Investigating the Relationship Between Cystic Fibrosis Transmembrane Conductance Regulator and Colorectal Cancer Under Endoplasmic Reticulum Stress

University of Minnesota M.S. thesis.April 2018. Major: Integrated Biosciences. Advisors: Patricia Scott, Robert Cormier. 1 computer file (PDF); vii, 64 pages.Our group has identified Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) as a tumor suppressor for colorectal cancer (CRC) (Than et al., 2016). However, the mechanism by which CFTR protects against CRC is unknown. Determining this mechanism may lead to new therapies for CRC. Gene Set Enrichment Analysis (GSEA) pathway analysis showed that CFTR-deficient normal and tumor tissues from mice and humans were enriched in genes involved in the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Altered genes, environmental stress and increased protein synthesis induce UPR activation in cancer cells (Kaufman & Wang, 2014; Ma & Hendershot, 2004; Dejeans, Barroso, Fernandez-Zapico, Samali, Chevet, 2015). The UPR has two potentially opposing effects on cancer development: on the one hand, some cancer cells require a higher level of UPR to support their growth and survival (Corazzari, Gagliardi, Fimia & Piacentini, 2017); on the other hand, increased UPR leads to loss of stemness which could inhibit cancer development (Heijmans et al., 2013). To identify the correlation between UPR and cancer-supporting effects of CFTR-deficiency, a pair of CFTR knockout (KO) and wildtype (WT) Caco2 cell lines created by CRISPR-Cas9 engineering were utilized to test the working model that UPR activation might be enhanced in CFTR KO cells, and this activation might support CRC cell survival under ER stress. To test the working model, the Caco2 cell lines were treated with chemical inducers to trigger ER stress. To evaluate the UPR activities, we used Thapsigargin (TG) to induce ER stress in CFTR KO and WT cell lines and compared iii mRNA expression changes of key genes in the two cell lines by performing RT- qPCR. The heat shock protein A member 5 (HSPA5) gene encodes the protein that initiates all three UPR sub-pathways. Although the expression of HSPA5 increased after TG treatment in both CFTR WT and CFTR KO cell lines, the TG treatment had more of an effect on HSPA5 expression in CFTR KO cells, suggesting a trend that CFTR deficiency might enhance the effect of ER stress on the expression of UPR gatekeeper HSPA5, the common regulator for initiating three UPR sub-pathways. The ATF4 expression levels in both CFTR WT and CFTR KO cell lines increased to the same extent, which reflects that the loss of CFTR might not promote the activation of PERK signaling. The decreased total XBP1 mRNA expression levels in CFTR KO cells suggests that CFTR deficiency might not activate the specific IRE1α-XBP1 signaling pathway. The TXNIP, which is part of an apoptotic pathway, is down- regulated in CFTR KO cells, which might provide protection from apoptosis. These studies suggest that UPR activation might be enhanced in CFTR KO cells, but the specific pathway might not be the one detected. Additionally, the down regulation of TXNIP might offer protection for CRC cells from apoptosis. Although many cancer cells require an enhanced UPR (Corazzari, Gagliardi, Fimia & Piacentini, 2017), excessive, prolonged activation triggers a switch to an apoptotic pathway (Wang & Kaufman, 2016; Niederreiter, L. et al., 2013). To examine this possibility, TG and Tunicamycin (TM) were used to induce ER stress in cells, and cell viability in CFTR KO and WT cell lines over time was compared by conducting MTT assay. Although the results showed decreased cell viability after treatment in both iv CFTR WT and CFTR KO cell lines, the ratios of treated over untreated viability at each point after TG and TM treatment was higher in CFTR KO cells, indicating that the loss of CFTR might cause TG-induced ER stress to have less of an effect on cell viability. These data suggest that the loss of CFTR increases protection from ER stress, and that this protection supports the survival of CRC cells. Based on the fact that CFTR deficiency can activate the NF-κB signaling pathway (Crites et al., 2015), and that UPR can activate the NF-κB (Grootjans, Kaser, Kaufman & Blumberg, 2016), the NF-κB activity in CFTR KO cells in response to UPR was also examined. RT-qPCR analysis was performed to compare the gene expression changes relating to NF-κB signaling pathway in CFTR WT and CFTR KO Caco2 cell lines treated with TG to trigger UPR activation. Although the results showed enhanced expression of IL8 and TNF in both cell lines, the TG treatment had less of an effect on the expression of these genes in CFTR KO cells. This suggests that CFTR deficiency might cause TG to produce less of an effect on the expression of NF-κB signaling target genes IL8 and TNF. These data suggest that the loss of CFTR might cause TG-induced UPR to have less of an effect on NF-κB activation. The enhanced basal expression levels of the NF-κB target gene IL8 in CFTR KO cells might suggest an increased activation of NF-κB signaling in CFTR KO cells and this activation might support the growth of cancer cells. In conclusion, in Caco2 CRC cells, CFTR deficiency may alter the response to UPR in several pathways, which may promote cancer cell survival. Additional experiments v must be conducted to determine which pathways among those that are altered are vital for cancer development

