Search results for: X inactivate-specific transcript; hsa-miRNA-34a-5p

Authors: Nehal Adel Khalil, Amel Foad Ketat, Fairouz Elsayed Mohamed Ali, Nahla Abdelmoneim Hamid, Hazem Farag Manaa

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Background: Chronic myeloid leukemia (CML) represents 15% of adult leukemias. Imatinib Mesylate (IM) is the gold standard treatment for new cases of CML. Treatment with IM results in improvement of the majority of cases. However, about 25% of cases may develop resistance. Sensitive and specific early predictors of IM resistance in CML patients have not been established to date. Aim: To investigate the value of miR-451 in CML as an early predictor for IM resistance in Egyptian CML patients. Methods: The study employed Real time Polymerase Reaction (qPCR) technique to investigate the leucocytic expression of miR-451 in fifteen newly diagnosed CML patients (group I), fifteen IM responder CML patients (group II), fifteen IM resistant CML patients (group III) and fifteen healthy subjects of matched age and sex as a control group (group IV). The response to IM was defined as < 10% BCR-ABL transcript level after 3 months of therapy. The following parameters were assessed in subjects of all the studied groups: 1- Complete blood count (CBC). 2- Measurement of plasma level of miRNA 451 using real-time Polymerase Chain Reaction (qPCR). 3- Detection of BCR-ABL gene mutation in CML using qPCR. Results: The present study revealed that miR-451 was significantly down-regulated in leucocytes of newly diagnosed CML patients as compared to healthy subjects. IM responder CML patients showed an up-regulation of miR- 451 compared with IM resistant CML patients. Conclusion: According to the data from the present study, it can be concluded that leucocytic miR- 451 expression is a useful additional follow-up marker for the response to IM and a promising prognostic biomarker for CML.

Keywords: chronic myeloid leukemia, imatinib resistance, microRNA 451, Polymerase Chain Reaction

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Authors: Mona Vijayaran, Gurleen Oberoi, Sanjay Mishra

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Introduction: p190 BCR‐ABL1 in CML is often associated with monocytosis. In the case described here, monocytosis is associated with coexisting p210 BCR‐ABL and CMML clones. Mutation analysis using next‐generation sequence (NGS) in our case showed TET2 and SRSF2 mutations. Aims & Objectives: A 75-year male was evaluated for monocytosis and thrombocytopenia. CBC showed Hb-11.8g/dl, TLC-12,060/cmm, Monocytes-35%, Platelets-39,000/cmm. Materials & Methods: Bone marrow examination showed a hypercellular marrow with myeloid series showing sequential maturation up to neutrophils with 30% monocytes. Immunophenotyping by flow cytometry from bone marrow had 3% blasts. Making chronic myelomonocytic leukemia as the likely diagnosis. NGS for myeloid mutation panel had TET2 (48.9%) and SRSF2 (32.5%) mutations. This report further supported the diagnosis of CMML. To fulfil the WHO diagnostic criteria for CMML, a BCR ABL1 by RQ-PCR was sent. The report came positive for p210 (B3A2, B2A2) Major Transcript (M-BCR) % IS of 38.418. Result: The patient was counselled regarding the unique presentation of the presence of 2 clones- P210 CML and CMML. After discussion with an international faculty with vast experience in CMML. It was decided to start this elderly gentleman on Imatinib 200mg and not on azacytidine, as ASXL1 was not present; hence, his chances of progressing to AML would be less and on the other end, if CML is left untreated then chances of progression to blast phase would always be a possibility. After 3 months on Imatinib his platelet count improved to 80,000 to 90,000/cmm, but his monocytosis persists. His 3rd month BCR-ABL1 IS% is 0.004%. Conclusion: After searching the literature, there were no case reports of a coexisting CML p210 with CMML. This case might be the first case report. p190 BCR ABL1 is often associated with monocytosis. There are few case reports of p210 BCR ABL1 positivity in patients with monocytosis but none with coexisting CMML. This case highlights the need for extensively evaluating patients with monocytosis with next-generation sequencing for myeloid mutation panel and BCR-ABL1 by RT-PCR to correctly diagnose and treat them.

Keywords: CMML, NGS, p190 CML, Imatinib

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Authors: Abhaydeep Pandey, Shweta Shukla, Saptamita Goswami, Bhaswati Bandyopadhyay, Vishnampettai Ramachandran, Sudhanshu Vrati, Arup Banerjee

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Background: Long non-coding RNAs (lncRNAs) are the important regulators of gene expression and play important role in viral replication and disease progression. The role of lncRNA genes in the pathogenesis of Dengue virus-mediated pathogenesis is currently unknown. Methods: To gain additional insights, we utilized an unbiased RNA sequencing followed by in silico analysis approach to identify the differentially expressed lncRNA and genes that are associated with dengue disease progression. Further, we focused our study on lncRNAs NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) as it was found to be differentially expressed in PBMC of dengue infected patients. Results: The expression of lncRNAs NEAT1, as compared to dengue infection (DI), was significantly down-regulated as the patients developed the complication. Moreover, pairwise analysis on follow up patients confirmed that suppression of NEAT1 expression was associated with rapid fall in platelet count in dengue infected patients. Severe dengue patients (DS) (n=18; platelet count < 20K) when recovered from infection showing high NEAT1 expression as it observed in healthy donors. By co-expression network analysis and subsequent validation, we revealed that coding gene; IFI27 expression was significantly up-regulated in severe dengue cases and negatively correlated with NEAT1 expression. To discriminate DI from dengue severe, receiver operating characteristic (ROC) curve was calculated. It revealed sensitivity and specificity of 100% (95%CI: 85.69 – 97.22) and area under the curve (AUC) = 0.97 for NEAT1. Conclusions: Altogether, our first observations demonstrate that monitoring NEAT1and IFI27 expression in dengue patients could be useful in understanding dengue virus-induced disease progression and may be involved in pathophysiological processes.

