and PEI-SLNs treated groups. Moreover, histopathological analysis of distal colon sections stained with hematoxylin/eosin and E-cadherin immunostaining revealed attenuated inflammation in an ES-PEI-SLNs-treated group. We also found that ES-PEI-SLNs markedly reduced the myeloperoxidase level and expression of TNF-alpha in colon tissue. Our results suggest that the pH-triggered charge reversal SLNs presented in this study would be a promising approach for ulcerative colitis therapy.

Keywords: solid lipid nanoparticles, stimuli-triggered charge-reversal, ulcerative colitis, methacrylate copolymer, budesonide

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Authors: Luiz Nolasco Jr. Rezende, Antonio Villar M. Sá, Claudia Marcia L. Pato

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The increasing urban violence in Brazil involves more and more infractions committed by children and youths. The challenges faced by the institutional environments responsible for the education and resocialization of adolescents in conflict with the law are enormous, especially of those deprived of their liberty. These institutions have an inadequate educational structure. They are characterized by a dirty and unhealthy environment without the minimum basic conditions for their activities, by frequent practices of degradation, humiliation, and the physical and psychological punishment of inmates. This mixed-method study investigated the personal values of adolescents with restriction of freedom in a socio-educational institutional environment aiming to contribute to the development of their morality through an educational process. For that, we used a survey and transdisciplinary play workshops involving thirty-two boys aged between 15 and 19 years old and at least two years out of school. To evaluate the survey the reduced version of the Portrait Questionnaire—PQ21—was used. The workshops happened once a week, lasting 80 minutes each, totaling twelve meetings. By using the game of chess and its metaphors, participants produced texts and engaged in critical brainstorming about their lives. The survey results pointed out that these young people showed a predominance of values of openness to change and self-transcendence, dissatisfaction with one's own reality and surroundings, not considering the consequences of their actions on themselves and others, difficulties in speaking and writing, and desire for changes in their lives. After the pedagogical interventions, these adolescents demonstrated an understanding of the implications of their actions for themselves, for their families, especially for the mothers, with whom they demonstrated stronger bonds. It was possible to observe evidence of improvement in the capacity of linguistic expression, more autonomy and critical vision, including about themselves and their respective contexts. These results demonstrated the educational potential of lively, symbolic, dynamic and playful activities that favor the mediation and identification of these adolescents with their lives, and contribute to the projection of dreams.

Keywords: adolescents arrested, human values, moral development, playful workshops

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Authors: Hacer Kobya Bulut, Kıymet Yeşilçiçek Çalık, Birsel Canan Demirbağ, Hacer Erdöl, Songül Aktaş

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Introduction: As an expression of an emotion which always exists in life, crying is regarded as one of the problematic behaviors of patients by nurses. Towards such patients, nurses may exhibit emotional and behavioral reactions such as feeling helpless, anger, indifferent, defense, and opposition. However crying either meets a need, reduces the tension to cope with problems or helps patient to gain strength. Therefore, nurses must accept that crying is a normal mechanism that reduces emotional tension and should approach a crying patient accordingly. Objective: This study was carried out to evaluate the communicational behaviors of the nurses towards ‘crying patient’. Methods: This descriptive study was conducted with the nurses working at a university hospital in a city in the Eastern Black Sea in June-September 2015. The entire universe was tried to be reached without sampling. 90% of the population was reached and the study was completed with 309 nurses who volunteered to participate in the study. Data were collected through a questionnaire which was prepared reviewing the literature by researchers. Data were evaluated in SPSS analysis program using percentages, numbers and chi-square test with the 95% confidence interval and p <0.05significance level. Findings: The findings showed that the average age of nurses was 31.52 ± 7.96, work experience was 10:09 ± 7.69 and only 22.7% had training about ‘approach to crying patient’ during their education. 97.1% of the nurses often faced with crying patients in their professional lives, 62.8% stated that they faced crying women patients. When they see crying patients, 84.8% of the nurses ‘do not want the patient to cry’, 80.9% wonder ‘why they are crying’, % 79.6 ‘feel uneasiness’,% 79.3 ‘feel sorry’ and 41.4% ‘ feel helpless’. The question ‘Why do you think the patient is crying?’ was answered by 93.5% nurses as ‘they are suffering’, by 86.1% ‘they are helpless’, 80.9% ‘they are sad’, 79.6% ‘they need help’, 54.4% ‘because they feel inadequate,’ and 44.7% ‘they fail to control their crying behavior. ‘How do you approach to your patient when she/he is crying?’ question was answered by 82.5% of nurses as ‘I would console’, 77.3% as ‘I would ask the reason’, 63.1% as ‘I would try to stop her from crying’ all of which are actually inappropriate nursing approaches. However, 92.2% of the nurses stated that ‘I do not judge the crying patient’, ‘87.1% said ‘I allocate time to crying patients’ and 85.8% said ‘ I ask patient whether they want to cry alone’. The study showed that educational background and work experience of the nurses affected the appropriate approach to crying patients (P <0.05). Conclusion: As a result of the study, it was found out that nurses do not want patients to cry, so they exhibit inappropriate approach such as consoling the patients and they have difficulty in approaching crying patients.

