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Identifying genetic determinants of reproductive success may highlight mechanisms underlying fertility and identify alleles under present-day selection. Using data in 785,604 individuals of European ancestry, we identified 43 genomic loci associated with either number of children ever born (NEB) or childlessness. These loci span diverse aspects of reproductive biology, including puberty timing, age at first birth, sex hormone regulation, endometriosis and age at menopause. Missense variants in ARHGAP27 were associated with higher NEB but shorter reproductive lifespan, suggesting a trade-off at this locus between reproductive ageing and intensity. Other genes implicated by coding variants include PIK3IP1, ZFP82 and LRP4, and our results suggest a new role for the melanocortin 1 receptor (MC1R) in reproductive biology. As NEB is one component of evolutionary fitness, our identified associations indicate loci under present-day natural selection. Integration with data from historical selection scans highlighted an allele in the FADS1/2 gene locus that has been under selection for thousands of years and remains so today. Collectively, our findings demonstrate that a broad range of biological mechanisms contribute to reproductive success.

In 1971 it was discovered by Kobayashi et al that ovarian activity of ornithine decarboxylase (ODC), the rate-limiting step of the polyamine pathway, increased just prior to ovulation in rats. Following the discovery in rats, this phenomenon of ovarian ODC activity increase was seen in several other mammal species, such as mice and pigs. However, despite this discovery occurring fifty years ago the purpose and function of this activity increase are still unknown. One factor of Odc which complicates studies is that whole-body knockouts have been found to be embryonic lethal, with the embryos not making it past embryonic day 3.5. In 2013 our lab found that older mice experienced a smaller ovarian ODC activity increase when compared to their younger counterparts. This decrease in ODC activity corresponded with the increase in reproductive complications typically seen in older mice, such as an increase in miscarriage, and an increased rate of aneuploidies. Furthermore, supplementing the drinking water of these old mice with putrescine, the product of ODC, was found to partially rescue some of these old-age symptoms, such as the rate of miscarriage. In order to further study the purpose of the ovarian ODC rise during the preovulatory period we have generated mice that have had Odc knocked out (Odc-KO) specifically in the oocytes, or in the granulosa cells. Double knockout mice which had Odc knocked out in both the oocytes and granulosa cells were also generated. With these tissue-specific Odc-KO mice, it was hypothesized that a lack of Odc in the oocytes or granulosa cells would negatively impact the follicular development and fertility of the mice. From the oocyte-specific Odc- KO mice we were able to demonstrate that Odc expression in the oocyte is not required for oogenesis. Knocking out Odc in the oocytes was found to have no effect on the development of the oocyte or the surrounding follicle and, no impact was seen on the fertility of the oocyte-specific Odc-KO mice when compared to wild-type controls. The granulosa cell-specific Odc-KO was found to only be a partial knockout. Due to this, the full effects of knocking out Odc in the granulosa cells remain inconclusive. More research is required to find a more reliable method of knocking out genes in the granulosa cells in order to study this further. Additionally, more work on the inhibition of gap junctions to prevent the transmission of polyamines throughout the follicle is required to better understand the effects of Odc expression and polyamines on reproduction.

Peer reviewed: True, Parthenogenesis has been documented in almost every phylum of animals, and yet this phenomenon is largely understudied. It has particular importance in dipterans since some parthenogenetic species are also disease vectors and agricultural pests. Here, we present a catalogue of parthenogenetic dipterans, although it is likely that many more remain to be identified, and we discuss how their developmental biology and interactions with diverse environments may be linked to different types of parthenogenetic reproduction. We discuss how the advances in genetics and genomics have identified chromosomal loci associated with parthenogenesis. In particular, a polygenic cause of facultative parthenogenesis has been uncovered in Drosophila mercatorum, allowing the corresponding genetic variants to be tested for their ability to promote parthenogenesis in another species, Drosophila melanogaster . This study probably identifies just one of many routes that could be followed in the evolution of parthenogenesis. We attempt to account for why the phenomenon has evolved so many times in the dipteran order and why facultative parthenogenesis appears particularly prevalent. We also discuss the significance of coarse genomic changes, including non-disjunction, aneuploidy, and polyploidy and how, together with changes to specific genes, these might relate to both facultative and obligate parthenogenesis in dipterans and other parthenogenetic animals.

The 7th phase of this longitudinal study, conducted in the United Kingdom, aimed to establish whether children born through third-party assisted reproduction experienced psychological problems, or difficulties in their relationship with their mothers, in early adulthood. The impact of disclosure of their biological origins, and of the quality of the mother-child relationship from age 3 onward, were also examined. Sixty-five assisted reproduction families, including 22 surrogacy families, 17 egg donation families, and 26 sperm donation families, were compared with 52 unassisted conception families when the children were aged 20. Less than half of the mothers had completed tertiary education and less than 5% were from ethnic minority backgrounds. Standardized interviews and questionnaires were administered to mothers and young adults. There were no differences between the assisted reproduction and unassisted conception families in mothers’ or young adults’ psychological wellbeing, or in the quality of family relationships. However, within the gamete donation families, egg donation mothers reported less positive family relationships than sperm donation mothers, and young adults conceived by sperm donation reported poorer family communication than those conceived by egg donation. Young adults who learned about their biological origins before age 7 had less negative relationships with their mothers, and their mothers showed lower levels of anxiety and depression. The associations between parenting and child adjustment did not differ between the assisted and unassisted reproduction families from age 3 to age 20. The findings suggest that the absence of a biological connection between children and their parents in assisted reproduction families does not interfere with the development of positive mother-child relationships or psychological adjustment in adulthood.

T cells represent the major component of tumor-infiltrating lymphocytes (TIL) in testicular germ cell tumors (TGCT). Whereas the roles of rarer subtypes, such as regulatory (Treg) and follicular helper T (Tfh) cells have not been investigated in TGCT, these have each been associated with tumor progression and prognosis in other tumor entities. This study particularly focused on these rare T cells in human TGCT and showed that both rare T cells are abundantly found in TGCT, providing the first evidence that rarer T cell subtypes and their signaling molecules are of functional importance in the TGCT immune environment.

Poster by Meritxell Espino-Guarch (Sidra Medicine), Rana Nahas (Sidra Medicine), Taushif Khan (Sidra Medicine), Susi Huang (Sidra Medicine), Anna Halama (Weill Cornell Medicine-Qatar), Khalid Hussain (Sidra Medicine), and Nicholas van Panhuys (Sidra Medicine) Background: The incidence of T2D has increased significantly in recent years and is characterized by impaired insulin secretion, glucose intolerance and hyperglycemia and is highly associated with a modern sedentary lifestyle and obesity. Despite having distinctly different pathologies, both types of diabetes are associated with defects in immune regulation that mediate sensitivity to insulin. Considering that T cells are an attractive target to modulate for therapeutic purposes and that the immunopathology of T2D in human subjects, has not been widely assessed. Objective: Based on next-generation tools we investigated the transcriptomics differences between Treg and Tcon cells and correlated these with the inflammatory proteins and metabolites present during the development of insulin resistance and T2D. Methods: Here we collected transcriptomic and metabolomic data from 86 children under 17 years-old to identify the regulatory events that participate in IR development. A comparative analysis of the transcriptome of the two major subtypes of CD4+ T cells, regulatory-T cells (Treg) and conventional T cells (Tcon) was conducted and compared with the plasma metabolome of 14 Control, 19 Obese, 17 Pre-diab, 16 T2D, and 20 T1D cases. Additionally, all samples were immunoprofiled by multi-parameter flow cytometry to assess the comparative levels of circulating adaptive and innate immune cell types present that potentially contribute to disease progression. Results: No significant differences between the absolute proportions of Treg and Tcon subsets present was observed in any of the groups studied. However, a differential analysis of CD49d expression on CD4+ T cells reveled distinct differences between the clinical phenotypes studied. CD49d (ITGA4) is a membrane protein active in pro-inflammatory T cells and participates in cellular migration to tissue. In all clinical phenotypes (obese, Pre-diab, T2D and T1D) CD4+ Treg cells showed increased expression of CD49d in comparison to controls, whereas in Tcon cells differences only were observed in the T2D group. In order to be functional, the CD49d integrin needs to form a heterodimer with ITGB1 protein. When transcriptomic data was interrogated, an imbalance between ITGA4/ ITGB1 was observed in all groups where CD49d showed increased expression. Associated with these findings, anti-inflammatory Treg cytokines including IL-10 were decreased in T2D patients and elevated plasma levels of IL-17, which contributes to the insulin resistance and inflammation were observed. Conclusion: Data generated phenotyping this cohort is currently being further integrated and investigated in order to better understand the immunopathology of T2D progression in children, with the future goal of modulating T cell functionality for therapeutic use.

When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (CaMKIIg), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy.

This is a graphical abstract of a published peer-reviewed article. It was created by the authors of the original article. For a full list of declarations, including funding and author disclosure statements, please read the full open-access article online.

Poster by Yasmin Abu Aqel (Hamad Bin Khalifa University), Racha Chouaib (Hamad Bin Khalifa University), Aldana I. Alnesf (Hamad Bin Khalifa University), Idil Ahmed (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Ahmed K. Elsayed (Hamad Bin Khalifa University), Zeyaul Islam (Hamad Bin Khalifa University), Sara Al Khawaga (Hamad Bin Khalifa University), Prasanna R. Kolatkar (Hamad Bin Khalifa University), Paul J. Thornalley (Hamad Bin Khalifa University), Khalid Hussain (Sidra Medicine), and Essam M. Abdelalim (Hamad Bin Khalifa University) Background: Glucokinase (GCK, hexokinase IV) phosphorylates glucose to glucose-6-phosphate during glycolysis process in pancreatic β-cells and hepatocytes. This is the rate limiting step in glucose metabolism and enables those cells to respond appropriately to blood glucose level. Mutations in the GCK gene can either result in hyperglycemia or hypoglycemia. Heterozygous loss of function mutation causes maturity‐onset diabetes of the young 2 (MODY2), while homozygous inactivating mutation leads to permanent neonatal diabetes mellitus (PNDM). The mechanism of diabetes development due to GCK mutations is not fully understood due to the lack of human models Objective: This study aims to understand the effect of GCK mutations on both pancreatic β-cells and mature hepatocytes to identify the molecular mechanisms underlying the defects associated with GCK mutations by using human iPSCs models. Methods: We generated induced pluripotent stem cells (iPSCs) from blood cells of two patients diagnosed with heterozygous and homozygous mutations in the GCK gene. The mutations were confirmed in patient’s samples using whole exome sequencing (WES) and Sanger sequencing. The iPSC lines were extensively characterized using different approaches. Several experiments were done to understand the effect of this mutation. CRISPR/Cas9 knock-in approach was used to generate isogenic controls by correcting the GCK mutation in the generated iPSCs. Mutated and corrected iPSC lines were differentiated into pancreatic and hepatic lineages to understand the effect of GCK mutations in the development of MODY2 and PNDM. in silico study was done to capture the protein’s 3D structure of GCK and predict the effect of this GCK mutation on protein level. Results: Our findings showed that the generated iPSC lines displayed pluripotency characteristics, were able to differentiate into the three germ layers spontaneously and showed normal karyotypes. Our enzymatic preliminary results showed that the mutated GCK protein is less stable compared to the wild type (WT) controls although they are both well folded. Furthermore, it showed higher binding ability and affinity to glucose. Moreover, in silico analysis suggested that this GCK mutation may affect the binding affinity of GCK with glucokinase regulatory protein (GKRP). All generated iPSCs and their isogenic controls were successfully differentiated into pancreatic β-cells and mature hepatocytes. Additional functional studies were performed on both pancreatic β-cells and mature hepatocytes to understand the molecular mechanisms underlying the defects associated with GCK mutations. Conclusion: In conclusion, these human iPSC models can provide valuable insights into the underlying mechanisms of monogenic diabetes, which will pave the way towards personalized treatment.