Keywords: dengue, lncRNA, NEAT1, transcriptome

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Authors: Neslihan Şahin Çelik, Ali Eraslan

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In recent years, mathematics educators have frequently stressed the necessity of instructing students about models and modeling approaches that encompass cognitive and metacognitive thought processes, starting from the first years of school and continuing on through the years of higher education. The purpose of this study is to examine the thought processes of 4th-grade primary school students in their modeling activities and to explore the difficulties encountered in these processes, if any. The study, of qualitative design, was conducted in the 2015-2016 academic year at a public state-school located in a central city in the Black Sea Region of Turkey. A preliminary study was first implemented with designated 4th grade students, after which the criterion sampling method was used to select three students that would be recruited into the focus group. The focus group that was thus formed was asked to work on the model eliciting activity of the Paper Airplane Problem and the entire process was recorded on video. The Paper Airplane Problem required the students to determine the winner with respect to: (a) the plane that stays in the air for the longest time; (b) the plane that travels the greatest distance in a straight-line path; and (c) the overall winner for the contest. A written transcript was made of the video recording, after which the recording and the students' worksheets were analyzed using the Blum and Ferri modeling cycle. The results of the study revealed that the students tested the hypotheses related to daily life that they had set up, generated ideas of their own, verified their models by making connections with real life, and tried to make their models generalizable. On the other hand, the students had some difficulties in terms of their interpretation of the table of data and their ways of operating on the data during the modeling processes.

Keywords: primary school students, model eliciting activity, mathematical modeling, modeling process, paper airplane problem

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Authors: Krizette Ladera

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Fathers are there not only to provide the financial stability of a family but a father is also there to provide the love and support that usually people would see as the mother’s responsibility. To describe the lived experiences and how fathers make sense of their lived experiences with their children who have cerebral palsy is the main objective of the study. A qualitative research using a thematic analysis was used for the study. The qualitative research focused on the personal narratives, self-report and expression of the participant’s memory in terms of how they tell their stories. The interpretative phenomenological analysis was used to focus on the experience of the participants on how they will describe their experiences, and to also add on that the IPA will also attempt to describe and explain the meaning of human experiences using interview, specifically on the father who have a child that suffers from cerebral palsy. For the sampling technique, the snowball technique was used to gather participants from the referral of other participants. The five non-randomly selected fathers will be served as the participants for the research. A self-made interview with an open-ended question was used as the research instrument; it includes profiling of the respondent as well as their experiences in taking care of their child that suffers from cerebral palsy. In analyzing a data, the researcher used the thematic analysis where in the interview was made into a transcript, then it was organized and divided themes. After that, the relations of each themes, was identified and it was later documented and translated into written text format using thematic grouping. Finally, the researcher analyzed each data according to its themes and put it in a table to be presented in the result section of the study And as for the result of the study, the researcher was able to come up with the four (4) main themes that most of the participants experienced and those are: The experiences in finding out about the condition of the Child, disclosing the condition of the child to the family and its emotional effect, The experiences of living the day of day realities in providing the physical, financial, emotional and a well balanced environment to the child, and the religious perspectives of the fathers. Along with those four (4) themes comes the subtheme which explains the themes in a more detailed explanation.

Keywords: cerebral palsy, children, fathers, lived experiences

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Authors: M. P. Dandekar, A. P. Bharne, P. T. Borkar, D. M. Kokare, N. K. Subhedar

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Ethanol ingestion by the mother ensue adverse consequences for her offspring. Herein, we examine the behavioral phenotype and neural substrate of the offspring of the mother on ethanol. Female rats were fed with ethanol-containing liquid diet from 8 days prior of conception and continued till 25 days post-parturition to coincide with weaning. Behavioral changes associated with anxiety, depression and learning and memory were assessed in the offspring, after they attained adulthood (day 85), using elevated plus maze (EPM), forced swim (FST) and novel object recognition tests (NORT), respectively. The offspring of the alcoholic mother, compared to those of the pair-fed mother, spent significantly more time in closed arms of EPM and showed more immobility time in FST. Offspring at the age of 25 and 85 days failed to discriminate between novel versus familiar object in NORT, thus reflecting anxiogenic, depressive and amnesic phenotypes. Neuropeptide cocaine- and amphetamine-regulated transcript peptide (CART) is known to be involved in central effects of ethanol and hence selected for the current study. Twenty-five days old pups of the alcoholic mother showed significant augmentation in CART-immunoreactivity in the cells of Edinger-Westphal (EW) nucleus and lateral hypothalamus. However, a significant decrease in CART-immunoreactivity was seen in nucleus accumbens shell (AcbSh), lateral part of bed nucleus of the stria terminalis (BNSTl), locus coeruleus (LC), hippocampus (CA1, CA2 and CA3), and arcuate nucleus (ARC) of the pups and/or adults offspring. While no change in the CART-immunoreactive fibers of AcbSh and BNSTl, CA2 and CA3 was noticed in the 25 days old pups, the CART-immunoreactive cells in EW and paraventricular nucleus (PVN), and fibers in the central nucleus of amygdala of 85 days old offspring remained unaffected. We suggest that the endogenous CART system in these discrete areas, among other factors, may be a causal to the abnormalities in the next generation of an alcoholic mother.