Keywords: approach to patient, communication, crying patient, nurse, Turkey

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Authors: S. Kozlovski, S.W. Feigelson, R. Alon

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The b2 integrin ligands ICAM-1 ICAM-2 and the endothelial VLA-4 integrin ligand VCAM-1 are constitutively expressed on different lung vessels and on high endothelial venules (HEVs), the main portal for lymphocyte entry from the blood into lung draining lymph nodes. ICAMs are also ubiquitously expressed by many antigen-presenting leukocytes and have been traditionally suggested as critical for the various antigen-specific immune synapses generated by these distinct leukocytes and specific naïve and effector T cells. Loss of both ICAM-1 and ICAM-2 on the lung vasculature reduces the ability to patrol monocytes and Tregs to patrol the lung vasculature at a steady state. Our new findings suggest, however, that in terms of innate leukocyte trafficking into the lung lamina propria, both constitutively expressed and virus-induced vascular VCAM-1 can functionally compensate for the loss of these ICAMs. In a mouse model for influenza infection, neutrophil and NK cell recruitment and clearance of influenza remained normal in mice deficient in both ICAMs. Strikingly, mice deficient in both ICAMs also mounted normal influenza-specific CD8 proliferation and differentiation. In addition, these mice normally combated secondary influenza infection, indicating that the presence of ICAMs on conventional dendritic cells (cDCs) that present viral antigens are not required for immune synapse formation between these APCs and naïve CD8 T cells as previously suggested. Furthermore, long-lasting humoral responses critical for protection from a secondary homosubtypic influenza infection were also normal in mice deficient in both ICAM-1 and ICAM-2. Collectively, our results suggest that the expression of ICAM-1 and ICAM-2 on lung endothelial and epithelial cells, as well as on DCs and B cells, is not critical for the generation of innate or adaptive anti-viral immunity in the lungs. Our findings also suggest that endothelial VCAM-1 can substitute for the functions of vascular ICAMs in leukocyte trafficking into various lung compartments.

Keywords: emigration, ICAM-1, lymph nodes, VCAM-1

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Authors: M. M. Essa, S. Subash, M. Akbar, S. Al-Adawi, A. Al-Asmi, G. J. Guillemein

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Transgenic (tg) mice which contain an amyloid precursor protein (APP) gene mutation, develop extracellular amyloid beta (Aβ) deposition in the brain, and severe memory and behavioral deficits with age. These mice serve as an important animal model for testing the efficacy of novel drug candidates for the treatment and management of symptoms of Alzheimer's disease (AD). Several reports have suggested that oxidative stress is the underlying cause of Aβ neurotoxicity in AD. Date palm fruits contain very high levels of antioxidants and several medicinal properties that may be useful for improving the quality of life in AD patients. In this study, we investigated the effect of dietary supplementation of Omani date palm fruits on the memory, anxiety and learning skills along with inflammation in an AD mouse model containing the double Swedish APP mutation (APPsw/Tg2576). The experimental groups of APP-transgenic mice from the age of 4 months were fed custom-mix diets (pellets) containing 2% and 4% Date palm fruits. We assessed spatial memory and learning ability, psychomotor coordination, and anxiety-related behavior in Tg and wild-type mice at the age of 4-5 months and 18-19 months using the Morris water maze test, rota rod test, elevated plus maze test, and open field test. Further, inflammatory parameters also analyzed. APPsw/Tg2576 mice that were fed a standard chow diet without dates showed significant memory deficits, increased anxiety-related behavior, and severe impairment in spatial learning ability, position discrimination learning ability and motor coordination along with increased inflammation compared to the wild type mice on the same diet, at the age of 18-19 months In contrast, PPsw/Tg2576 mice that were fed a diet containing 2% and 4% dates showed a significant improvements in memory, learning, locomotor function, and anxiety with reduced inflammatory markers compared to APPsw/Tg2576 mice fed the standard chow diet. Our results suggest that dietary supplementation with dates may slow the progression of cognitive and behavioral impairments in AD. The exact mechanism is still unclear and further extensive research needed.