Conservation translocations are becoming an increasingly necessary tool to stem the decline of threatened species globally. The greater bilby (Macrotis lagotis) is a nationally threatened species in Australia. While bilby translocations are expected to contribute to the species’ persistence, the scarcity of information on their behaviour and ecology prevents informed-management. By intensively studying a population of bilbies both prior to, and following reintroduction, and subsequent reinforcements to a fenced sanctuary, I aimed to (1) advance knowledge of bilby behaviour and examine behaviours potentially relevant to fitness (i.e. survival and breeding success), (2) improve ecological knowledge of bilbies within understudied (temperate) climates, and (3) use this knowledge to suggest and develop effective tools for their conservation. Chapter 1 describes the current state of research in applied conservation research, and how increased behavioural data could help address some of the current knowledge gaps for bilby conservation. In Chapter 2, I examined patterns in bilby resource selection, finding that selection changed between seasons and years due to the environmental conditions and resources available. I also found that resource requirements are likely to be behavioural-state dependent and sex-specific. In Chapter 3, I constructed social networks to examine nocturnal proximity of bilbies and concurrent burrow sharing and found that associations were non-random. Expanding on this, in Chapter 4, I found that burrow sharing was likely to help describe breeding strategies, as males strongly avoided other males, and mixed-sex dyads exhibited kin-avoidance when mate choice was more limited. In Chapter 5, I developed a test to screen personality traits in bilbies, finding links between male response to handling and relative breeding success post-release. Lastly, in Chapter 6, I described a method to collect detailed movement data on the bilby, and discussed some of the practical and animal welfare constraints for its application. My thesis provides new insights into the behavioural ecology of the bilby with potential implications for future management of the species. With further translocations necessary for long-term persistence of the bilby, this research is highly relevant to current and future management of this ecologically important species, with potential applications to other similarly at-risk species.

Poster by Zahra Morshed (Royan Institute for Reproductive Biomedicine), Mohamad Rezaie (Royan Institute for Reproductive Biomedicine), Behrouz Asgari Abibeiglou (Royan Institute for Stem Cell Biology and Technology) , Zahra Ghezelatagh (Royan Institute for Reproductive Biomedicine) , and Yaser Tahamtani (Royan Institute for Reproductive Biomedicine, Royan Institute for Stem Cell Biology and Technology) Background: Human pluripotent stem cells (hPSC) derived pancreatic cells (PCs) has been recently introduced as a promising cell source for cell replacement therapy for type I diabetic patients. Before conducting clinical trials, laboratory made pancreatic cells need to be characterized through preclinical evaluations. Mice, rats and higher non-human primates have been common models for evaluating toxicity, feasibility and efficacy of this lab-made cells. Despite having many similarities to humans, these models have limitations However, since the technology of differentiating hPSC into different PCs is still under development, introducing new methods that enable us to faster and cheaper compare our generated cells in-vivo is necessary. Zebrafish have been introduced as a proper animal model for evaluating functionality of human cells due to its fast breeding, transparent larvae, and lack of acquired immune system in the first 14 days of its life. High-throughput zebrafish cell injection platform is also available for screening different cell types. Objective: The aim is to examine zebrafish as a model for hPSCs-PPs injection and to evaluate their viability, integration into the pancreatic niche, further differentiation and functionality. Methods: Using our previously published differentiation protocol, hPSCs were differentiated into PDX1+/NKX6.1+ PPs within 14 days in 37°C. Afterward, the cells marked with the vital dye pkh26 (red). The transgenic zebrafish model Tg (ins: Kaede-NTR) larvae (kept at 28°C water) were used, which have a green fluorescent pancreas that can be abolished by metronidazole (MTZ). For cell injection, the 3-day-old larvae anesthetized with Tricain, then transferred to the PDMS mold plate, and microinjected under the fluorescent microscope. To check the viability of zebrafish after injection, images taken every day using an optical microscope. Also, to determine the viability of the injected cells, every second day from day 3 to 14, the pancreatic portion of the larvae, were detected by confocal microscopy. Results: As the first step of this project, to optimize the injection site, obtain the correct number of cells for injection, and evaluate the survival rate of larvae and cells after injection, hESCs were injected into the pancreatic niche of Tg (ins: Kaede-NTR) zebrafish. Results showed that the best injection site was pancreatic niche which is labeled by kaede, and the optimum number of cells injected was a volume of 1 µl containing 50-250 cells. larvae viability of 80% ± 20% was observed after injection which was probably due to larvae maintenance at 32°C. Larvae remained viable until 14 days and red hESCs were traceable up to 7 days after injection in tissues near and also far from the injection site which is representative of cell migration after transplantation. In the next step, we intend to evaluate the survival, differentiation, and function of hESCs-PPs in our zebrafish model by injecting hESCs-PPs into zebrafish larvae with/without distraction of their endogenous pancreatic beta cells. Conclusion: This method can be used to follow further differentiation and functionality of hPSCs-PPs in zebrafish larvae as a good assay for evaluating and comparing different differentiation protocols in vivo.

Poster by Olfa Khalifa (Hamad Bin Khalifa University), Khaoula Errafii (Mohammed VI Polytechnic University), Sama Ayoub (Weill Cornell Medicine - Qatar), Abdelilah Arredouani (Hamad Bin Khalifa University) Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide in part due to the obesity epidemic and insulin resistance. The mechanisms underlying the non-alcoholic steatosis are poorly understood and little information is available on the pathways responsible for the progressive hepatocellular damage that follows lipids accumulation. NAFLD research is incredibly important because its diagnosis requires the conduction of a biopsy. Accordingly, more recently biomarkers including noncoding RNAs have been investigated for their involvement in the disease. There are several types of noncoding RNAs associated with NAFLD. Long noncoding RNAs (LncRNAs), which include more than 200 nucleotides, have a role in a range of biological processes including cell proliferation, death, and differentiation. MALAT1 (metastasis associated with lung adenocarcinoma transcript 1) is a well-conserved lncRNA that has been linked to a variety of illnesses, including cancer. However, its role in hepatic steatosis and lipid accumulation is unknown. MALAT1 has recently attracted attention for its role in the evolution of diabetic complications, since it has been demonstrated that MALAT1 dysregulation contributes to the pathogenesis of diabetes and liver disease. Objective: The purpose of this study was to investigate MALAT1's effects on hepatic lipid accumulation and potential targets in presence of the GLP-1R agonist Exendin-4 (Ex-4), which has the potential to improve steatosis and even steatohepatitis. Methods: A steatosis cell model was established using HepG2 cells by treating them overnight with 400 mM oleic acid (OA). We determined the optimal concentration of OA needed to obtain saturating levels of triglycerides. Then starved the cells for 6h in DMEM containing 1% fatty-acid-free bovine serum albumin. Following the starvation, a 16h incubation in DMEM containing increasing concentrations of OA (0–500 µM) at 37 °C was performed, and steatosis was quantified by triglycerides quantification, Bodipy staining, and quantification of the expression of perilipin genes family. The transcriptomic profiling was performed using total RNA extracted from untreated, steatotic and Ex-4-treated steatotic, cells. We validated a subset of differentially expressed LncRNAs with qRT-PCR and identified the most significantly enriched cellular functions associated with the relevant LncRNAs, among them MALAT1. Finally, CD36 was silenced, and lipogenesis genes expression were quantified using q-PCR and/or western blots. Results: Treatment with 200nM Ex-4 for 3h decreased the OA-induced lipid buildup significantly (p

Poster by Noura Aldous, Ahmed Elsayed, Nehad Alajez, and Essam M. Abdelalim (Hamad Bin Khalifa University) Background: Generation of pancreatic beta cells from human pluripotent stem cells (hPSCs) is a promising approach for studying diabetes pathogenesis and transplantation therapy. Our recent study showed that FOXA2 is crucial for human alpha and beta cell development during pancreatic islet differentiation. However, whether miRNAs are regulated by FOXA2 or not is largely unknown. Objective: In this report, we aimed to capture the dysregulated miRNAs associated with the absence of FOXA2 and identify their pancreatic-specific target genes in the iPSC-derived pancreatic progenitors (iPSC-PPs). Methods: Two different FOXA2 knockout (FOXA2–/–) hiPSC lines were generated using CRISPR/Cas9. FOXA2–/–hiPSCs and their isogenic controls were differentiated into PPs in vitro using our protocol. PPs generated from FOXA2–/–hiPSCs and WT controls were exposed to RNA-sequencing and miRNA-sequencing. Gene ontology (GO) and KEGG pathways analyses were performed on the differentially expressed genes (DEGs). For differentially expressed miRNAs, a fold-change of >2 with a P-value 1.0, p < 0.05) DEGs. Several important transcription factors (TFs) required for pancreatic islet development and function were significantly downregulated in FOXA2–/–PPs such as PDX1, NKX6.1, SOX9, PAX4, GATA6, NEUROG3, NEUROD1, and PTF1A, which were also validated by RT-qPCR. Furthermore, genes associated with MODY, pancreatic development, Notch and WNT signaling pathways were downregulated, while glucose and cholesterol homeostasis associated genes were upregulated. miRNA-seq data analysis on FOXA2–/–PPs showed 107 downregulated (Log2 FC < −1.0, p < 0.05) and 111 upregulated (Log2 FC > 1.0, p < 0.05) differentially expressed miRNAs. Integrating miRNA and mRNA profiling using IPA showed that 190 miRNAs target 2654 mRNAs. Our analysis showed that the upregulated miRNAs (miR-92a-2-5p, miR-92b-5p, and miR-184) targeted both FOXA2 and NKX6.1, while miR-124-3p targeted multiple important pancreatic TFs including FOXA2, NGN3, NEUROD1, GATA6, SOX9, INSM1, and RFX6. miR-291a-3p is another miRNA that was found to have several target genes including NGN3, GLIS3, ARX, ONECUT1, and NEUROD1. Multiple upregulated miRNAs targeted key pancreatic TFs in FOXA2–/–PPs including NKX6.1, NEUROD1, ONECUT1, NGN3, PTF1A, GATA6, GATA4, RFX6, PDX1, and PAX4. miR-122-5p, miR-184, miR-194-5p, miR-373-3p, miR-885-5p, and miR-371a-3p were validated for upregulated miRNAs, while miR-493-3p was validated for downregulated miRNAs. Conclusion: These findings indicate that the expression of FOXA2 during pancreatic islet development is crucial in maintaining the normal expression of the key miRNAs that target pancreatic endocrine TFs.

This review propose to assess one of the strategies to improve cryopreservation of gonadal tissues (either ovarian or testicle). This technique is important in as when pre-pubertal human patients begin gonadotoxic treatments (example, in cancer cases), patients that will begin cancer treatment immediately or that have hormone sensitive tumors and can not cryopreserve oocytes. In this sense, advances on research that can answer not only if, but which antioxidant would be more itneresting to use in tissue cryopreservation, can help at the development of new strategies in improvement of ovarian and/or testicle tissues cryopreservation and thus, to preserve fertility of patients that need to use this strategy.

Poster by Muneera Vakayil (Weill Cornell Medicine - Qatar, Hamad Bin Khalifa University), Aisha Madani (Weill Cornell Medicine - Qatar), Yasser Majeed (Weill Cornell Medicine - Qatar), and Nayef Mazloum (Weill Cornell Medicine - Qatar) Background: The increased prevalence of obesity has led to a concomitant increase in the incidence of type 2 diabetes (T2D). Obesity-linked inflammation is the major cause of insulin resistance, contributing to the onset of T2D. Therefore, understanding the cellular mechanisms responsible for adipogenesis, the conversion of preadipocytes to adipocytes, is critical for identifying anti-diabetic therapeutic targets. Uhrf1 is a key epigenetic regulator that coordinates DNA methylation and histone modifications; however, the function of Uhrf1 in metabolic diseases is not known. Objective: Therefore, here, we aimed to understand the role of Uhrf1 in adipogenesis by studying Uhrf1 loss of function cells. Methods: We performed CRISPR/ Cas9 and shRNA-mediated gene silencing in addition to whole transcriptomics analysis on Uhrf1 KO cells. Results: We identified altered molecular and cellular pathways indicating that Uhrf1 is a critical factor required for adipocyte metabolism, and in its absence, cells exhibit adipocyte dysfunctions with higher levels of inflammatory markers. Conclusion: Our results highlight the possibility of Uhrf1 and its targets to have clinical applications in obesity and T2D treatment.