Keywords: anxiety, depression, CART, ethanol, immunocytochemistry

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Authors: Akunna Agunwah, Kevin Gallimore, Kathryn Kinmond

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Entrepreneurship is widely associated and seen as a vehicle for economic growth; however, different scholars have studied entrepreneurship from various perspectives, resulting in multiple definitions. It is surprising to know most entrepreneurship definition does not incorporate growth as part of their definition of entrepreneurship. Economic growth is engineered by the activities of the entrepreneurs. The purpose of the present theoretical study is to explore the working practices of the successful entrepreneurs towards achieving business growth by understanding the experiences of the entrepreneur using the Theory of Planned Behaviour (TPB) as a lens. Ten successful entrepreneurs in the North West of England in various business sectors were interviewed using semi-structured interview method. The recorded audio interviews transcribed and subsequently evaluated using the thematic deductive technique (qualitative approach). The themes were examined using Theory of Planned Behaviour to ascertain the presence of the three intentional antecedents (attitude, subjective norms, and perceived behavioural control). The findings categorised in two folds, firstly, it was observed that the three intentional antecedents, which make up Theory of Planned Behaviour were evident in the transcript. Secondly, the entrepreneurs are most concerned with achieving a state of freedom and realising their visions and ambitions. Nevertheless, the entrepreneur employed these intentional antecedents to enhance business growth. In conclusion, the work presented here showed a novel way of understanding the working practices and experiences of the entrepreneur using the theory of planned behaviour in qualitative approach towards enhancing business growth. There exist few qualitative studies in entrepreneurship research. In addition, this work applies a novel approach to studying the experience of the entrepreneurs by examining the working practices of the successful entrepreneurs in the North-West England through the lens of the theory of planned behaviour. Given the findings regarding TPB as a lens in the study, the entrepreneur does not differentiate between the categories of the antecedents reasonably sees them as processes that can be utilised to enhance business growth.

Keywords: business growth, experience, interpretive, theory of planned behaviour

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Authors: Parvaneh Mahmoudi, Ahmad Moeni, Seyed Mojtaba Khayam Nekoei, Mohsen Mardi, Mehrshad Zeinolabedini, Ghasem Hosseini Salekdeh

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Saffron (Crocus sativus L.) is one of the most important spice and medicinal plants. The three-branch style of C. sativus flowers are the most important economic part of the plant and known as saffron, which has several medicinal properties. Despite the economic and biological significance of this plant, knowledge about its molecular characteristics is very limited. In the present study, we, for the first time, constructed a comprehensive dataset for C. sativus stigma through de novo transcriptome sequencing. We performed de novo transcriptome sequencing of C. sativus stigma using the Illumina paired-end sequencing technology. A total of 52075128 reads were generated and assembled into 118075 unigenes, with an average length of 629 bp and an N50 of 951 bp. A total of 66171unigenes were identified, among them, 66171 (56%) were annotated in the non-redundant National Center for Biotechnology Information (NCBI) database, 30938 (26%) were annotated in the Swiss-Prot database, 10273 (8.7%) unigenes were mapped to 141 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, while 52560 (44%) and 40756 (34%) unigenes were assigned to Gen Ontology (GO) categories and Eukaryotic Orthologous Groups of proteins (KOG), respectively. In addition, 65 candidate genes involved in three stages of crocin biosynthesis were identified. Finally, transcriptome sequencing of saffron stigma was used to identify 6779 potential microsatellites (SSRs) molecular markers. High-throughput de novo transcriptome sequencing provided a valuable resource of transcript sequences of C. sativus in public databases. In addition, most of candidate genes potentially involved in crocin biosynthesis were identified which could be further utilized in functional genomics studies. Furthermore, numerous obtained SSRs might contribute to address open questions about the origin of this amphiploid spices with probable little genetic diversity.

Keywords: saffron, transcriptome, NGS, bioinformatic

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Authors: Abhaydeep Pandey, Shweta Shukla, Saptamita Goswami, Bhaswati Bandyopadhyay, Vishnampettai Ramachandran, Sudhanshu Vrati, Arup Banerjee

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Dengue virus infection is now considered as one of the most important mosquito-borne infection in human. The virus is known to promote vascular permeability, cerebral edema leading to Dengue hemorrhagic fever (DHF) or Dengue shock syndrome (DSS). Dengue infection has known to be endemic in India for over two centuries as a benign and self-limited disease. In the last couple of years, the disease symptoms have changed, manifesting severe secondary complication. So far, Delhi has experienced 12 outbreaks of dengue virus infection since 1997 with the last reported in 2014-15. Without specific antivirals, the case management of high-risk dengue patients entirely relies on supportive care, involving constant monitoring and timely fluid support to prevent hypovolemic shock. Nonetheless, the diverse clinical spectrum of dengue disease, as well as its initial similarity to other viral febrile illnesses, presents a challenge in the early identification of this high-risk group. WHO recommends the use of warning signs to identify high-risk patients, but warning signs generally appear during, or just one day before the development of severe illness, thus, providing only a narrow window for clinical intervention. The ability to predict which patient may develop DHF and DSS may improve the triage and treatment. With the recent discovery of high throughput RNA sequencing allows us to understand the disease progression at the genomic level. Here, we will collate the results of RNA-Sequencing data obtained recently from PBMC of different categories of dengue patients from India and will discuss the possible role of deregulated genes and long non-coding RNAs NEAT1 for development of disease progression.