Keywords: Alzheimer's disease, date palm fruits, Oman, cognitive decline, memory loss, anxiety, inflammation

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Authors: S. Junaid S. Qazi, Denice C. Bay, Raymond Chew, Raymond J. Turner

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EmrE, a member of the small multidrug resistance protein family in bacteria is considered to be the archetypical member of its family. It confers host resistance to a wide variety of quaternary cation compounds (QCCs) driven by proton motive force. Generally, purification yield is a challenge in all membrane proteins because of the difficulties in their expression, isolation and solubilization. EmrE is extremely hydrophobic which make the purification yield challenging. We have purified EmrE protein using two different approaches: organic solvent membrane extraction and hexahistidine (his6) tagged Ni-affinity chromatographic methods. We have characterized changes present between ligand affinity of untagged and his6-tagged EmrE proteins in similar membrane mimetic environments using biophysical experimental techniques. Purified proteins were solubilized in a buffer containing n-dodecyl-β-D-maltopyranoside (DDM) and the conformations in the proteins were explored in the presence of four QCCs, methyl viologen (MV), ethidium bromide (EB), cetylpyridinium chloride (CTP) and tetraphenyl phosphonium (TPP). SDS-Tricine PAGE and dynamic light scattering (DLS) analysis revealed that the addition of QCCs did not induce higher multimeric forms of either proteins at all QCC:EmrE molar ratios examined under the solubilization conditions applied. QCC binding curves obtained from the Trp fluorescence quenching spectra, gave the values of dissociation constant (Kd) and maximum specific one-site binding (Bmax). Lower Bmax values to QCCs for his6-tagged EmrE shows that the binding sites remained unoccupied. This lower saturation suggests that the his6-tagged versions provide a conformation that prevents saturated binding. Our data demonstrate that tagging an integral membrane protein can significantly influence the protein.

Keywords: small multidrug resistance (SMR) protein, EmrE, integral membrane protein folding, quaternary ammonium compounds (QAC), quaternary cation compounds (QCC), nickel affinity chromatography, hexahistidine (His6) tag

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Authors: Konstantin S. Vainutis, Mark E. Andreev, Anastasia N. Voronova, Mikhail Yu. Shchelkanov

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Introduction: The trematodes from the family Cyclocoelidae (cyclocoelids) belong to the superfamily Echinostomatoidea infecting air sacs and trachea of wild birds. At present, the family Cyclocoelidae comprises nine valid genera in three subfamilies: Cyclocoelinae (type taxon), Haematotrephinae, and Typhlocoelinae. To our best knowledge, in this study, molecular genetic methods were used for the first time for studying cyclocoelids from the Russian Far East. Here we provide the data on the morphology and phylogeny of cyclocoelids from gadwall from the Russian Far East. The morphological and genetic data obtained for cyclocoelids indicated the necessity to revise the previously proposed classification within the family Cyclocoelidae. Objectives: The first objective was performing the morphological study of cyclocoelids found in M. strepera from the Russian Far East. The second objective is to reconstruct the phylogenetic relationships of the studied trematodes with other cyclocoelids using the 28S gene. Material and methods: During the field studies in the Khasansky district of the Primorsky region, 21 cyclocoelids were recovered from the air sacs of a single gadwall Mareca strepera. Seven samples of cyclocoelids were overstained in alum carmine, dehydrated in a graded ethanol series, cleared in clove oil, and mounted in Canada balsam. Genomic DNA was extracted from four cyclocoelids using the alkaline lysis method HotShot. The 28S rDNA fragment was amplified using the forward primer Digl2 and the reverse primer 1500R. Results: According to morphological features (ovary intratesticular, forming a triangle with the testes), the studied worms belong to the subfamily Cyclocoelinae Stossich, 1902. In particular, the highest morphological similarity was observed in relation to the trematodes of the genus Cyclocoelum Brandes, 1892 – genital pores are pharyngeal. However, the genetic analysis has shown significant discrepancies between the trematodes studied regarding the genus Cyclocoelum. On the phylogenetic tree, these trematodes took the sister position in relation to the genus Morishitium (previously considered in the subfamily Szidatitrematinae). Conclusion: Based on the results of the morphological and genetic studies, cyclocoelids isolated from Mareca strepera are suggested to be described in the previously unknown genus and differentiated from the type genus Cyclocoelum of the type subfamily Cyclocoelinae. Considering the available molecular data, including described cyclocoelids, the family Cyclocoelidae comprises ten valid genera in the three subfamilies mentioned above.