Poster by Ikhlak Ahmed (Sidra Medicine), Mubarak Ziab (Sidra Medicine), Sahar Da’as (Sidra Medicine, Hamad Bin Khalifa University), Waseem Hasan (Sidra Medicine), Sujitha P. Jeya (Sidra Medicine), Elbay Aliyev (Sidra Medicine), Sabah Nisar (Sidra Medicine), Ajaz A. Bhat (Sidra Medicine), Khalid Fakhro (Sidra Medicine, Hamad Bin Khalifa University, Weill Cornell Medicine College - Qatar), Ammira S. Alshabeeb Akil (Sidra Medicine) Background: Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that gene susceptibility to DN plays a critical role in disease pathophysiology. Therefore, many genetic studies have been performed to categorize candidate genes in prominent diabetic cohorts, aiming to investigate DN pathogenesis and etiology. Objective: The objective of this study is to identify critical transcriptional factors associated with diabetic nephropathy (DN) progression and validate their role in DN development using a hyperglycemic zebrafish model. Methods: In this study, we performed a meta-analysis on the expression profiles of GSE1009, GSE30122, GSE96804, GSE99340, GSE104948, GSE104954, and GSE111154 to identify critical transcriptional factors associated with DN progression. The analysis was conducted for all individual datasets for each kidney tissue (glomerulus, tubules, and kidney cortex). Results: We identified distinct clusters of susceptibility genes that were dysregulated in a renal compartment- specific pattern. Further, we recognized a small but a closely connected set of these susceptibility genes enriched for podocyte differentiation, several of which were characterized as genes encoding critical transcriptional factors (TFs) involved in DN development and podocyte function. To validate the role of identified TFs in DN progression, we functionally validated the three main TFs (DACH1, LMX1B, and WT1) identified through differential gene expression and network analysis using the hyperglycemic zebrafish model. We report that hyperglycemia-induced altered gene expression of the key TF genes leads to morphological abnormalities in zebrafish glomeruli, pronephric tubules, proximal and distal ducts. Conclusion: This study demonstrated that altered expression of these TF genes could be associated with hyperglycemia-induced nephropathy and, thus, aids in understanding the molecular drivers, essential genes, and pathways that trigger DN initiation and development.

This thesis examines the effects of an essential micronutrient – a fat-like molecule called cholesterol – in mediating the beneficial effects of a low protein diet on fruit fly lifespan. It shows that low protein diets extend lifespan by inhibiting reproductive output, this rescues flies from committing limited cholesterol reserves towards reproduction. In turn, if high protein diets are supplemented with cholesterol, both lifespan and reproduction can be maximised simultaneously. These findings help us to better understand how changes in diet influence lifespan.

Marjan Nouri-Keshtkar (University of Science and Culture, Royan Institute for Stem Cell Biology and Technology), Hassan Ketabi (MCI), Maryam Alasadi (University of Science and Culture, Royan Institute for Stem Cell Biology and Technology), Roya Hossein (Royan Institute for Reproductive Medicine), Reza Homayounfar (Fasa University of Medical Sciences, Shahid Beheshti University of Medical Sciences) Mehdi Totonchi (Royan Institute for Reproductive Medicine), Yaser Tahamtani (University of Science and Culture, Royan Institute for Stem Cell Biology and Technology, Roya Institute for Reproductive Medicine) Background: Metformin is the first line antidiabetic drug for type 2 diabetics. Despite its widespread use, it doesn't work optimally for all and about 30% of patients are resistant to metformin. Different studies showed that there are genetic factors affecting this phenomenon. However, due to many influencing environmental factors, developing a cell-based modeling system can be utilized as a tool to better understand the nature of metformin response. Objective: In this study, we selected our experimental sources (metformin responders/non-responders) from Fasa PERSIAN cohort study which is a population-based cohort of >10K individuals aged 35-70 years established in Fasa city in IRAN. For exact selection of patients, we developed a visual search tool (VST). Fibroblast cells were collected from selected patients to be used for further experiments. Methods: To develop the VST, each patients’ data was indexed as one record in elastic search. The user interface is a customized Kibana dashboard that enhances Lucene based full-text search of associations with interactive aggregation visualizations. All patients entered this study based on informed consent and skin biopsies were collected by taking a punch-size piece of skin. Fibroblast cells were isolated by enzymatic digestion, expanded and adequately banked. To detect metformin effects at the cellular level, at first step, fibroblasts were treated by 10 to 100 mM metformin for 72 hours to find the optimal dose and the phosphorylation of AMP-activated protein kinase (AMPK) was measured by western blotting technique. Next steps of this study will include generation of fibroblast derived induced pluripotent stem cells (iPSCs) and iPSCs -derived hepatocytes. Results: At the first step, among the population of the Fasa PERSIAN cohort, 252 patients were selected using VST for HbA1c test, among them 62 patients were available and tested for HbA1c. Accordingly, six patients who had a HbA1c level higher than 7.5%, and 19 patients who had a HbA1c level less than 7.5% were selected as non-responders and responder patients respectively. After being interviewed by an endocrinologist, two patients were selected for each groups. The isolated Fibroblasts were cultured up to passage number 15 and they were morphologically fibroblast-like cells. The western blot results for 10 mM to 100 mM metformin-treated fibroblasts showed that 10mM of metformin leads to the phosphorylation of APMK as a cell energy sensor through the change in AMP/ATP ratio. Conclusion: Most metformin-resistant patients are unaware of their drug resistance, so we required a protocol to identify them. Identifying these patients is a specialized and time-consuming process, which requires interpretation and analysis tasks. However, by using VST, which provides a user-friendly platform, medical researchers were enabled to perform complex analyses by entering a few queries in a short time; the capability that can be also used for other cohort-driven patient selection studies. By having access to the patient-derived iPSCs, we would be able to differentiate them toward all target cell types such as intestinal epithelial cells and hepatocytes, and explore the differences between different groups at cellular level. Such a cell-based modeling platform also can be an effective tool for conducting drug discovery studies.

This is pre-publication preview introduces the major questions, methods, and insights of my book When a Human Gives Birth to a Raven: Rabbis & the Reproduction of Species (UC Press, 2023).

Poster by Imran Ramzan, Moira Taylor, Kenneth Smith, Dan Wilkinson, Philip Atherton, and Iskandar Idris (University of Nottingham) Background: Recent studies have identified branched chain amino acids (BCAAs; isoleucine, leucine and valine) as potential biomarkers of, and being involved in, the pathogenesis of type 2 diabetes mellitus (T2DM), insulin resistance (IR) and obesity. Reducing circulatory BCAAs by dietary restriction was suggested to mitigate these risks in rodent models, but this is a challenging paradigm to deliver in humans. Objective: We aimed to design and assess the feasibility of a diet aimed at reducing circulating BCAA concentrations in humans, while maintaining energy balance and overall energy/protein intake. Methods: Twelve healthy individuals were assigned to either a 7-day BCAA-restricted diet or a 7-day control diet. Diets were iso-nitrogenous and iso-caloric, with only BCAA levels differing between the two. Results: The BCAA-restricted diet significantly reduced circulating BCAA concentrations by ~50% i.e., baseline 437 ± 60 to 217 ± 40 µmol/L (p < 0.005). Individually, both valine (245 ± 33 to 105 ± 23 µmol/L; p < 0.0001), and leucine (130 ± 20 to 75 ± 13 µmol/L; p < 0.05), decreased significantly in response to the BCAA-restricted diet. The BCAA-restricted diet marginally lowered Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) levels: baseline 1.5 ± 0.2 to 1.0 ± 0.1; (p = 0.096). Conclusion: We successfully lowered circulating BCAAs by 50% while maintaining iso-nitrogenous, iso-caloric dietary intakes, and while meeting the recommended daily allowances (RDA) for protein requirements. The present pilot study represents a novel dietary means by which to reduce BCAA, and as such, provides a blueprint for a potential dietary therapeutic in obesity/diabetes.

Poster by Houda Yasmine Ali Moussa (Hamad Bin Khalifa University) , Nimshitha Manaph (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Selma Maacha (Sidra Medicine), Kyung Chul Shin (Hamad Bin Khalifa University),, Samia M. Ltaief (Hamad Bin Khalifa University), Vijay Gupta (Hamad Bin Khalifa University), Yongfeng Tong (Hamad Bin Khalifa University), Janarthanan Ponraj (Hamad Bin Khalifa University), Salam Salloum-Asfar (Hamad Bin Khalifa University), Said Mansour (Hamad Bin Khalifa University), Fouad A. Al-Shaban (Hamad Bin Khalifa University), Hyung-Goo Kim (Hamad Bin Khalifa University), Lawrence W Stanton (Hamad Bin Khalifa University), Jean-Charles Grivel (Sidra Medicine), Sara A Abdulla (Hamad Bin Khalifa University), Abeer R. Al-Shammari (Hamad Bin Khalifa University), Yongsoo Park (Hamad Bin Khalifa University) Background: Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. Objective: We aim to identify specific markers for brain-derived EVs isolated from plasma for biomarkers of neuronal disorders. Methods: Using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. We have also used human induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids to study neuronal EVs. Results: Neural cell adhesion molecule (NCAM) is highly expressed and enriched in EVs released from human iPSCs-derived cortical neurons and organoids. Subpopulations of blood plasma exosomes contain NCAM, suggesting its origin from neuronal cells. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Conclusion: Our data propose that NCAM-positive neuronal exosomes isolated from blood plasma can be detected and analyzed using flow cytometry. Neuronal EVs identified here will pave the way for further studies to discover biomarkers for neurological disorders.

Poster by Neha Gopinath (Sidra Medicine), Ishita Gupta (Sidra Medicine), Alice Turdo (Sidra Medicine), Rebecca Mathew (Sidra Medicine), Harshitha Shobha Manjunath (Sidra Medicine), Mohammed Toufiq (Sidra Medicine), Ola Hussein (Sidra Medicine), Evonne Chin-Smith (Sidra Medicine), Matilde Todaro (Sidra Medicine), Giorgio Stassi (University of Palermo), Sara Tomei (Sidra Medicine), Ira Skvortsova (Medical University of Innsbruck and Tyrolean Cancer Research Institute), Soldano Ferrone (Harvard Medical School), Cristina Maccalli (Sidra Medicine) Background: Cancer stem cells (CSCs) represent a rare subpopulation of cells within the tumor that are considered responsible of metastatization and resistance to therapies, including immunotherapy. Objective: The aim of this study is to identify the molecular mechanisms regulating the immunological properties of cancer stem cells (CSCs) isolated from colorectal cancer (CRC) and breast cancer (BC) using integrative approaches including flow cytometry, miRNAs and RNA seq-based transcriptomic profile and in vitro functional assays. Methods: Colorectal cancer (CRC; N=15) and breast cancer (BC; N=21) cell lines, including differentiated tumor cells and CSCs, and, for BC, selected in vitro for radioresistance or invasiveness were used for this study. The baseline expression of HLA molecules and the components involved in the antigen processing machinery (APM) following or not the treatment with immunomodulatory or epigenetic agents was assessed through flow cytometry. Tumor cells were co-cultured with HLA-matched peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to assess the efficiency of the CRC and BC cells endowed with stemness properties to elicit antigen-specific T cell responses. The nCounter platform (Nanostring) was utilized to assess the hybridization with 800 probes for miRNAs and the RNA seq-based transcriptomic profile (Lexogen) was also assessed. Results: A general impairment of the expression of HLA and APM molecules was observed in CRC and BC lines. The down-modulation of the expression of HLA and APM was superior in “stem-like” cells as compared to the differentiated tumor cells. The treatment of these cells with immunomodulating or epigenetic agents could only partially restore the expression of these molecules. The induction in vitro of anti-tumor T cell responses through the co-culture of tumor cells with PBMCs, was associated with the levels of the expression HLA and APM molecules by the tumor cells and on the efficiency of their up-modulation by either immunomodulating or epigenetic agents. Differential miRNA profiles (p