Acupuncture for migraine: a protocol for a meta-analysis and meta-regression of randomised controlled trials

INTRODUCTION Although the effectiveness of acupuncture for episodic migraine has been confirmed by multiple clinical trials and Cochrane systematic reviews, the mechanisms underlying the specific effect of acupuncture for migraine remain controversial. We aim to evaluate the effectiveness and safety of acupuncture for both episodic migraine and chronic migraine by meta-analysis and explore the possible factors influencing the specific effect of acupuncture for migraine by meta-regression. METHODS AND ANALYSIS We will search for randomised control trials of acupuncture for migraine in the following eight databases: Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, AMED (via OVID) and four Chinese databases (Chinese Biomedical Literature Database, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database and Wanfang Database) from inception to 31 December 2017. We will also search OpenSIGLE (opensigle.inist.fr) for conference abstracts. No language restriction will be applied. The selection of studies, data extraction and coding and assessment of risk of bias of the included studies will be conducted independently by two reviewers. Standard meta-analysis and, if appropriate, meta-regression will be performed using the R packages Meta and Metafor. ETHICS AND DISSEMINATION The results of this meta-analysis and meta-regression will be disseminated through publication in a peer-reviewed journal and presented at a relevant conference. The data used in this meta-analysis will not contain individual patient data; therefore, ethical approval is not required. PROSPERO REGISTRATION NUMBER CRD42018087270

Epigenetic stress memory: A new approach to study cold and heat stress responses in plants

Understanding plant stress memory under extreme temperatures such as cold and heat could contribute to plant development. Plants employ different types of stress memories, such as somatic, intergenerational and transgenerational, regulated by epigenetic changes such as DNA and histone modifications and microRNAs (miRNA), playing a key role in gene regulation from early development to maturity. In most cases, cold and heat stresses result in short-term epigenetic modifications that can return to baseline modification levels after stress cessation. Nevertheless, some of the modifications may be stable and passed on as stress memory, potentially allowing them to be inherited across generations, whereas some of the modifications are reactivated during sexual reproduction or embryogenesis. Several stress-related genes are involved in stress memory inheritance by turning on and off transcription profiles and epigenetic changes. Vernalization is the best example of somatic stress memory. Changes in the chromatin structure of the Flowering Locus C (FLC) gene, a MADS-box transcription factor (TF), maintain cold stress memory during mitosis. FLC expression suppresses flowering at high levels during winter; and during vernalization, B3 TFs, cold memory cis-acting element and polycomb repressive complex 1 and 2 (PRC1 and 2) silence FLC activation. In contrast, the repression of SQUAMOSA promoter-binding protein-like (SPL) TF and the activation of Heat Shock TF (HSFA2) are required for heat stress memory. However, it is still unclear how stress memory is inherited by offspring, and the integrated view of the regulatory mechanisms of stress memory and mitotic and meiotic heritable changes in plants is still scarce. Thus, in this review, we focus on the epigenetic regulation of stress memory and discuss the application of new technologies in developing epigenetic modifications to improve stress memory.Peer reviewe

The Measurement of rho‐independent Transcription Terminator Efficiency

The purpose of this RFC is to provide standard methodology for the measurement of the absolute strength of terminators in bacteria. Because we have characterized the performance of terminator in E. coli and used a simple equation model, it can be expressed in PoPS

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A\u2013C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A\u2013C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity

Epigenetic stress memory: A new approach to study cold and heat stress responses in plants

Understanding plant stress memory under extreme temperatures such as cold and heat could contribute to plant development. Plants employ different types of stress memories, such as somatic, intergenerational and transgenerational, regulated by epigenetic changes such as DNA and histone modifications and microRNAs (miRNA), playing a key role in gene regulation from early development to maturity. In most cases, cold and heat stresses result in short-term epigenetic modifications that can return to baseline modification levels after stress cessation. Nevertheless, some of the modifications may be stable and passed on as stress memory, potentially allowing them to be inherited across generations, whereas some of the modifications are reactivated during sexual reproduction or embryogenesis. Several stress-related genes are involved in stress memory inheritance by turning on and off transcription profiles and epigenetic changes. Vernalization is the best example of somatic stress memory. Changes in the chromatin structure of the Flowering Locus C (FLC) gene, a MADS-box transcription factor (TF), maintain cold stress memory during mitosis. FLC expression suppresses flowering at high levels during winter; and during vernalization, B3 TFs, cold memory cis-acting element and polycomb repressive complex 1 and 2 (PRC1 and 2) silence FLC activation. In contrast, the repression of SQUAMOSA promoter-binding protein-like (SPL) TF and the activation of Heat Shock TF (HSFA2) are required for heat stress memory. However, it is still unclear how stress memory is inherited by offspring, and the integrated view of the regulatory mechanisms of stress memory and mitotic and meiotic heritable changes in plants is still scarce. Thus, in this review, we focus on the epigenetic regulation of stress memory and discuss the application of new technologies in developing epigenetic modifications to improve stress memory