Keywords: new species, trematoda, phylogeny, cyclocoelidae

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Authors: Kai Pai Huang

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Background: There are several types of plasma cell neoplasms including multiple myeloma, plasmacytoma, lymphoplasmacytic lymphoma, and monoclonal gammopathy of undetermined significance (MGUS) are found in our lab. Today, we want to compare with two cases using the method of capillary electrophoresis. Method: Serum is prepared and electrophoresis is performed at alkaline PH in a capillary using the Sebia® Capillary 2. Albumin and globulins are detected by the detector which is located in the cathode of the capillary and the signals are transformed to peaks. Serum was treated with beta-mercaptoethanol which reducing the polymerized immunoglobulin to monomer immunoglobulin to clarify two M-protein are secreted from the same plasma cell clone in bone marrow. Result: Case 1: A 78-year-old female presenting dysuria, oliguria and leg edema for several months. Laboratory data showed proteinuria, leukocytosis, results of high serum IgA and lambda light chain. A renal biopsy found amyloid fibrils in the glomerular mesangial area. Serum protein electrophoresis shows a major monoclonal peak in the β region and minor small peak in gamma region, and the immunotyping studies for serum showed two IgA/λ type. Case 2: A 55-year-old male presenting abdominal distension and low back pain for more than one month. Laboratory data showed T12 T8 compression fracture, results of high serum IgM and kappa light chain. Bone marrow aspiration showed the cells from the bone marrow are B cells with monotypic kappa chain expression. Bone marrow biopsy found this is lymphoplasmacytic lymphoma (Waldenstrom macroglobulin). Serum protein electrophoresis shows a monoclonal peak in the β region and the immunotyping studies for serum showed IgM/κ type. Conclusion: Plasma cell neoplasm can be diagnosed by many examinations. Among them, using capillary electrophoresis by a lab can separate several types of gammopathy and the quantification of a monoclonal peak can be used to evaluate the patients’ prognosis or treatment.

Keywords: plasma cell neoplasm, capillary electrophoresis, serum protein electrophoresis, immunotyping

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Authors: Neha Toppo

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Method and strategies of teaching instruction highly influence learning of students. In second language teaching, number of ways and methods has been suggested by different scholars and researchers through times. The present article deals with the role of teaching instruction in developing compositional ability of students in writing. It focuses on the secondary level students of rural areas, whose exposure to English language is limited and they face challenges even in simple compositions. The students till high school suffer with their disability in writing formal letter, application, essay, paragraph etc. They face problem in note making, writing answers in examination using their own words and depend fully on rote learning. It becomes difficult for them to give language to their own ideas. Teaching writing composition deserves special attention as writing is an integral part of language learning and students at this level are expected to have sound compositional ability for it is useful in numerous domains. Effective method of instruction could help students to learn expression of self, correct selection of vocabulary and grammar, contextual writing, composition of formal and informal writing. It is not limited to school but continues to be important in various other fields outside the school such as in newspaper and magazine, official work, legislative work, material writing, academic writing, personal writing, etc. The study is based on the experimental method, which hypothesize that guided instruction will be more effective in teaching writing compositions than usual instruction in which students are left to compose by their own without any help. In the test, students of one section are asked to write an essay on the given topic without guidance and another section are asked to write the same but with the assistance of guided instruction in which students have been provided with a few vocabulary and sentence structure. This process is repeated in few more schools to get generalize data. The study shows the difference on students’ performance using both the instructions; guided and unguided. The conclusion of the study is followed by the finding that writing skill of the students is quite poor but with the help of guided instruction they perform better. The students are in need of better teaching instruction to develop their writing skills.