Poster by Abdoulaye Diane, Razik Bin Abdul Mu-u-min, Asma Allouch, and Heba H. Al-Siddiqi (Hamad Bin Khalifa University) Background: Impaired insulin secretion contributes to the pathogenesis of diabetes mellitus type 1 (T1DM) through autoimmune destruction of pancreatic β-cells and the pathogenesis of diabetes mellitus type 2 (T2DM) through β-cell dedifferentiation and other mechanisms. Emerging β-cell replacement with human pluripotent stem cell (hPSC)–derived β-cells may provide remedial cell therapy. Most in vitro differentiation protocols have generated hPSC-derived β-cells with either immature phenotype such as impaired or weakened glucose-stimulated insulin secretion (GSIS) relative to primary β-cells. Evidence has shown that β-cell mitochondria play a central role in coupling glucose metabolism to insulin exocytosis. Therefore, the impairment of GSIS in hPSC-derived β-cells may be attributed to impaired glucose sensing and/or mitochondrial dysfunction. Objective: The aim of this study is to investigate the effect of Exendin-4 (GLP-1 receptor analog), known to promote mitochondrial function, on enhancing maturation and functionality of hPSC-derived β-cells. Methods: Differentiation of hPSC into β-cells was carried out in a stepwise 3D differentiation protocol using 30 ml spinner flask adapted from the protocol of Veres et al. (2019). This consists of 6 stages: S1, definitive endoderm; S2, gut tube endoderm; S3, pancreatic progenitors 1; S4, pancreatic progenitors 2; S5, endocrine precursors; and S6, β-like cells. To determine the differentiation efficiency, relevant stage-specific marker expression was assessed at each stage using flow cytometry. Finally, to further test the effect of GLP-1 receptor analog on the functionality of hPSC-derived β-cells, 50 nM exendin-4 was added to the suspension culture during the last three days of differentiation and GSIS was performed. Gene expression in cell clusters was determined by RT-PCR. Data are mean ± SEM (n=6) Results: Flow cytometry data for relevant stage-specific markers showed 96 % OCT4 positive, 89% SOX17 and 83% PDX1 positive cells, indicating good pluripotency, high definite endoderm and pancreatic progenitor induction, respectively. As for β-cell markers, we found 41.4% NKX6.1/insulin double positive cells at final stage 6, indicating a generation of β-cells. Expression profiling during differentiation confirmed the generation of insulin-expressing β-cells. However, GSIS data showed no difference in c-peptide secretion between low (2.8mM) and high (20 mM) glucose but high response to direct cellular depolarization-mediated c-peptide by KCl; suggesting a lack of functional β-cells. Interestingly, addition of Exendin-4 (50 nM) during the last 3 days of the differentiation significantly enhanced GSIS associated with increased expression of glucose-sensing apparatus genes (Glut2, G6P2C, GCK) and genes encoding mitochondrial oxidative phosphorylation machinery. Conclusion: Our data demonstrated for the first time in 3D differentiation of hPSC-derived β-cells that addition of Exendin-4 promotes GSIS through upregulation of both glucose-sensing apparatus and mitochondrial oxidative phosphorylation machinery genes without increasing β-cell number.

In modern society, continuous light exposure and obesity are increasingly prevalent, especially in women of childbearing age. Circadian, metabolic, and reproductive systems have a complex, inter-regulated relationship. Thus, the disruption of one system likely impedes another. Excessive adiposity and circadian disruption alter normal behavior and physiology and disrupt the endocrine milieu. The overall goal of the studies described in this thesis was to develop and test a model system that could tease apart the influence of prepregnancy obesity and circadian disruption, as well as study the combined effects on female reproductive competence. The first study focuses on the prepregnancy period and aims to determine the effect of high fat diet feeding on diurnal eating pattern, body weight over the four-week period, the body composition at the end of the four-week period, hair corticosterone levels, and circadian fecal corticosterone patterns on female ICR mice. Five-week-old female ICR mice were randomly assigned to control (CON; 10% fat) or high fat (HF; 60%) diets and fed for four weeks to achieve adequate adiposity. During this four-week time period, mice had routine light exposure of 12h light and 12h dark. Feed was weighed at 0600 and 1745 Monday-Friday to determine diurnal feed intake. The mice were weighed on a weekly basis. After four weeks on respective diets, mice were anesthetized with isoflurane to measure crown-rump length to calculate BMI and hair was shaved for corticosterone extraction. Once mice recovered from anesthesia, body composition was measured with EchoMRI. After 1 week on diets, HF mice consumed more (P

Ο καρκίνος του πνεύμονα είναι ένας από τους συνηθέστερους τύπους καρκίνου παγκοσμίως, σύμφωνα με τα επιδημιολογικά στατιστικά του Παγκόσμιου Οργανισμού Υγείας. To γονίδιο RAS (Rat sarcoma) αποτελεί το πιο σύνηθες μεταλλαγμένο ογκογονίδιο στον ανθρώπινο καρκίνο, με το KRAS να καθιστά την πιο συχνά μεταλλαγμένη ισομορφή του. Οι πιο συχνές μορφές καρκίνου στις οποίες το γονίδιο KRAS εμφανίζεται με πολύ μεγάλη συχνότητα είναι ο καρκίνος του πνεύμονα, ο καρκίνος του παγκρέατος και ο καρκίνος του παχέος εντέρου και ορθού. Πιο συγκεκριμένα, όσον αφορά στον καρκίνο του πνεύμονα, οι μεταλλάξεις KRAS εμφανίζονται κυρίως στα αδενοκαρκινώματα του πνεύμονα, τον πιο κοινό ιστολογικό υπότυπο του μη μικροκυτταρικού καρκίνου του πνεύμονα (NSCLC), με συχνότητες που κυμαίνονται έως και 40%. Παρόλο που η χρήση διάφορων στοχευμένων θεραπειών και των αναστολέων Ανοσολογικών Σημείων Ελέγχου (ΑΣΕ) έχει αλλάξει δραστικά το τοπίο στη θεραπεία του μη μικροκυτταρικου καρκίνου του πνεύμονα τα τελευταία χρόνια, διάφορες προσπάθειες για στόχευση του KRAS γονιδίου είχαν ελάχιστη επιτυχία. Από την άλλη, η πρόσφατη έγκριση του Sotorasib και του Adagrasib από τον FDA για τη θεραπεία του τοπικά προχωρημένου ή μεταστατικού NSCLC με μετάλλαξη KRAS G12C ως τα πρώτα μικρά μόρια που λειτουργούν σαν άμεσοι αναστολείς της KRAS πρωτεΐνης έδωσε νέα πνοή στην εξέλιξη των κλινικών μελετών με άλλους άμεσους αναστολείς, αλλά και με συνδυασμένες θεραπείες. Η συγκεκριμένη βιβλιογραφική ανασκόπηση εξετάζει τον ρόλο των KRAS μεταλλάξεων στον μη μικροκυτταρικό καρκίνο του πνεύμονα και τις προκλήσεις στην στόχευσή του. Με βάση την βιολογία του KRAS, αναφέρουμε μία ενδιαφέρουσα πρόοδο, εξερευνώντας τη χρήση πρωτοποριακών αναστολέων της KRAS πρωτεΐνης ως μονοθεραπεία ή σε συνδυασμό με άλλες στοχευμένες θεραπείες, χημειοθεραπεία και ανοσοθεραπεία, Lung cancer is one of the most common types of cancer worldwide, according to the epidemiological studies of the World Health Organization. Rat sarcoma (RAS) is the most frequently mutated oncogene in human cancer, with Kirsten rat sarcoma (KRAS) being the most commonly mutated RAS isoform. The most common types of cancer in which the KRAS gene is overexpressed are lung cancer, pancreatic cancer and colorectal cancer. More specifically, regarding lung cancer, KRAS mutations occur mainly in lung adenocarcinomas, the most common histological subtype of non-small cell lung cancer (NSCLC), with frequencies ranging up to 40%. While the use of targeted therapies and immune checkpoint inhibitors (CPIs) has drastically changed the treatment landscape of advanced non-small-cell lung cancer (NSCLC) in recent years, historic attempts to target KRAS have had little success. On the other hand, the recent FDA approval of Sotorasib and Adagrasib, for the treatment of locally advanced and metastatic NSCLC with KRAS G12C mutation as the first small molecules acting as direct inhibitors of the KRAS protein, has given new life to the development of clinical trials with other direct inhibitors, but also with combined treatments. This review examines the role of KRAS mutations in NSCLC and the challenges in targeting KRAS. Based on specific KRAS biology, it reports exciting progress, exploring the use of novel direct KRAS inhibitors as monotherapy or in combination with other targeted therapies, chemotherapy, and immunotherapy

Red blood cells (RBCs) have been extensively studied as a potential biotherapeutic delivery system for several decades. This research has been prompted by these cells' remarkable properties. RBCs are incredibly tenacious and robust, possessing the high mechanical flexibility needed to flow through the human vasculature for 120 days. Their unusual biconcave shape gives them a high surface-area-to-volume ratio, and the expression of a variety of immunomodulatory markers on the RBC membrane allows for excellent biocompatibility and low immunogenicity. Coupling a therapeutic protein to the RBC surface has the potential to enhance its pharmacokinetics as well as modulate its immune response. The goal of this work was to explore a chemistry-oriented approach to synthesizing RBC-based drug delivery systems. Covalent crosslinking to the RBC membrane is ideal for prolonging the circulation of biotherapeutics with intravascular drug targets. Donor or autologous blood can be modified ex vivo with a method that is rapid, tunable, and cell-tolerated. In Chapter 2, we synthesized an RBC platform that could stably display almost any therapeutic antibody. Our system consisted of an RBC covalently coupled to Protein A (SpA) via a polyethylene glycol (PEG) crosslinker. The antibody was then presented on the RBC surface by SpA binding at its Fc region. In Chapter 3, we directly tethered butyrylcholinesterase (BChE) via PEG to the RBC membrane to develop an improved prophylactic treatment against organophosphate nerve agents. After the synthesis of each RBCPEG-protein construct, we confirmed that the therapeutic was still active once displayed on the surface and performed a series of in vitro assays to assess the impact of engineering on cell health. Finally, in Chapter 4, we began preliminary studies exploring the use of atom-transfer radical polymerization (ATRP) for RBC-biotherapeutic coupling. Our results provide early evidence that chemical conjugation can be a viable approach for RBC-based drug delivery systems. In future work, we plan to use an animal model to test the safety, toxicity, and efficacy of our RBC-biotherapeutic platforms.

The National Survey of Australian Secondary Students and Sexual Health (SSASH) is a periodic survey of sexual health among school-aged young people in Australia. The survey began in 1992 and, in this report, we present findings from the seventh iteration. The SSASH survey reports on a range of issues related to sexual identity and experiences, STI and HIV prevention, and relationships and sexuality education (RSE) and provides information about the progress of key priority areas in the Fourth National Sexually Transmissible Infections Strategy (Australian Department of Health, 2018).

Poster by Maram Hasan and Abdelali Agouni (Qatar University) Background: Diabetes is highly prevalent worldwide and is associated with several complications that predispose to a high risk of mortality. Endothelial dysfunction is a hallmark cellular process associated with diabetes. Mesenchymal stem cells (MSCs) possess specific properties including the inhibition of apoptosis, oxidative stress, and secretion of trophic factors. These unique properties prompted us to investigate the effect of MSCs conditioned medium (MSCs-CM) on preventing the noxious effects of methylglyoxal (MGO), a precursor of advanced glycated end-products (AGEs) that play a key role in the pathophysiology of diabetes, on endothelial cells with respect to oxidative stress and cell survival. Additionally, we investigated certain cellular alterations associated with oxidative damage such as the Nrf2 pathway, as a major system involved in cellular defense mechanisms against oxidative stress. Objective: To assess the protective effect of (MSCs-CM) against MGO-mediated injury of cultured endothelial cells in terms of ROS generation, cell survival, and Nrf2 antioxidant pathway. Methods: Endothelial EA.hy926 cells were exposed or not to MGO(1mM) in the presence or absence of (MSCs-CM)(50% in α-MEM). Then, oxidative stress was quantified by DCF-DA assay and cell viability and apoptosis quantification via Annexin V & caspase 3/7 activity were assessed using flowcytometry. The activation of the Nrf2 pathway was evaluated by western blot and qPCR methods. Results: Our results showed that MGO (1 mM) induced substantial ROS generation in EA.hy926 cells after 6 hours of exposure as measured by DCF-DA assay; this effect was attenuated by treating cells with MSCs-CM (50%). Moreover, Ea.hy926 cell viability was significantly reduced by 35% when treated with MGO; while cells treated with MSCs-CM (50%) were protected. We also investigated the protective effects of MSCs-CM against MGO-induced apoptosis in EA.hy926 endothelial cells using the Annexin V/propidium iodide assay and the quantification of caspases 3/7 activity. Our findings showed a significant protective effect of MSCs-CM (50%) on MGO-mediated cell death and caspases 3/7 activation in endothelial cells. Nrf2 controls the transcription of several ROS-detoxifying enzymes including NQO1, and GSTA gene expression, thus the effect of MSCs-CM (50%) on the mRNA expression of Nrf2, NQO1, and GSTA was assessed in MGO-injured cells. We noted that only the mRNA expression of Nrf2 was increased in cells treated with MSCs-CM (50%). At the protein expression level, our results demonstrated that MSCs-CM (50%) enhanced the expression of the detoxifying NQO1in MGO-injured cells. Conclusion: Altogether, these findings underscore the promising therapeutic potential of MSC-CM in fighting oxidative stress and molecular alterations associated with AGEs, a major molecular hallmark of diabetes, in endothelial cells. The protective effects of MSC-CM against endothelial dysfunction, the first step in atherogenesis, may contribute to the reduction of the risk of cardiovascular complications associated with diabetes.