Keywords: long non-coding RNA (lncRNA), dengue, peripheral blood mononuclear cell (PBMC), nuclear enriched abundant transcript 1 (NEAT1), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS)

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Authors: Aditya Jayadas

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During a pandemic like the one we are in with COVID-19, older adults, especially those who live in a senior retirement facility, experience even bigger challenges as they are often dependent on other individuals for care. Many older adults are dependent on caregivers to assist with their instrumented activities of daily living (IADL). With travel restrictions imposed during a pandemic, there is a critical need to ensure that older adults who are homebound continue to be able to participate in physical exercise, cognitive exercise, and social interaction programs. The objective of this study was to better understand the challenges that older adults faced during the pandemic and what they were doing specifically to cope with the pandemic physically, mentally, and through social interaction. A focus group was conducted with ten older adults (age: 82.70 ± 7.81 years; nine female and one male) who resided in a senior retirement facility. During the course of one hour, seven open-ended questions were posed to the participants: a) What has changed in your life since the start of the pandemic, b) What has been most challenging for you, c) What are you doing to take care of yourself, d) Are you doing anything specifically as it relates to your physical health, e) Are you doing anything specifically as it relates to your mental health, f) What did you do for social interaction during the pandemic, g) Is there anything else you would like to share as it relates to your experience during the pandemic. The focus group session was audio-taped, and verbatim transcripts were created to evaluate the responses of the participants. The transcript consisted of 4,698 words and 293 lines of text. The data was analyzed using content analysis. The unit of analysis was the text from the audio recordings that were transcribed. From the review of the transcribed text, themes and sub-themes were identified, along with salient quotes under each sub-theme. The major themes that emerged from the data were: having a routine, engaging in activities, attending exercise classes, use of technology, family, community, and prayer. The quotes under the sub-themes provided compelling evidence of how older adults coped during the pandemic while addressing the challenges they faced and developing strategies to address their physical and mental health while interacting with others. Lessons learned from this focus group can be used to develop specific physical exercise, cognitive exercise, and social interaction programs that benefit the health and well-being of older adults.

Keywords: cognitive exercise, pandemic, physical exercise, social interaction

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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: David Sarpong, Waleed Khursheed, Ernest Danquah, Erin Murphy

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Shigella is a pathogenic bacterium responsible for shigellosis, a severe diarrheal disease that claims the lives of immunocompromised individuals worldwide. To develop therapeutics against this disease, an understanding of the molecular mechanisms underlying the pathogen’s physiology is crucial. Small non-coding RNAs (sRNAs) have emerged as important regulators of bacterial physiology, including as components of toxin-antitoxin systems. In this study, we investigated the role of RyfA in S. flexneri physiology and virulence. RyfA, originally identified as an sRNA in Escherichia coli, is conserved within the Enterobacteriaceae family, including Shigella. Whereas two copies of ryfA are present in S. dysenteriae, all other Shigella species contain only one copy of the gene. Additionally, we identified a putative open reading frame within the RyfA transcript, suggesting that it may be a dual-functioning gene encoding a small protein in addition to its sRNA function. To study ryfA in vitro, we cloned the gene into an inducible plasmid and observed the effect on bacterial growth. Here, we report that RyfA production inhibits the growth of S. flexneri, and this inhibition is dependent on the contained open reading frame. In-silico analyses have revealed the presence of two divergently transcribed sRNAs, RyfB1 and RyfB2, which share nucleotide complementarity with RyfA and thus are predicted to function as anti-toxins. Our data demonstrate that RyfB2 has a stronger antitoxin effect than RyfB1. This regulatory pattern suggests a novel form of a toxin-antitoxin system in which the activity of a single toxin is inhibited to varying degrees by two sRNA antitoxins. Studies are ongoing to investigate the regulatory mechanism(s) of the antitoxin genes, as well as the downstream targets and mechanism of growth inhibition by the RyfA toxin. This study offers distinct insights into the regulatory mechanisms underlying Shigella physiology and may inform the development of new anti-Shigella therapeutics.

Keywords: sRNA, shigella, toxin-antitoxin, Type 1 toxin antitoxin

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Authors: S. Ibrahim, D. Salilew-Wondim, M. Hoelker, C. Looft, E. Tholen, C. Grosse-Brinkhaus, K. Schellander, C. Neuhoff, D. Tesfaye

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Bovine endometrial cells appear to have a key role in innate immune defense of the female genital tract. A better understanding of molecular changes in microRNAs (miRNAs) and their target genes expression may identify reliable prognostic indicators for cows that will resolve inflammation and resume cyclicity. In the current study, we hypothesized that let-7 miRNAs family has a primary role in the innate immune defence of the endometrium tissue against bacterial infection, which is partly achieved via regulating mRNA stability of pro-inflammatory cytokines at the post-transcriptional level. Therefore, we conducted two experiments. In the first experiment, primary bovine endometrial cells were challenged with clinical (3.0 μg/ml) and sub-clinical (0.5 μg/ml) doses of lipopolysaccharide (LPS) for 24h. In the 2nd experiment, we have investigated the potential role of let-7 miRNAs (let-7a and let-7f) using gain and loss of function approaches. Additionally, tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 induced transcript 1 (TGFB1I1) and serum deprivation response (SDPR) genes were validated using reporter assay. Here we addressed for the first time that let-7 miRNAs have a precise role in bovine endometrium, where LPS dysregulated let-7 miRNAs family expression was associated with an increased pro-inflammatory cytokine level by directly/indirectly targeting the TNFα, interleukin 6 (IL6), nuclear factor kappa-light-chain enhancer of activated B cells (NFκB), TGFβ1I1 and SDPR genes. To our knowledge, this is the first study showing that TNFα, TGFβ1I1 and SDPR were identified and validated as novel let-7 miRNAs targets and could have a distinct role in inflammatory immune response of LPS challenged bovine endometrial cells. Our data represent a new finding by which uterine homeostasis is maintained through functional regulation of let-7a by down-regulation of pro-inflammatory cytokines expression (TNFα and IL6) at the mRNA and protein levels. These findings suggest that LPS serves as a negative regulator of let-7 miRNAs expression and provides a mechanism for the persistent pro-inflammatory phenotype, which is a hallmark of bovine subclinical endometritis.