Keywords: composition, essay, guided instruction, writing skill

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Authors: Bruna Lima Gomes, Vanessa Caroline De Barros Bonato, Luciano Freschi, Eduardo Purgatto

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Plant hormones are long known to be tightly associated with fruit development and are involved in controlling various aspects of fruit ripening. For fleshy fruits, ripening is characterized for changes in texture, color, aroma and other parameters that markedly contribute to its quality. Ethylene is one of the major players regulating the ripening-related processes, but emerging evidences suggest that auxin is also part of this dynamic control. Thus, the aim of this study was providing new insights into the auxin role during ripening and the hormonal interplay between auxin and ethylene. For that, tomato fruits (Micro-Tom) were collected at mature green stage and separated in four groups: one for indole-3-acetic acid (IAA) treatment, one for ethylene, one for a combination of IAA and ethylene, and one for control. Hormone solution was injected through the stylar apex, while mock samples were injected with buffer only. For ethylene treatments, fruits were exposed to gaseous hormone. Then, fruits were left to ripen under standard conditions and to assess ripening development, hue angle was reported as color indicator and ethylene production was measured by gas chromatography. The transcript levels of three ripening-related ethylene receptors (LeETR3, LeETR4 and LeETR6) were evaluated by RT-qPCR. Results showed that ethylene treatment induced ripening, stimulated ethylene production, accelerated color changes and induced receptor expression, as expected. Nonetheless, auxin treatment showed the opposite effect once fruits remained green for longer time than control group and ethylene perception has changed, taking account the reduced levels of receptor transcripts. Further, treatment with both hormones revealed that auxin effect in delaying ripening was predominant, even with higher levels of ethylene. Altogether, the data suggest that auxin modulates several aspects of the tomato fruit ripening modifying the ethylene perception. The knowledge about hormonal control of fruit development will help design new strategies for effective manipulation of ripening regarding fruit quality and brings a new level of complexity on fruit ripening regulation.

Keywords: ethylene, auxin, fruit ripening, hormonal crosstalk

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Authors: Francesco Pantano, Marta Fogolari, Michele Iuliani, Sonia Simonetti, Silvia Cavaliere, Marco Russano, Fabrizio Citarella, Bruno Vincenzi, Silvia Angeletti, Giuseppe Tonini

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Background: Although immune checkpoint inhibitors (ICIs) have changed the treatment paradigm of non–small cell lung cancer (NSCLC), these drugs fail to elicit durable responses in the majority of NSCLC patients. The gut microbiota, able to regulate immune responsiveness, is emerging as a promising, modifiable target to improve ICIs response rates. Since the oral microbiome has been demonstrated to be the primary source of bacterial microbiota in the lungs, we investigated its composition as a potential predictive biomarker to identify and select patients who could benefit from immunotherapy. Methods: Thirty-five patients with stage IV squamous and non-squamous cell NSCLC eligible for an anti-PD-1/PD-L1 as monotherapy were enrolled. Saliva samples were collected from patients prior to the start of treatment, bacterial DNA was extracted using the QIAamp® DNA Microbiome Kit (QIAGEN) and the 16S rRNA gene was sequenced on a MiSeq sequencing instrument (Illumina). Results: NSCLC patients were dichotomized as “Responders” (partial or complete response) and “Non-Responders” (progressive disease), after 12 weeks of treatment, based on RECIST criteria. A prevalence of the phylum Candidatus Saccharibacteria was found in the 10 responders compared to non-responders (abundance 5% vs 1% respectively; p-value = 1.46 x 10-7; False Discovery Rate (FDR) = 1.02 x 10-6). Moreover, a higher prevalence of Saccharibacteria Genera Incertae Sedis genus (belonging to the Candidatus Saccharibacteria phylum) was observed in "responders" (p-value = 6.01 x 10-7 and FDR = 2.46 x 10-5). Finally, the patients who benefit from immunotherapy showed a significant abundance of TM7 Phylum Sp Oral Clone FR058 strain, member of Saccharibacteria Genera Incertae Sedis genus (p-value = 6.13 x 10-7 and FDR=7.66 x 10-5). Conclusions: These preliminary results showed a significant association between oral microbiota and ICIs response in NSCLC patients. In particular, the higher prevalence of Candidatus Saccharibacteria phylum and TM7 Phylum Sp Oral Clone FR058 strain in responders suggests their potential immunomodulatory role. The study is still ongoing and updated data will be presented at the congress.

Keywords: oral microbiota, immune checkpoint inhibitors, non-small cell lung cancer, predictive biomarker

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