Poster by Ahmed K. Elsayed, Nehad M. Alajez, and Essam M. Abdelalim (Hamad Bin Khalifa University) Background: Our recent studies showed that FOXA2 plays a key role in the development of endocrine and exocrine pancreas. Downregulation of FOXA2 expression during differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets leads to a significant reduction in α-and β-cell masses. However, whether these changes are associated with alterations in the expression profile of the long non-coding RNAs (lncRNAs) remains unclear. Objective: the objective of this study is to investigate the alterations in the lncRNA profile and its relationship with the dysregulated mRNAs at the pancreatic progenitors (PPs) and pancreatic islet stages lacking FOXA2. Methods: Therefore, here, we used our recently established iPSCs lacking FOXA2 (FOXA2-/- iPSCs). we screened the dysregulated lncRNAs in the pancreatic progenitors (PPs) and β-cell stage derived from FOXA2-/- iPSCs in comparison to its control. Results: PPs and islet cells lacking FOXA2 showed several dysregulated lncRNAs in comparison to controls. Differential expression analysis identified 442 downregulated and 114 upregulated lncRNAs in PPs and 177 downregulated and 59 upregulated in the β-cell stage. Correlation analysis revealed 12 DE-lncRNAs, which were strongly correlated to key downregulated pancreatic genes in both pancreatic progenitors and beta cell stages (MEG3, H19, ZNF667-AS1, LINC00543, LINC00261, AC097639.1, AL035661.1, SLC25A25-AS1, U73166.1, ZNF790-AS1, MNX1-AS2, and AC091563.1). Conclusion: Our results showed a strong correlation of lncRNAs to several key pancreatic genes and TFs during pancreatic differentiation. Furthermore, these data suggest that the FOXA2-related defective pancreatic islet development is associated with significant alterations in the expression profile of lncRNAs.

Η αντίσταση στα χημειοθεραπευτικά φάρμακα αποτελεί ένα πρόβλημα που συμβάλει στη μη αποτελεσματική θεραπευτική αντιμετώπιση πολλών νεοπλασιών, συμπεριλαμβανομένων του καρκίνου του παγκρέατος. Εκτός από την κατανόηση του καρκινικού μικροπεριβάλλοντος και του γενετικού υπόβαθρου του καρκίνου του παγκρέατος, φαρμακογονιδιωματική μελέτη της πακλιταξέλης και εξαγωγή συσχετίσεων μεταξύ αλλαγών στο γονιδίωμα ασθενών και της αποτελεσματικότητά της, μπορεί να συμβάλουν στη μείωση του φαινομένου αυτού, καθώς η πακλιταξέλη συνδεδεμένη με λευκωματίνη υπό μορφή νανοσωματιδίων (nab-paclitaxel) σε συνδυασμό με την γεμσιταβίνη αποτελούν ένα από τα πιο συχνά θεραπευτικά σχήματα γι’ αυτήν την ασθένεια. Αξιοποιώντας πληροφορίες από βάσεις δεδομένων φαρμακογονιδιωματικού και γονιδιωματικού ενδιαφέροντος και αναλύοντας τα με τη βοήθεια της βιοπληροφορικής, μελετήσαμε το γονιδίωμα ανθρώπινων κυτταρικών σειρών παγκρεατικού αδενοκαρκινώματος που εμφανίζουν ανθεκτικότητα και ευαισθησία στη θεραπεία με πακλιταξέλη. Από την ανάλυση προέκυψε πως 19 γονίδια παρουσιάζουν ιδιαίτερες μεταβολές στο γονιδίωμα και στα επίπεδα έκφρασής τους, αποκλειστικά και μόνο στις κυτταρικές σειρές που εμφανίζουν είτε ανθεκτικότητα, είτε ευαισθησία στο φάρμακο, επηρεάζοντας άμεσα και έμμεσα γονίδια του μονοπατιού – στόχου της πακλιταξέλης, μονοπάτι χασμοσυνδέσμων, τα οποία συμμετέχουν και σε σημαντικά σηματοδοτικά μονοπάτια που ρυθμίζουν τον πολλαπλασιασμό, την επιβίωση, την απόπτωση και τη μετάσταση των καρκινικών κυττάρων. Περαιτέρω μελέτη αυτών των πιθανών μοριακών βιοδεικτών μπορεί να οδηγήσει στην καλύτερη κατανόηση των μοριακών μηχανισμών ανάπτυξης ανθεκτικότητας των κυττάρων παγκρεατικού αδενοκαρκινώματος στην πακλιταξέλη και να συμβάλει στην επίτευξη της βέλτιστης αποτελεσματικότητας της θεραπείας, προσφέροντας πολλαπλά οφέλη στους ασθενείς., Chemoresistance is a critical problem contributing to the ineffectiveness of therapies in several cancers, including pancreatic adenocarcinoma. In addition to understanding the tumor microenvironment and genetic background of pancreatic cancer, a pharmacogenomic study of the drug paclitaxel would also be of great importance. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) in combination with gemcitabine is a gold standard therapy for pancreatic adenocarcinoma, and detecting correlations between genetic variations and drug efficacy could also help to reduce the resistance phenomenon. We examined the genome of human pancreatic adenocarcinoma cell lines resistant and sensitive to paclitaxel, utilizing data from pharmacogenomic and genomic databases and analyzing them bioinformatically. The analysis revealed 19 genes with unique features in their mutational status and gene expression, exclusively in the resistant or sensitive cell lines. These genes directly or indirectly affect a variety of genes that are part of the paclitaxel target pathway, gap junction, and are also involved in critical signaling pathways that regulate the proliferation, survival, apoptosis, and metastasis of cancer cells. Further investigation of these potential molecular biomarkers may lead to a better understanding of the molecular mechanisms underlying the development of resistance of pancreatic adenocarcinoma cells to nab-paclitaxel therapy and help to achieve optimal treatment efficacy, which would provide multiple benefits to patients.

Poster by Kyung Chul Shin, Gowher Ali, Houda Yasmine Ali Moussa, Lawrence W. Stanton, and Yongsoo Park (Hamad Bin Khalifa University) Background: Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder, mainly caused by rare and de novo genetic variants and mutations. ASD co-occurs with comorbid attention-deficit hyperactivity disorder (ADHD) and epilepsy, which are associated with hyperexcitability of neurons. TRPC6 is a candidate risk factor for ASD and implicated in ASD etiology; de novo missense and nonsense mutations in TRPC6 associated with ASD etiology have been reported. Loss-of-function mutations in TRPC6 reduce calcium influx in human induced pluripotent stem cell (hiPSC)‐derived neurons cell and TRPC6 knockdown (KD) in Drosophila as a ASD animal model causes autism-like behavioral deficits and leads to the hyperactivity phenotype. However, the pathophysiology underlying hyperactivity phenotype caused by TRPC6 mutations in ASD is unclear and mains poorly understood. Objective: We aim to study novel pathophysiological mechanisms of ASD using human pluripotent stem cell technology that offer a platform to model pathology of ASD. Methods: Using hiPSC‐derived cortical neurons, single cell calcium imaging, and electrophysiological recording, we study how TRPC6 knockout (KO) leads to hyperactivity of neurons. Results: We show that TRPC6 KO reduces store-operated Ca2+ entry (SOCE) signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data show the molecular and cellular pathophysiology underlying hyperactivity of ASD individuals, and TRPC6 KO hiPSC-derived cortical neurons can be a good model to study hyperactive behavior of ASD. TRPC6 KO neurons will be used to screen therapeutics that rescue SOCE and reverse hyperexcitability. Conclusion: Our data unveil the molecular and cellular pathophysiology underlying hyperactivity of ASD individuals and TRPC6 KD hiPSC-derived cortical neurons as a platform to model neuropathology of ASD will pave the way for further studies to discover therapeutics for intervention of ASD.

In many vertebrate societies dominant individuals breed at substantially higher rates than subordinates, but whether this hastens ageing remains poorly understood. While frequent reproduction may trade off against somatic maintenance, the extraordinary fecundity and longevity of some social insect queens highlight that breeders need not always suffer more rapid somatic deterioration than their nonbreeding subordinates. Here, we used extensive longitudinal assessments of telomere dynamics to investigate the impact of dominance status on within-individual age-related changes in somatic integrity in a wild social bird, the white-browed sparrow-weaver (Plocepasser mahali). Dominant birds, who monopolise reproduction, had neither shorter telomeres nor faster telomere attrition rates over the long-term (1-5 years) than their subordinates. However, over shorter (half-year) time intervals dominants with shorter telomeres showed lower rates of telomere attrition (and evidence suggestive of telomere lengthening), while the same was not true among subordinates. Dominants may therefore invest more heavily in telomere length regulation (and/or somatic maintenance more broadly); a strategy that could mitigate the long-term costs of reproductive effort, leaving their long-term telomere dynamics comparable to those of subordinates. Consistent with the expectation that reproduction entails short-term costs to somatic integrity, telomere attrition rates were most severe for all birds during the breeding seasons of wetter years (rainfall is the key driver of reproductive activity in this arid-zone species). Our findings suggest that, even in vertebrate societies in which dominants monopolise reproduction, dominants may experience long-term somatic integrity trajectories indistinguishable from those of their nonreproductive subordinates.

Peer reviewed: True, We present a method for rapid calculation of coronavirus growth rates and [Formula: see text]-numbers tailored to publicly available UK data. We assume that the case data comprise a smooth, underlying trend which is differentiable, plus systematic errors and a non-differentiable noise term, and use bespoke data processing to remove systematic errors and noise. The approach is designed to prioritize up-to-date estimates. Our method is validated against published consensus [Formula: see text]-numbers from the UK government and is shown to produce comparable results two weeks earlier. The case-driven approach is combined with weight-shift-scale methods to monitor trends in the epidemic and for medium-term predictions. Using case-fatality ratios, we create a narrative for trends in the UK epidemic: increased infectiousness of the B1.117 (Alpha) variant, and the effectiveness of vaccination in reducing severity of infection. For longer-term future scenarios, we base future [Formula: see text] on insight from localized spread models, which show [Formula: see text] going asymptotically to 1 after a transient, regardless of how large the [Formula: see text] transient is. This accords with short-lived peaks observed in case data. These cannot be explained by a well-mixed model and are suggestive of spread on a localized network. This article is part of the theme issue 'Technical challenges of modelling real-life epidemics and examples of overcoming these'.

Peer reviewed: True, Funder: Diabetes UK RDLF, Funder: BBSRC iCASE PhD studentship partnered with AstraZeneca, Substantial evidence suggests crosstalk between reproductive and gut-axis but mechanisms linking metabolism and reproduction are still unclear. The present study evaluated the possible role of glucose-dependent-insulinotropic-polypeptide (GIP) and glucagon-like-peptide-1 (GLP-1) in reproductive function by examining receptor distribution and the effects of global GIPR and GLP-1R deletion on estrous cycling and reproductive outcomes in mice. GIPR and GLP-1R gene expression were readily detected by PCR in female reproductive tissues including pituitary, ovaries and uterine horn. Protein expression was confirmed with histological visualisation of incretin receptors using GIPR-Cre and GLP1R-Cre mice in which the incretin receptor expressing cells were fluorescently tagged. Functional studies revealed that female GIPR-/- and GLP-1R-/- null mice exhibited significantly (p < 0.05 and p < 0.01) deranged estrous cycling compared to wild-type controls, indicative of reduced fertility. Furthermore, only 50% and 16% of female GIPR-/- and GLP-1R-/- mice, respectively produced litters with wild-type males across three breeding cycles. Consistent with a physiological role of incretin receptors in pregnancy outcome, litter size was significantly (p < 0.001-p < 0.05) decreased in GIPR-/- and GLP-1R-/- mice. Treatment with oral metformin (300 mg/kg body-weight), an agent used clinically for treatment of PCOS, for a further two breeding periods showed no amelioration of pregnancy outcome except that litter size in the GIPR-/- group was approximately 2 times greater in the second breeding cycle. These data highlight the significance of incretin receptors in modulation of female reproductive function which may provide future targets for pharmacological intervention in reproductive disorders.