Keywords: bovine endometrial cells, let-7, lipopolysaccharide, pro-inflammatory cytokines

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Authors: Emanuela Chiarella, Clelia Nisticò, Nicola Lombardo, Giovanna Lucia Piazzetta, Nadia Lobello, Maria Mesuraca

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Killian’s Antrochoanal Polyp is a benign lesion of the maxillary sinus characterized by unilateral nasal obstruction, pus discharge, and headache. It affects, more commonly children and young adults. Although its etiology still remains unclear, chronic inflammation, autoreactivity, allergies, and viral infections are strongly associated with its formation and development, resulting in nasal tissue remodeling. We aimed to investigate the stem cells components which reside in this pathological tissue. In particular, we adopted a protocol for the isolation and culturing of mesenchymal stem cells from surgical biopsies of three Killian nasal polyp patients (KNP-MSCs) as well as from their healthy nasal tissue (HNT-MSCs) that were used as controls. The immunophenotype profile of HNT-MSCs and KNP-MSCs was more similar, with a marked positivity for CD73, CD90, and CD105 expression, while being negative for CD34 and CD14 haematopoietic genes. Cell proliferation assay showed that KNP-MSCs had a replicative disadvantage compared to HNT-MSCs, as evidenced by the significantly lower number of cells in the S-phase of the cell cycle. KNP-MSCs also took longer to close a wound than HNT-MSCs, indicating a partial epithelial phenotype in which low levels of ICAM-1 mRNA and a significant increase in E-CAD transcript were detectable. Subsequently, the differentiation potential of both MSCs populations was analyzed by inducing osteoblastic or adipocyte differentiation for up to 20 days. KNP-MSCs showed the ability to differentiate into osteoblasts, although ALP activity as well as the number and size of calcium deposits were lower than osteogenic induced-HNT-MSCs. Also, mRNA levels of osteoblastic marker genes (OCN, OPN, OSX, RUNX2) resulted lower compared to control cell population. Instead, the analysis of the adipogenic differentiation potential showed a similar behavior between KNP-MSCs and HNT-MSCs considering that the amount of lipid droplets, the expression of adipocyte-specific genes (FABP4, AdipoQ, PPARγ2, LPL) and the content of triacylglycerols were almost overlapping. Taken together, these results first demonstrated that Killian's nasal polyp is a source of mesenchymal stem cells with self-renewal and multi-differentiative capabilities.

Keywords: Mesenchymal stem cells, adipogenic differentiation, osteogenic differentiation, EMT

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Authors: Diana K. Kwok

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Despite the fact that Hong Kong government aims to develop an inclusive society, sexual minority students continue to encounter sexual prejudice without legal protection. They also have difficulties accessing relevant services from mental health and educational professionals, who do not receive systematic training to work with sexual minority students. Informed by the literature on sexual prejudice, heterosexual hegemony, genderism, as well as code of practice for frontline practitioners, the authors explored self-perceived knowledge of teachers and sexual minorities on sexuality and sexual prejudice, and how they perceive prejudice towards sexual minorities in Chinese cultural context. Semi-structure qualitative interviews were carried out with 31 school personnel informants (school teachers and counseling team members) and 25 sexual minority informants on their understanding of sexuality knowledge, their perception of sexual prejudice within school context in Hong Kong, as well as their suggested themes on teachers training on sexual prejudice reduction. This presentation specifically focuses on transcripts from sexual minority informants. Data analysis was carried out through NVivo, and followed the procedures spelt out in the qualitative research literature. Trustworthiness of the study was addressed through various strategies. Preliminary themes emerged from transcript content analysis: 1) A gap of knowledge between sexual minority informants and teachers; 2) Perception on sexual prejudice within cultural context; 3) Heterosexual hegemony and genderism within school system; 4) Needs for mandatory training: contents and strategies. The sexual minority informants found that teachers they encountered were predominantly adopted concepts of binary sex and dichotomous gender. Informants also indicated that the teachings of Confucianism cultural values, religiosity in Hong Kong might well be important cultural forces contributing to sexual prejudice manifested in school context. Although human rights and social justice concepts were embedded in professional code of practice of teachers and school helping professionals, informants found that teachers they encountered may face a dilemma when supporting sexual minority students navigating heterosexual hegemony and genderism in, as a consequence of their personal, institutional, cultural and religious backgrounds. Acknowledgments: The sexual prejudice project was funded by the Hong Kong Research Grant Council (ECS28401614), 2015 to 2017.

Keywords: sexual prejudice, Chinese teachers, Chinese sexual minorities, teacher training

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Authors: Elizabeth Grier, Meg Gemmill, Mary Martin, Leora Reiter, Herman Tang, Alexandra Donaldson, Isis Lunsky, Mia Wu