An improved contrastive learning network for semi-supervised multi-structure segmentation in echocardiography

Cardiac diseases have high mortality rates and are a significant threat to human health. Echocardiography is a commonly used imaging technique to diagnose cardiac diseases because of its portability, non-invasiveness and low cost. Precise segmentation of basic cardiac structures is crucial for cardiologists to efficiently diagnose cardiac diseases, but this task is challenging due to several reasons, such as: (1) low image contrast, (2) incomplete structures of cardiac, and (3) unclear border between the ventricle and the atrium in some echocardiographic images. In this paper, we applied contrastive learning strategy and proposed a semi-supervised method for echocardiographic images segmentation. This proposed method solved the above challenges effectively and made use of unlabeled data to achieve a great performance, which could help doctors improve the accuracy of CVD diagnosis and screening. We evaluated this method on a public dataset (CAMUS), achieving mean Dice Similarity Coefficient (DSC) of 0.898, 0.911, 0.916 with 1/4, 1/2 and full labeled data on two-chamber (2CH) echocardiography images, and of 0.903, 0.921, 0.928 with 1/4, 1/2 and full labeled data on four-chamber (4CH) echocardiography images. Compared with other existing methods, the proposed method had fewer parameters and better performance. The code and models are available at https://github.com/gpgzy/CL-Cardiac-segmentation

Biodegradable double-network GelMA-ACNM hydrogel microneedles for transdermal drug delivery

As a minimally invasive drug delivery platform, microneedles (MNs) overcome many drawbacks of the conventional transdermal drug delivery systems, therefore are favorable in biomedical applications. Microneedles with a combined burst and sustained release profile and maintained therapeutic molecular bioactivity could further broaden its applications as therapeutics. Here, we developed a double-network microneedles (DN MNs) based on gelatin methacrylate and acellular neural matrix (GelMA-ACNM). ACNM could function as an early drug release matrix, whereas the addition of GelMA facilitates sustained drug release. In particular, the double-network microneedles comprising GelMA-ACNM hydrogel has distinctive biological features in maintaining drug activity to meet the needs of application in treating different diseases. In this study, we prepared the double-network microneedles and evaluated its morphology, mechanical properties, drug release properties and biocompatibility, which shows great potential for delivery of therapeutic molecules that needs different release profiles in transdermal treatment

Nanotechnology in peripheral nerve repair and reconstruction

The recent progress in biomaterials science and development of tubular conduits (TCs) still fails in solving the current challenges in the treatment of peripheral nerve injuries (PNIs), in particular when disease-related and long-gap defects need to be addressed. Nanotechnology-based therapies that seemed unreachable in the past are now being considered for the repair and reconstruction of PNIs, having the power to deliver bioactive molecules in a controlled manner, to tune cellular behavior, and ultimately guide tissue regeneration in an effective manner. It also offers opportunities in the imaging field, with a degree of precision never achieved before, which is useful for diagnosis, surgery and in the patientâ s follow-up. Nanotechnology approaches applied in PNI regeneration and theranostics, emphasizing the ones that are moving from the lab bench to the clinics, are herein overviewed.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the financial support provided to Joaquim M. Oliveira (IF/01285/2015) and Joana Silva-Correia (IF/00115/2015) under the program “Investigador FCT”.info:eu-repo/semantics/publishedVersio

Implicating genes, pleiotropy, and sexual dimorphism at blood lipid loci through multi-ancestry meta-analysis

Publisher Copyright: © 2022, The Author(s).Background: Genetic variants within nearly 1000 loci are known to contribute to modulation of blood lipid levels. However, the biological pathways underlying these associations are frequently unknown, limiting understanding of these findings and hindering downstream translational efforts such as drug target discovery. Results: To expand our understanding of the underlying biological pathways and mechanisms controlling blood lipid levels, we leverage a large multi-ancestry meta-analysis (N = 1,654,960) of blood lipids to prioritize putative causal genes for 2286 lipid associations using six gene prediction approaches. Using phenome-wide association (PheWAS) scans, we identify relationships of genetically predicted lipid levels to other diseases and conditions. We confirm known pleiotropic associations with cardiovascular phenotypes and determine novel associations, notably with cholelithiasis risk. We perform sex-stratified GWAS meta-analysis of lipid levels and show that 3–5% of autosomal lipid-associated loci demonstrate sex-biased effects. Finally, we report 21 novel lipid loci identified on the X chromosome. Many of the sex-biased autosomal and X chromosome lipid loci show pleiotropic associations with sex hormones, emphasizing the role of hormone regulation in lipid metabolism. Conclusions: Taken together, our findings provide insights into the biological mechanisms through which associated variants lead to altered lipid levels and potentially cardiovascular disease risk.Peer reviewe