Consumption of areca nut alone, or in the form of betel quid (BQ), has negative health effects and is carcinogenic to humans. Indonesia is one of the largest producers of areca nuts worldwide, yet little is known about the biomolecular composition of Indonesian areca nuts and BQs. We have recently shown that phenolic and alkaloid content of Indonesian BQs exhibits distinct geographical differences. Here, we profiled for the first time the metabolomics of BQ constituents from four regions of Indonesia using non-targeted gas chromatography–mass spectrometry (GC–MS) analysis. In addition to well-known alkaloids, the analysis of small-molecule profiles tentatively identified 92 phytochemicals in BQ. These included mainly benzenoids and terpenes, as well as acids, aldehydes, alcohols, and esters. Safrole, a potentially genotoxic benzenoid, was found abundantly in betel (Piper betle) inflorescence from West Papua and was not detected in areca nut samples from any Indonesian region except West Papua. Terpenes were mostly detected in betel leaves and inflorescence/stem. Areca nut, husk, betel leaf, the inflorescence stem, and BQ mixture expressed distinctive metabolite patterns, and a significant variation in the content and concentration of metabolites was found across different geographical regions. In summary, this was the first metabolomic study of BQs using GC–MS. The results demonstrate that the molecular constituents of BQs vary geographically and suggest that the differential disease-inducing capacity of BQs may reflect their distinct chemical composition.

Funder: University of Zurich; Id: http://dx.doi.org/10.13039/501100006447, Funder: MAVA Foundation, Funder: Mammal Research Institute at the University of Pretoria, The ability of dispersing individuals to adjust their behaviour to changing conditions is instrumental in overcoming challenges and reducing dispersal costs, consequently increasing overall dispersal success. Understanding how dispersers' behaviour and physiology change during the dispersal process, and how they differ from resident individuals, can shed light on the mechanisms by which dispersers increase survival and maximise reproduction. By analysing individual behaviour and concentrations of faecal glucocorticoid metabolites (fGCM), a stress-associated biomarker, we sought to identify the proximate causes behind differences in survival and reproduction between dispersing and resident meerkats Suricata suricatta. We used data collected on 67 dispersing and 108 resident females to investigate (a) which individual, social and environmental factors are correlated to foraging and vigilance, and whether the role of such factors differs among dispersal phases, and between dispersers and residents; (b) how time allocated to either foraging or vigilance correlated to survival in dispersers and residents and (c) the link between aggression and change in fGCM concentration, and their relationship with reproductive rates in dispersing groups and resident groups with either long-established or newly established dominant females. Time allocated to foraging increased across dispersal phases, whereas time allocated to vigilance decreased. Time allocated to foraging and vigilance correlated positively and negatively, respectively, with dispersers' group size. We did not find a group size effect for residents. High proportions of time allocated to foraging correlated with high survival, and more so in dispersers, suggesting that maintaining good physical condition may reduce mortality during dispersal. Furthermore, while subordinate individuals rarely reproduced in resident groups, the conception rate of subordinates in newly formed dispersing groups was equal to that of their dominant individuals. Mirroring conception rates, in resident groups, fGCM concentrations were lower in subordinates than in dominants, whereas in disperser groups, fGCM concentrations did not differ between subordinates and dominants. Our results, which highlight the relationship between behavioural and physiological factors and demographic rates, provide insights into some of the mechanisms that individuals of a cooperative species can use to increase overall dispersal success.

Peasant women in Cajamarca, Peru, who were sterilized by the Peruvian government in the 1990s, narrate their experiences of reproductive abuse using Andean medical principles of debilidad and fuerza (debility and strength) (Tapias 2006). In their narratives, many describe a generalized sense of loss of strength resulting from the procedure. This contrasts with the reproductive rights framework's emphasis on infertility as the main harm. In this article, I ponder the dissonance between these two frameworks and propose the concept of debilitated lifeworlds as decolonial feminist delinking (Mignolo 2007) from human fertility-centric narratives. This concept is methodologically significant as a decolonial attunement to local motifs to talk about abuse and for weaving a constellation of embodied, emotional, social, and family harms. This article contributes to the emerging field of "decolonial reproductive studies" (Smietana et al. 2018: 117).