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Background: Individuals with Cerebral Palsy (CP) and/or IDD face numerous physical and mental health challenges, including difficulty accessing effective palliative care. The aim of this study is to assess the knowledge and comfort of healthcare providers in providing community-based palliative care for patients with Cerebral Palsy (CP) and severe to profound Intellectual and Developmental Disabilities (IDD). Methods: This study includes a mixed methods approach obtaining both quantitative and qualitative data. Quantitative data from palliative care practitioners was obtained through an online survey assessing comfort in symptom management, grief assessment, and goals of care discussion. This survey was distributed to physicians and allied health practitioners across Canada through the College of Family Physicians of Canada Member Interest Groups for Palliative Care and for IDD. Survey results guided the development of a semi-structured interview template, which was used to conduct a focus group on the same topic. Participants were four palliative care providers (3 physicians and one spiritual care practitioner). The focus group transcript is currently undergoing thematic analysis using NVivo 12 software. Results: 57 palliative care practitioners completed the survey. 87% of participants indicated they have provided palliative care services for persons with CP and/or IDD. Findings suggest practitioners are somewhat confident in identifying specific physical symptoms (dyspnea, pressure ulcers) but less confident in identifying physical/emotional pain, addressing grief, and prognosticating life expectancy in this population. 54% of responses indicated they had little/no training on palliating those with CP or IDD, and 45% somewhat or strongly disagree members of their profession can manage symptoms for this population. Focus group analysis is underway, and results will be available at the time of the poster presentation. Conclusion: Persons with CP and IDD are more likely to experience severe health inequities when accessing palliative care. Results of this study suggest further education is needed for palliative care professionals to address the barriers and challenges in providing palliative care to this patient population.

Keywords: palliative care, symptom management, health equity, community healthcare, intellectual and developmental disabilities

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Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

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Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

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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: Gunta Resevica, Nikita Zrelovs, Ivars Silamikelis, Ieva Kalnciema, Helvijs Niedra, Gunārs Lācis, Toms Bartulsons, Inga Moročko-Bičevska, Arturs Stalažs, Kristīne Drevinska, Andris Zeltins, Ina Balke

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Newly discovered viruses provide novel knowledge for basic phytovirus research, serve as tools for biotechnology and can be helpful in identification of epidemic outbreaks. Blackcurrant-associated rhabdovirus (BCaRV) have been discovered in USA germplasm collection samples from Russia and France. As it was reported in one accession originating from France it is unclear whether the material was already infected when it entered in the USA or it became infected while in collection in the USA. Due to that BCaRV was definite as non-EU viruses. According to ICTV classification BCaRV is representative of Blackcurrant betanucleorhabdovirus specie in genus Betanucleorhabdovirus (family Rhabdoviridae). Nevertheless, BCaRV impact on the host, transmission mechanisms and vectors are still unknown. In RNA-seq data pool from Ribes plants resistance gene study by high throughput sequencing (HTS) we observed differences between sample group gene transcript heat maps. Additional analysis of the whole data pool (total 393660492 of 150 bp long read pairs) by rnaSPAdes v 3.13.1 resulted into 14424 bases long contig with an average coverage of 684x with shared 99.5% identity to the previously reported first complete genome of BCaRV (MF543022.1) using EMBOSS Needle. This finding proved BCaRV presence in EU and indicated that it might be relevant pathogen. In this study leaf tissue from twelve asymptomatic blackcurrant cv. Mara Eglite plants (negatively tested for blackcurrant reversion virus (BRV)) from Dobele, Latvia (56°36'31.9"N, 23°18'13.6"E) was collected and used for total RNA isolation with RNeasy Plant Mini Kit with minor modifications, followed by plant rRNA removal by a RiboMinus Plant Kit for RNA-Seq. HTS libraries were prepared using MGI Easy RNA Directional Library Prep Set for 16 reactions to obtain 150 bp pair-end reads. Libraries were pooled, circularized and cleaned and sequenced on DNBSEQ-G400 using PE150 flow cell. Additionally, all samples were tested by RT-PCR, and amplicons were directly sequenced by Sanger-based method. The contig representing the genome of BCaRV isolate Mara Eglite was deposited at European Nucleotide Archive under accession number OU015520. Those findings indicate a second evidence on the presence of this particular virus in the EU and further research on BCaRV prevalence in Ribes from other geographical areas should be performed. As there are no information on BCaRV impact on the host this should be investigated, regarding the fact that mixed infections with BRV and nucleorhabdoviruses are reported.

Keywords: BCaRV, Betanucleorhabdovirus, Ribes, RNA-seq

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Authors: Kim Toyer, Belinda Thompson, Louise Koelmeyer

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Lymphoedema is a chronic condition caused by dysfunction of the lymphatic system, which limits the drainage of fluid and tissue waste from the interstitial space of the affected body part. The normal physiological changes in pregnancy cause an increased load on a normal lymphatic system which can result in a transient lymphatic overload (oedema). The interaction between lymphoedema and pregnancy oedema is unclear. Women with pre-existing lymphoedema require accurate information and additional strategies to manage their lymphoedema during pregnancy. Currently, no resources are available to guide women or their healthcare providers with accurate advice and additional management strategies for coping with lymphoedema during pregnancy until they have recovered postnatally. This study explored the experiences of Australian women with pre-existing lymphoedema during recent pregnancy and the early postnatal period to determine how their usual lymphoedema management strategies were adapted and what were their additional or unmet needs. Interactions with their obstetric care providers, the hospital maternity services, and usual lymphoedema therapy services were detailed. Participants were sourced from several Australian lymphoedema community groups, including therapist networks. Opportunistic sampling is appropriate to explore this topic in a small target population as lymphoedema in women of childbearing age is uncommon, with prevalence data unavailable. Inclusion criteria were aged over 18 years, diagnosed with primary or secondary lymphoedema of the arm or leg, pregnant within the preceding ten years (since 2012), and had their pregnancy and postnatal care in Australia. Exclusion criteria were a diagnosis of lipedema and if unable to read or understand a reasonable level of English. A mixed-method qualitative design was used in two phases. This involved an online survey (REDCap platform) of the participants followed by online semi-structured interviews or focus groups to provide the transcript data for inductive thematic analysis to gain an in-depth understanding of issues raised. Women with well-managed pre-existing lymphoedema coped well with the additional oedema load of pregnancy; however, those with limited access to quality conservative care prior to pregnancy were found to be significantly impacted by pregnancy, including many reporting deterioration of their chronic lymphoedema. Misinformation and a lack of support increased fear and apprehension in planning and enjoying their pregnancy experience. Collaboration between maternity and lymphoedema therapy services did not happen despite study participants suggesting it. Helpful resources and unmet needs were identified in the recent Australian context to inform further research and the development of resources to assist women with lymphoedema who are considering or are pregnant and their supporters, including health care providers.