This is the protocol of the systematic review. The structure of the protocol is based on the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 ADMINISTRATIVE INFORMATION 1. Tittle 1.a. Identification: Analyses of published methods for the isolation and characterization of salivary extracellular vesicles from the MISEV 2018 perspective. Protocol for a systematic review. 1.b. Update: First SR on the subject. 2. Registration: The protocol of this systematic review will be registered on Open Science Framework (www.osf.io9). 3. Authors 3.a. Contact · Felipe Martí (FM) · Universidad de Los Andes, Santiago, Chile · Email address: fmartic@gmail.com · Cristián Rojas (CR) · Universidad de Los Andes, Santiago, Chile · Email address: cristianrojas21@gmail.com · José Tomás Rivera (JTR) · Universidad de Los Andes, Santiago, Chile · Email address: jtrivera@miuandes.cl · Fernanda Jadue (FJ) · Universidad de Los Andes, Santiago, Chile · Email address: fernandajadue@gmail.com · Mailing address: El Peumo 271-20, Lo Barnechea, Santiago, Chile. 3.b. Contributions: FJ is the guarantor. FJ developed the selection criteria and data extraction criteria. All authors contributed to the development of the risk of bias assessment strategy. All authors read, provided feedback and approved the final manuscript. 4. Amendments: In the event of protocol amendments, the date of each amendment will be accompanied by a description of the change and rationale and uploaded to Open Science Framework. 5. Support: This systematic review does not receive funding. INTRODUCTION 6. Rationale There is consensus among the scientific community about a lack of standardization in the development and reporting of extracellular vesicle (EV) research. In general, each group of investigators uses its own method to collect, pre-process and store biofluid samples. And later on, uses its own method to isolate the EVs and characterize them. This has turned into a problem, because generally the methods used are not reported with enough detail, making studies incomparable and impossible to be replicated by other investigators. Besides this, a lack of rigor when characterizing the obtained EVs has been observed. The characterization procedures done by investigators do not cover all of the items that have to be evaluated to assure the obtained EV preparation is free of contaminants (non-EV components), affecting the validity of the conclusions reported by the authors. To address this problem, on 2018 the International Society for Extracellular Vesicles (ISEV) published the Minimal Information for Studies of Extracellular Vesicles (MISEV 2018); a list of minimal requirements studies of extracellular vesicles have to comply with and report to be considered reproducible and that the reported conclusions are valid. Even though this requirements were updated recently (there had previously been a MISEV 2014) and one could think it is not fair to judge previous publications under this new parameters, all points stated are of great relevance, and not complying with them may mean that the study conclusions are not valid. For example, if a publication claims they found an oral cancer biomarker on salivary EVs, but they didn’t assess the purity of the EV preparation they obtained from saliva, it could be that the cancer biomarker they found was actually bound to a compound contaminating the EV preparation (e.g., a lipoprotein) and not really associated with salivary EVs. For this reason, this systematic review aims to analyze publications were they have isolated salivary EVs, from the MISEV 2018 perspective, to know if research in this field complies with the defined standards, and thus if studies are reproducible and the conclusions they have reached are valid. This analysis will establish the strengths of the investigations published until know, so that they can be used by investigators interested in this field. It will also identify weaknesses; which will be a starting point for new research questions that will strengthen this area of knowledge. 7. Objectives Scientific question: ¿What is the degree of compliance that the published methods for the isolation and characterization of salivary extracellular vesicles have with the MISEV 2018 requirements? General objective: To describe the degree of compliance that the published methods for the isolation and characterization of salivary extracellular vesicles have with the MISEV 2018 requirements METHODS 8. Elegibility criteria We will include experimental studies were human salivary EVs have been obtained. Studies in animal saliva, other oral fluids and revisions were no experimental procedures have been done will be excluded. We will consider studies published in English and Spanish until August 20, 2020, with no past time limit. Since one of the purposes of the systematic review is to assess the quality of reporting of the experiments, we will only consider published studies. 9. Information sources Literature search strategies will be developed using medical subject headings (MeSH), Emtree terms and text words related to salivary EVs. We will search MEDLINE (PubMed interface), EMBASE, BIREME and EV-TRACK (knowledgebase created to record experimental parameters of EV-related studies). The literature search will be limited to English and Spanish languages and types of studies that are not reviews. The final search will be done independently by two authors (FJ and FM) on August 20, 2020. Exclusion of studies that don’t fulfill the eligibility criteria will be done by reading the titles, abstracts and/or full texts. 10. Search strategy PubMed search strategy: ((((((("saliva"[MeSH Terms] OR "saliva"[All Fields]) OR "salivas"[All Fields]) OR "saliva s"[All Fields]) OR "salivary"[All Fields]) OR ((("mouth"[MeSH Terms] OR "mouth"[All Fields]) OR "oral"[All Fields]) AND (("fluid"[All Fields] OR "fluid s"[All Fields]) OR "fluids"[All Fields]))) AND (((((((((("exosomal"[All Fields] OR "exosomes"[MeSH Terms]) OR "exosomes"[All Fields]) OR "exosome"[All Fields]) OR "exosomic"[All Fields]) OR (((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "microvesicle"[All Fields]) OR "microvesicles"[All Fields])) OR (((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "microparticle"[All Fields]) OR "microparticles"[All Fields])) OR (((("extracellular vesicles"[MeSH Terms] OR ("extracellular"[All Fields] AND "vesicles"[All Fields])) OR "extracellular vesicles"[All Fields]) OR ("extracellular"[All Fields] AND "vesicle"[All Fields])) OR "extracellular vesicle"[All Fields])) OR ((((("cell-derived microparticles"[MeSH Terms] OR ("cell derived"[All Fields] AND "microparticles"[All Fields])) OR "cell derived microparticles"[All Fields]) OR "ectosome"[All Fields]) OR "ectosomes"[All Fields]) OR "ectosomal"[All Fields])) OR "oncosome"[All Fields]) OR (((("extracellular vesicles"[MeSH Terms] OR ("extracellular"[All Fields] AND "vesicles"[All Fields])) OR "extracellular vesicles"[All Fields]) OR ("apoptotic"[All Fields] AND "body"[All Fields])) OR "apoptotic body"[All Fields]))) AND ("english"[Language] OR "spanish"[Language])) NOT "review"[All Fields] EMBASE search strategy: (('saliva' OR 'saliva'/exp OR saliva OR salivary OR 'oral fluid'/exp OR 'oral fluid') AND ('exosome' OR 'exosome'/exp OR exosome OR 'membrane microparticle'/exp OR 'membrane microparticle' OR 'platelet microparticle'/exp OR 'platelet microparticle' OR 'endothelial microparticle'/exp OR 'endothelial microparticle' OR 'microparticle' OR 'microparticle'/exp OR microparticle OR 'microparticles' OR 'microparticles'/exp OR microparticles OR 'ectosome' OR 'ectosome'/exp OR ectosome OR 'oncosome' OR 'oncosome'/exp OR oncosome OR 'apoptotic body'/exp OR 'apoptotic body') AND ([english]/lim OR [spanish]/lim) AND [embase]/lim) AND ('article'/it OR 'article in press'/it OR 'chapter'/it OR 'conference abstract'/it OR 'conference paper'/it OR 'editorial'/it OR 'letter'/it OR 'note'/it OR 'short survey'/it) BIREME search strategy: tw:((tw:(saliva OR "fluido oral")) AND ((tw:(exosoma)) OR (tw:(exosomas)) OR (tw:("vesícula extracelular")) OR (tw:("vesículas extracelulares")) OR (tw:("partícula extracelular")) OR (tw:("partículas extracelulares")) OR (tw:(microvesícula)) OR (tw:(microvesículas)) OR (tw:(micropartícula)) OR (tw:(micropartículas)) OR (tw:("cuerpo apoptótico")) OR (tw:("cuerpos apoptóticos")) OR (tw:(oncosoma)) OR (tw:(oncosomas)) OR (tw:(ectosoma)) OR (tw:(ectosomas)))) AND ( la:("en")) EV-TRACK search strategy: Biofluid: saliva 11. Study records 11.a. Data management: Literature search results will be entered by two authors independently (FJ and FM) in an excel spreadsheet previously designed. One sheet will contain included studies, while another sheet will contain excluded studies with reason for exclusion. Full texts (pdf. files) will be uploaded to a shared file on Zotero reference management software. Because one of the aims of the systematic review is to asses the quality of reporting, experiments that are published more than once (for example in two different journals) will be analyzed individually, because the details reported in each publication could be different. 11.b. Selection process: Two authors (FJ and FM) will independently conduct the search and screen titles and abstracts for the evaluation of the eligibility criteria. If there is any uncertainty, full texts will be read. Any disagreement between the authors will be solved by discussion. If there is no agreement, a third author (JTR) will decide. 11.c. Data collection process: An excel spreadsheet containing all the MISEV 2018 requirements will be created for data extraction. Extraction of the data from the articles will be done in duplicate by two authors independently (FJ y CR). Results of both authors will be compared and any disagreement will be solved by discussion. If there is no agreement, a third author (JTR) will decide. It is possible that some studies work with EVs from other body fluids, tissues or conditioned media, besides saliva. In that case only data concerning saliva will be extracted. We will consider the information published in the articles and supplementary material the authors may cite. In cases were a methodology is not described in detail, but reference is made to it being cited in a previous publication, we will consider the information obtained from the cited publication. If there is missing information for studies with data uploaded on EV-TRACK, the information will be searched there. If there is any difference between the information published in the article and the data entered on EV-TRACK, we will consider the information contained in the article. In case of information not entered on EV-TRACK and articles that are not uploaded on EV-TRACK, we will not contact the authors for missing information, because quality of reporting is one of the items being analyzed. We will only contact authors to clarify information that generated discrepancies between the two authors analyzing the data and that could not be resolved by the third author (JTR). In case we are not able to contact the authors of the article, we will consider the data is missing, because it will be assumed that in the case we wanted to replicate the experiment, not being able to contact the author would have prevented us form doing it. 12. Data items The data that will be extracted corresponds to the items contained in the 6 domains that the MISEV 2018 encompasses: nomenclature, collection and pre-processing, EV separation and concentration, EV characterization, functional studies and reporting. Specific items that will be extracted for each domain are the following and can be classified as mandatory (***), mandatory if applicable (**) and encouraged (*): Nomenclature • Uses the generic term “extracellular vesicle” with demonstration of extracellular (no intact cells) and vesicular (complies with characterization requirements) nature. Or uses the term “extracellular particle” if the extracellular origin can be demonstrated, but the characterization to demonstrate it’s vesicular nature is not satisfied.*** • Complements the name “extracellular vesicle” with some specification about size, density or other parameter of the studied vesciles.* • Specifies the subcellular origin (“microvesicle” from plasma membrane, “exosome” from endosome) demonstrating the subcellular origin.* Collection and pre-processing • Specifies donor status if available (sex, gender, previous consumption of food/water, medication, time of collection, etc.)** • Quantifies the volume of biofluid sample collected by donor.*** • If individual donor biofluid was pooled, specifies the total volume of biofluid used for EV isolation.** • Reports all known collection conditions (time, collection method, if samples were kept on ice, etc.), including additives al time of collection.*** • Specifies pre-treatment to separate major fluid-specific contaminants before EV isolation.*** • Reports temperature and time of biofluid handling before and during pre-treatment.*** • Reports storage and recovery procedures (e.g., thawing) of biofluid before EV isolation (storage temperature, vessel, time; method of thawing or other simple preparation).*** • Reports storage and recovery of EVs after isolation (Temperature, vessel, time, additives, etc.).*** EV separation and concentration • Specifies experimental details of the method used.*** · Centrifugation: reference number of tube(s), rotor(s), adjusted k-factor(s) of each centrifugation step, temperature, freak settings. · Density gradient: nature of matrix, method of generating gradient, reference (and size) of tubes, bottom-up (simple at bottom, high density) or top-bottom (simple on top, low density), centrifugation speed and time (with brake specified), method and volume of fraction recovery. · Chromatography: matrix (nature, pore size), loaded simple volume, fraction volume and number of fractions. · Precipitation: reference of polymer, ratio vol/vol or weight/vol polymer/fluid, time and temperature of incubation, centrifugation time, speed and temperature. · Filtration: reference of filter type (nature of membrane, pore size), time and speed of centrifugation, initial and final volume (in case of concentration). · Antibody-based: reference of antibodies, mass of antibodies per amount of EVs, nature of antibody carrier (bead, surface, etc.), and amount of antibodies per carrier surface. · Other necessary details to allow replication. · Additional steps to concentrate, if any. · Additional steps to wash matrix and/or simple, if any. • Reports category of the chosen EV separation/ concentration method.* · High recovery, low specificity: mixed EVs and non-EV components. · Intermediate recovery, intermediate specificity: mixed EVs with limited non-EV components. · Low recovery, high specificity: subtype(s) of EVs with as little non-EV as possible. · High recovery, high specificity: subtype(s) of EVs with as little non-EV as possible. EV Characterization • Quantification · Reports the volume of biofluid used to isolate EVs.*** · Performs global quantification by at least 2 methods: total protein amount, particle number, lipid amount, expressed per volume of initial fluid.*** · Reports the ratio of the 2 quantification figures.*** • Global characterization · Evaluates the presence of at least one transmembrane or GP-anchored protein localized at plasma membrane or endosomes.*** · Evaluates the presence of at least one cytosolic protein with membrane-binding or association capacity.*** · Evaluates the presence/ absence of at least on expected contaminant.*** · If the study investigates specifically small EVs (less than 200nm), it evaluates the presence of proteins associated with other cellular compartments (different from the plasma membrane and endosomes).** · If the study identifies a functional soluble protein on EVs, in addition to identifying that component, it evaluates it’s possible transmembrane ligands (receptors on EVs).** · Evaluates the topology of the relevant functional components.* • Individual characterization of EVs · Reports images of single EVs by wide-field and close-up (e.g., electron microscopy, scanning probe microscopy, super-resolution fluorescence microscopy, etc.).*** · Uses a non-image-based method that analyses large numbers of single EVs (e.g., NTA, high-resolution flow cytometry, Raman spectroscopy, etc.).*** Functional studies • Performs a dose-response assessment.*** • Performs a negative control: Evaluates function in the biofluid of control donors, as applicable.*** • Performs a quantitative comparison of the functional activity of total biofluid, versus EV-depleted biofluid, versus EVs (after high recovery/low specificity separation) (always considering that contaminants could have been co-isolated).*** • Performs a quantitative comparison of the functional activity of EVs, versus other extracellular particles or fractions (contaminants) (after low recovery/high specificity separation).*** • If EV subtype-specific function is being claimed, they perform the quantitative comparison of the functional activity of the different subtypes of EVs.* • Demonstrates that the functional activity exists in the absence of contact between EV donor and EV recipient cells.* Reporting • Submits methodological details to EV-TRACK (evtrack.org) with EV-TRACK number provided.* • Submits data (proteomic, sequencing, other) to relevant public, curated databases or open-access repositories.*** • Submits data to EV specific databases (e.g., EVpedia, Vesiclepedia, exRNA atlas).* • Tempers EV-specific claims when MISEV requirements cannot be entirely satisfied.** 13. Outcomes y prioritization All outcomes will be assessed by two independent authors (FJ y CR). Any disagreement will be solved by discussion. If there is no agreement, a third author (JTR) will decide. Primary outcome The primary outcome will be the degree of compliance of the studies with the MISEV 2018 requirements. All mandatory (*** and **) items of each domain will be evaluated and classified as “accomplished” or “not accomplished”. When an item is classified as “not accomplished” the author will specify the reason for that classification. Depending on how many items are classified al “accomplished”, each of the 6 domains will be classified as: • Accomplished: All of the items of the domain are accomplished. • Partially accomplished: Some of the items of the domain are accomplished. • Not accomplished: None of the items of the domain are accomplished. To represent the information in a graphic form and thus make visualization of the results easier, a color code will be used: • Accomplished: Green • Partially accomplished: Yellow • Not accomplished: Red Results will be presented for each study and also for the proportion of studies classified in each category (accomplished, partially accomplished, not accomplished) for each of the 6 MISEV 2018 domains. Secondary outcomes Secondary outcomes will be evaluated with the intention of knowing more in detail the characteristics of publications that have studied salivary EVs (types of EVs studied, types of saliva samples that have been used, methods of EV isolation and characterization, etc.). The outcomes that will be evaluated are: • Subtypes of EVs that have been studied: · Exosomes · Microvesicles · Microparticles · Small EVs · Large EVs · Ectosomes · Oncosomes · Apoptotic bodies • Types of saliva that have been studied · Unstimulated whole saliva · Stimulated whole saliva · Unstimulated saliva from a specific salivary gland · Stimulated saliva from a specific salivary gland. • Pre-treatments used to lower the viscosity of saliva prior to EV isolation · Centrifugation · Sonication · Filtration · Dilution · Combination of methods • EV separation and concentration methods that have been used · Differential ultracentrifugation · Density gradient centrifugation · Filtration · Size exclusion chromatography · Precipitation · Immunoaffinity capture · Combination of methods • EV global quantification methods that have been used · Total protein concentration · Particle concentration · Lipid concentration • Protein markers that have been used to characterize salivary EVs. • Contaminants that have been analyzed on salivary EV preparations. • Single EV characterization methods that have been used · Transmission electron microscopy · Scanning electron microscopy · Super-resolution fluorescence microscopy · Nanoparticle tracking analyses (NTA) · High-resolution flow cytometry · Raman spectroscopy 14. Risk of bias in individual studies MISEV 2018 requirements seek that conclusions reported by studies on EVs are supported by the experimental procedures that were done (are valid) and that the reported experimental parameters allow other investigators to replicate the experiments. Because of this, as more MISEV 2018 requirements are met, it is considered that the study has less risk of bias. Thus, it can be argued that the main outcome of this systematic review is assessing the risk of bias of the published studies. For this reason, a separated risk of bias assessment will not be done. The primary outcome results will be extrapolated into a risk of bias classification for each study as follows: • Low risk of bias: All MISEV 2018 domains classified as accomplished (green). • Unclear risk of bias: 1 or more MISEV 2018 domains classified as partially accomplished (yellow), with no domains classified as not accomplished (red). • High risk of bias: 1 or more MISEV 2018 domains not accomplished (red). Results will be presented for each study and globally; presenting the proportion of studies that is classified in each risk of bias category. A graphic representation will be made. 15. Data synthesis 15.a. No quantitative synthesis will be done. 15.b. Doesn’t apply. 15.c. Doesn’t apply. 15.d. A narrative qualitative synthesis will be provided, were results will be presented starting with the primary outcome and continuing with the secondary outcomes in the order they appear in this protocol. The primary outcome will be presented in narrative form and graphically, as explained above. The secondary outcomes will be presented in tables as general results and as a narrative synthesis for a more detailed analysis. We will report results for all included studies irrespective of their risk of bias classification. 16. Meta-bias(es): Doesn’t apply. 17. Confidence in cumulative evidence: Doesn’t apply, because the aim of the systematic review is to evaluate the quality of the studies that have been published on this subject.

Despite recent advances in psychiatric treatment, addictive disorders remain a major challenge to healthcare systems, causing significant morbidity and mortality. With improved understanding of the pathophysiology of addiction and innovative neuromodulatory technologies, deep brain stimulation (DBS) research have shown encouraging early results in the treatment of refractory addictive disorders. Here we will systematically review preclinical DBS studies and discuss its translational potential to clinical practice.

Since the term artificial intelligence (AI) was first coined in 1956 by McCarthy and Minsky, its wide-reaching applications to medicine and research has grown in recent years. Developments in artificial intelligence in ophthalmology have utilized machine learning algorithms, whereby computers are manually ‘taught’ to diagnose conditions via feeding of massive amounts of information into computers under specialist supervision. To date, several studies on the use of artificial intelligence in ophthalmology has utilized deep learning technology discussed above, which allows for unsupervised programming and training of computer algorithms to make diagnosis of common eye diseases including diabetic retinopathy (DR), macular degeneration, retinopathy of prematurity (ROP), and glaucoma. Much of this technology is focused on using artificial intelligence to remotely image, triage, and diagnose disease before specialist administered treatment. AI use in diabetic retinopathy diagnosis has specifically seen much development in recent years, with a recent study of DL software developed for diagnosis of DR demonstrating a sensitivity of 96.8% and a specificity of 87.0%. A bibliometric analysis is a statistical analysis of a large set of research pertaining to a chosen topic. This evaluation can be performed from a number of standpoints which will be later discussed in the methods section and allows accurate measurement on the impact of a body of research on a scientific community. Within ophthalmology, bibliometric analyses have been conducted on the general body of ophthalmologic literature and some subspecialties such as glaucoma (4). Currently, there is no existing bibliometric analysis on the topic of artificial intelligence in ophthalmology. A better understanding of the existing literature on AI will provide insight into the growing influence and importance of artificial intelligence on the field of ophthalmology. This will allow the medical community to adapt to and anticipate new technology as it comes, so it may be better integrated into the future model of patient care.

Statins are the most widely used lipid-lowering drugs across the globe for the treatment of hyperlipidemia. The aims of this scoping review are to: (i) identify the genetic variants associated with statins treatment responses among Asian populations, and (ii) explore the effects of these genetic variants on drug efficacy and susceptibility to statin-induced adverse drug reactions (ADR).

Manuscript review about the current knowledge in the pharmacogenomics of myofascial pain syndrome. This will serve as an initial framework in conducting future studies.

Topical ozonated virgin coconut oil improves wound healing and increases HSP90α, VEGF-A, EGF, bFGF and CD34 in diabetic ulcer mouse model of wound healing

Wearable and mobile health applications are postulated to be promising remote monitoring approach in patients with heart failure. However, till today it remains unclear if monitoring of patients with heart failure could be sufficiently enabled by non-invasive wearables. We aime to review all currently available trials for wearable-based remote monitoring of heart failure patient's including their applied methods, sensors, sensor locations, analysis algorithms and results.

Osteoporosis is the most prevalent bone disease and represents a major public health burden. Resulting from a reduction in bone quality and quantity, it can lead to fragility fractures with significant associated morbidity and mortality. Mechanical loading is a potent, albeit incompletely understood, anabolic stimulus that could lead to novel therapies for osteoporosis. With weight bearing, mechanosensing osteocytes will transduce this physical stimulus into trophic factors that will facilitate the strengthening of bone at the site of loading. It is understood that this process will involve the recruitment, proliferation, and differentiation of progenitors thereby enhancing bone formation. The overall goal of this thesis was to investigate the application of biophysical forces that are experienced by primary cells in vivo and apply them to cellular cultures in vitro with the objective of encouraging more physiological responses. Given the critical role of mechanical loading and fluid flow stimulation in bone, the first aim of the thesis was to apply established fluid flow stimulation techniques to mechanosensitive osteocytes and osteoblasts in order to observe the secretion of bioactive soluble factors that could, in turn, modulate the effects of osteoblasts and stem cells. Significantly, paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and also significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors (Chapter 3). One such important soluble factor regulating bone anabolism is sclerostin, produced by osteocytes. Osteocyte physiology has traditionally been challenging to investigate due to its uniquely mineralized extracellular matrix (ECM) leading to the development of cell lines. Importantly, the most widely researched osteocyte cell line: MLO-Y4, does not express sclerostin (Sost) in typical in vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact Sost expression. This study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells when cultured on a hydroxyapatite (HA) containing two-dimensional (2D) film analogues expressed Sost. Three-dimensional (3D) culture upon HA-containing scaffolds significantly further increased Sost expression. Importantly, Sost expression in MLO-Y4 cells cultured on a 3D collagen-HA scaffold was found to be comparable to physiological levels. Moreover, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in Sost expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, and therefore overcoming a main limitation of this model (Chapter 4). With mechanical loading it is expected that stem cells would be recruited to a site that they would be required, thereby experiencing a change in their microenvironment from a soft marrow to an organic osteoid with stiff mineralising components which may in turn influence their osteogenic capacity. We therefore investigated the effect of ECM analogues, of different compositions and dimensions, in directing stem cell osteogenic lineage commitment in the absence of exogenous osteoinductive factors. A significant finding was that stem cells cultured upon a collagen HA 3D scaffold demonstrated osteogenesis with the absence of exogenous growth factors (Chapter 5). It is hoped that a greater understanding of the processes occurring in mechanical loading-induced bone formation can then be used to help us understand the pathology underpinning bone diseases such as osteoporosis and develop better models for the investigation of bone biology in future.

There is a strong consensus that selection for fungicide resistant pathogen strains can be most effectively limited by using applications of mixtures of fungicides designed to balance disease control against selection. However, how to do this in practice is not entirely characterized. Previous work indicates optimal mixtures of pairs of fungicides which are both at a high risk of resistance can be constructed using pairs of doses that select equally for both single resistant strains in the first year of application. What has not been addressed thus far is the important real-world case in which the initial levels of resistance to each fungicide differ, for example because the chemicals have been available for different lengths of time. We show how recommendations based on equal selection in the first year can be suboptimal in this case. We introduce a simple alternative approach, based on equalizing the frequencies of single resistant strains in the year that achieving acceptable levels of control is predicted to become impossible. We show that this strategy is robust to changes in parameters controlling pathogen epidemiology and fungicide efficacy. We develop our recommendation using a preexisting, parameterized model of Zymoseptoria tritici (the pathogen causing Septoria leaf blotch on wheat), which exemplifies the range of plant pathogens that predominantly spread clonally, but for which sexual reproduction forms an important component of the life cycle. We show that pathogen sexual reproduction can influence the rate at which fungicide resistance develops but does not qualitatively affect our optimal resistance management recommendation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

This project aims to understand the impact of thermal variation on the behaviour, physiology and fitness of ectothermic animals. Here, we consider thermal variation to include fluctuations in temperature within a daily timescale. We do not consider seasonal thermal variation nor acute changes in temperature. We address our aim by conducting a systematic review and meta-analysis. The main hypotheses to be address by our analyses include: 1. Overall: Living in thermally variable environments will be more costly for ectothermic animals (in terms of behavioural and physiological performance and fitness) compared to ectotherms under constant temperatures. The non-linear relationship between temperature and ectotherm performance means that temperature variation above and below an average temperature will have disproportionate impacts (i.e., the fallacy of the average) on ectotherm performance and fitness (Denny 2017; Martin and Huey, 2008). 2. Magnitude of thermal variation: Reductions in ectotherm performance and fitness are expected to worsen with greater fluctuations around a mean temperature. Larger deviations from a mean temperature means that animals will spend a longer proportion of time above and below ‘optimal’ temperatures, leading to greater performance decrements. 3. Duration of exposure/acclimation/acclimatisation: Behavioural and physiological traits are expected to become less sensitive to thermal variation with longer exposure durations. Physiological acclimation/acclimatisation processes require time to develop, therefore, longer exposure durations allow for more time for acclimatory responses to take hold (Gunderson and Stillman, 2015). 4. Impact of thermal variation across functional traits: Exposure to thermal variation will negative impact behavioural, physiological, and reproductive traits, and lower survival. Behavioural, physiological, and reproductive traits will be impacted more than survival by increases in thermal variation. 5. Terrestrial versus aquatic: Terrestrial ectotherms are hypothesised to have greater capacity to cope with thermal variation than aquatic ectotherms. Terrestrial environments tend to be more thermally variable than aquatic environments because water has a higher heat capacity than land. As such, terrestrial ectotherms may be predisposed to dealing with greater thermal variation than aquatic ectotherms. The alternative hypothesis is that terrestrial ectotherms will be more sensitive to thermal variation compared to aquatic ectotherms because terrestrial animals have lower acclimatory capabilities (Seebacher et al., 2015), which may make terrestrial ectotherms more sensitive to thermal variation. 6. Sensitivity through ontogeny: Sensitivity to thermal variation is expected to change through ontogeny. Adults are hypothesised to be more tolerant of thermal variation compared to embryonic and reproductive adult life stages, which will be more sensitive. The thermal tolerance window of adults is theorised to be wider than that of embryonic and reproductive life stages (Dahlke et al., 2020), which will drive differences in sensitivity to thermal variation across ontogeny. 7. Wild versus Laboratory experiments: Wild-caught animals are expected to be more resilient to thermal variation than animals from captive populations. Maintaining phenotypic plasticity confers physiological costs (DeWitt et al., 1998). Animals maintained under laboratory or captive conditions experience shallower fluctuation in temperature than wild animals, which will make captive-sources animals more sensitive to thermal variation (Morgan et al., 2022).

Respiratory diseases and major airway trauma represent a global healthcare emergency with most treatment options providing symptomatic relief rather than curative treatment. In addition, they are associated with enormous socioeconomic burden. Respiratory tissue engineering commonly focuses on two broad areas: (i) the development of novel in vitro models for drug discovery and (ii) on artificial constructs for tissue repair and regeneration. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of novel inhaled therapies by accurately reflecting in vivo respiratory pharmacological and toxicological responses. On the other hand, effective restoration of extensive tracheal damage arising from cancer, stenosis, infection or congenital abnormalities remains an unmet clinical need in respiratory medicine. In this project, tissue-engineered (TE) collagen hyaluronic acid bilayered (CHyA-B) scaffolds were used to evaluate bacterial and drug induced toxicity and inflammation using Calu-3 bronchial epithelial cells and Wi38 lung fibroblast co-culture under air liquid interface (ALI) conditions as an in vitro model of the upper respiratory tract. After using lipopolysaccharide (LPS) and bleomycin to induce bacterial and drug responses in vitro; differential cell responses were seen between 2D and 3D airway models. Furthermore, the co-culture 3D model provided differential epithelial responses to a number of inhaled nanoparticles compared to 2D models. Based on our understanding of the cell-matrix interactions involved in supporting respiratory epithelial cell growth and differentiation, we sought to harness these CHyA-B scaffolds to develop tubular TE tracheal constructs. A 3Dprinted polymer mesh of thermoplastic polymers with tailored architecture was fabricated and characterised to provide the tubular backbone of the construct. Polyglobalide (PGL), an advanced synthetic polymer that allows for post-processing surface modifications in comparison to commonly used polymers such as polycaprolactone (PCL), was employed as a 3D printable material. This was then covered with CHyA-B scaffolds using a freeze-drying process and Calu-3 epithelial cells were seeded onto its luminal film surface resulting in the formation of a partial epithelial layer. 3D printed PCL- and PGL-CHyA tubular scaffolds are promising biocompatible prototypes for tracheal regeneration with adequate mechanical properties supporting epithelial growth.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an unprecedented challenge to global public health with researchers striving to find a possible therapeutic candidate that could limit the spread of the virus. In this context, the present study employed an in silico molecular interaction-based approach to estimate the inhibitory potential of the phytochemicals from ethnomedicinally relevant Indian plants including Justicia adhatoda, Ocimum sanctum and Swertia chirata, with reported antiviral activities against crucial SARS-CoV-2 proteins. SARS-CoV-2 proteins associated with host attachment and viral replication namely, spike protein, main protease enzyme Mpro and RNA-dependent RNA polymerase (RdRp) are promising druggable targets for COVID-19 therapeutic research. Extensive molecular docking of the phytocompounds at the binding pockets of the viral proteins revealed their promising inhibitory potential. Subsequent assessment of physicochemical features and potential toxicity of the compounds followed by robust molecular dynamics simulations and analysis of MM-PBSA energy scoring function revealed anisotine against SARS-CoV-2 spike and Mpro proteins and amarogentin against SARS-CoV-2 RdRp as potential inhibitors. It was interesting to note that these compounds displayed significantly higher binding energy scores against the respective SARS-CoV-2 proteins compared to the relevant drugs that are currently being targeted against them. Present research findings confer scopes to explore further the potential of these compounds in vitro and in vivo towards deployment as efficient SARS-CoV-2 inhibitors and development of novel effective therapeutics. Communicated by Ramaswamy H. Sarma