Keywords: lymphoedema, management strategies, pregnancy, qualitative

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Authors: Felice Elefant, Akanksha Bhatnaghar, Keegan Krick, Elizabeth Heller

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Context: The severity of Alzheimer’s Disease (AD) progression involves an interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT) mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Altered RNA splicing has recently been highlighted as a widespread hallmark in the AD transcriptome that is implicated in the disease. Research Aim: The aim of this study was to identify a novel RNA binding/splicing function for Tip60 in human hippocampus and impaired in brains from AD fly models and AD patients. Methodology/Analysis: The authors used RNA immunoprecipitation using RNA isolated from 200 pooled wild type Drosophila brains for each of the 3 biological replicates. To identify Tip60’s RNA targets, they performed genome sequencing (DNB-SequencingTM technology, BGI genomics) on 3 replicates for Input RNA and RNA IPs by Tip60. Findings: The authors' transcriptomic analysis of RNA bound to Tip60 by Tip60-RNA immunoprecipitation (RIP) revealed Tip60 RNA targets enriched for critical neuronal processes implicated in AD. Remarkably, 79% of Tip60’s RNA targets overlap with its chromatin gene targets, supporting a model by which Tip60 orchestrates bi-level transcriptional regulation at both the chromatin and RNA level, a function unprecedented for any HAT to date. Since RNA splicing occurs co-transcriptionally and splicing defects are implicated in AD, the authors investigated whether Tip60-RNA targeting modulates splicing decisions and if this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq data sets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs were bonafide Tip60-RNA targets enriched for in the AD-gene curated database, with some AS alterations prevented against by increasing Tip60 in fly brain. Importantly, human orthologs of several Tip60-modulated spliced genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60’s splicing function in AD pathogenesis. Theoretical Importance: The authors' findings support a novel RNA interaction and splicing regulatory function for Tip60 that may underlie AS impairments that hallmark AD etiology. Data Collection: The authors collected data from RNA immunoprecipitation experiments using RNA isolated from 200 pooled wild type Drosophila brains for each of the 3 biological replicates. They also performed genome sequencing (DNBSequencingTM technology, BGI genomics) on 3 replicates for Input RNA and RNA IPs by Tip60. Questions: The question addressed by this study was whether Tip60 has a novel RNA binding/splicing function in human hippocampus and whether this function is impaired in brains from AD fly models and AD patients. Conclusions: The authors' findings support a novel RNA interaction and splicing regulatory function for Tip60 that may underlie AS impairments that hallmark AD etiology.

Keywords: Alzheimer's disease, cognition, aging, neuroepigenetics

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Authors: H. Weigelt-Marom

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Over the years and up to the arousal of the COVID-19 pandemic, deaf or hard of hearing students studying in higher education institutions, participated lectures on campus using hearing aids and strategies adapted for frontal learning in a classroom. Usually, these aids were well known to them from their earlier study experience in school. However, the transition to online lessons, due to the latest pandemic, led deaf or hard of hearing students to study outside of their physical, well known learning environment. The change of learning environment and structure rose new challenges for these students. The present study examined the learning experience, limitations, challenges and benefits regarding learning online with lecture and classmates via the “Zoom” video conference program, among deaf or hard of hearing students in academia setting. In addition, emotional and social aspects related to learning in general versus the “Zoom” were examined. The study included 18 students diagnosed as deaf or hard of hearing, studying in various higher education institutions in Israel. All students had experienced lessons on the “Zoom”. Following allocation of the group study by the deaf and hard of hearing non-profit organization “Ma’agalei Shema”, and receiving the participants inform of consent, students were requested to answer a google form questioner and participate in an interview. The questioner included background information (e.g., age, year of studying, faculty etc.), level of computer literacy, and level of hearing and forms of communication (e.g., lip reading, sign language etc.). The interviews included a one on one, semi-structured, in-depth interview, conducted by the main researcher of the study (interview duration: up to 60 minutes). The interviews were held on “ZOOM” using specific adaptations for each interviewee: clear face screen of the interviewer for lip and face reading, and/ or professional sign language or live text transcript of the conversation. Additionally, interviewees used their audio devices if needed. Questions regarded: learning experience, difficulties and advantages studying using “Zoom”, learning in a classroom versus on “Zoom”, and questions concerning emotional and social aspects related to learning. Thematic analysis of the interviews revealed severe difficulties regarding the ability of deaf or hard of hearing students to comprehend during ”Zoom“ lessons without adoptive aids. For example, interviewees indicated difficulties understanding “Zoom” lessons due to their inability to use hearing devices commonly used by them in the classroom (e.g., FM systems). 80% indicated that they could not comprehend “Zoom” lessons since they could not see the lectures face, either because lectures did not agree to open their cameras or, either because they did not keep a straight forward clear face appearance while teaching. However, not all descriptions regarded learning via the “zoom” were negative. For example, 20% reported the recording of “Zoom” lessons as a main advantage. Enabling then to repeatedly watch the lessons at their own pace, mostly assisted by friends and family to translate the audio output into an accessible input. These finding and others regarding the learning experience of the group study on the “Zoom”, as well as their recommendation to enable deaf or hard of hearing students to study inclusively online, will be presented at the conference.

Keywords: deaf or hard of hearing, learning experience, Zoom, qualitative research

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Authors: Mukul Sharma, Madhusmita Das, Sundeep Chaitanya Vedithi

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Leprosy is a chronic human disease caused by Mycobacterium leprae, that cannot be cultured in vitro. Though treatable with multidrug therapy (MDT), recently, bacteria reported resistance to multiple antibiotics. Targeting DNA replication and repair pathways can serve as the foundation of developing new anti-leprosy drugs. Due to the absence of an axenic culture medium for the propagation of M. leprae, studying cellular processes, especially those belonging to DNA repair pathways, is challenging. Genomic understanding of M. Leprae harbors several protein-coding genes with no previously assigned function known as 'hypothetical proteins'. Here, we report identification and expression of known and hypothetical DNA repair genes from a human skin biopsy and mouse footpads that are involved in base excision repair, direct reversal repair, and SOS response. Initially, a bioinformatics approach was employed based on sequence similarity, identification of known protein domains to screen the hypothetical proteins in the genome of M. leprae, that are potentially related to DNA repair mechanisms. Before testing on clinical samples, pure stocks of bacterial reference DNA of M. leprae (NHDP63 strain) was used to construct standard graphs to validate and identify lower detection limit in the qPCR experiments. Primers were designed to amplify the respective transcripts, and PCR products of the predicted size were obtained. Later, excisional skin biopsies of newly diagnosed untreated, treated, and drug resistance leprosy cases from SIHR & LC hospital, Vellore, India were taken for the extraction of RNA. To determine the presence of the predicted transcripts, cDNA was generated from M. leprae mRNA isolated from clinically confirmed leprosy skin biopsy specimen across all the study groups. Melting curve analysis was performed to determine the integrity of the amplification and to rule out primer‑dimer formation. The Ct values obtained from qPCR were fitted to standard curve to determine transcript copy number. Same procedure was applied for M. leprae extracted after processing a footpad of nude mice of drug sensitive and drug resistant strains. 16S rRNA was used as positive control. Of all the 16 genes involved in BER, DR, and SOS, differential expression pattern of the genes was observed in terms of Ct values when compared to human samples; this was because of the different host and its immune response. However, no drastic variation in gene expression levels was observed in human samples except the nth gene. The higher expression of nth gene could be because of the mutations that may be associated with sequence diversity and drug resistance which suggests an important role in the repair mechanism and remains to be explored. In both human and mouse samples, SOS system – lexA and RecA, and BER genes AlkB and Ogt were expressing efficiently to deal with possible DNA damage. Together, the results of the present study suggest that DNA repair genes are constitutively expressed and may provide a reference for molecular diagnosis, therapeutic target selection, determination of treatment and prognostic judgment in M. leprae pathogenesis.

Keywords: DNA repair, human biopsy, hypothetical proteins, mouse footpads, Mycobacterium leprae, qPCR

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Authors: Brandy Yee, Dianne Yee

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Through the exploration of the lived experiences, beliefs and values of instructional leaders, teachers and students in Finland, Germany and Canada, we investigated the factors which contribute to developmentally responsive, intellectually engaging middle-level learning environments for early adolescents. Student-centred leadership dimensions, effective instructional practices and student agency were examined through the lens of current policy and research on middle-level learning environments emerging from the Canadian province of Manitoba. Consideration of these three research perspectives in the context of early adolescent learning, placed against an international backdrop, provided a previously undocumented perspective on leading, teaching and learning in the middle years. Aligning with a social constructivist, qualitative research paradigm, the study incorporated collective case study methodology, along with constructivist grounded theory methods of data analysis. Data were collected through semi-structured individual and focus group interviews and document review, as well as direct and participant observation. Three case study narratives were developed to share the rich stories of study participants, who had been selected using maximum variation and intensity sampling techniques. Interview transcript data were coded using processes from constructivist grounded theory. A cross-case analysis yielded a conceptual framework highlighting key factors that were found to be significant in the establishment of developmentally responsive, intellectually engaging middle-level learning environments. Seven core categories emerged from the cross-case analysis as common to all three countries. Within the visual conceptual framework (which depicts the interconnected nature of leading, teaching and learning in middle-level learning environments), these seven core categories were grouped into Essential Factors (student agency, voice and choice), Contextual Factors (instructional practices; school culture; engaging families and the community), Synergistic Factors (instructional leadership) and Cornerstone Factors (education as a fundamental cultural value; preservice, in-service and ongoing teacher development). In addition, sub-factors emerged from recurring codes in the data and identified specific characteristics and actions found in developmentally responsive, intellectually engaging middle-level learning environments. Although this study focused on 12 schools in Finland, Germany and Canada, it informs the practice of educators working with early adolescent learners in middle-level learning environments internationally. The authentic voices of early adolescent learners are the most important resource educators have to gauge if they are creating effective learning environments for their students. Ongoing professional dialogue and learning is essential to ensure teachers are supported in their work and develop the pedagogical practices needed to meet the needs of early adolescent learners. It is critical to balance consistency, coherence and dependability in the school environment with the necessary flexibility in order to support the unique learning needs of early adolescents. Educators must intentionally create a school culture that unites teachers, students and their families in support of a common purpose, as well as nurture positive relationships between the school and its community. A large, urban school district in Canada has implemented a school cohort-based model to begin to bring developmentally responsive, intellectually engaging middle-level learning environments to scale.

Keywords: developmentally responsive learning environments, early adolescents, middle level learning, middle years, instructional leadership, instructional practices, intellectually engaging learning environments, leadership dimensions, student agency

Procedia PDF Downloads 304