bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Novel Candidate to Promote Hepatic Maturation in HepG2 Cells In Vitro
Authors: De Vos Kristof , Annaert Pieter
Published: May 2022

Drug‐induced cholestasis (DIC) is characterized by impaired bile flow caused by exogenous compounds and remains a major culprit of attrition of drug candidates, as illustrated by liver toxicity representing 40% of all clinical trial terminations. Strategies for earlier (preclinical) detection of DIC‐inducing drug candidates has thus been an area of extensive research. Nevertheless, the currently available in vitro models for DIC suffer from major knowledge gaps in terms of underlying mechanisms. The crux is the inability to fully mature hepatic phenotypes. Previous studies show a different transcriptome, proteome and epigenetic profile between iPSC‐derived hepatocyte‐like cells (HLCs) and primary human hepatocytes (PHHs), with low levels of hepatic transcription factors (TFs) and biotransformation CYP450 enzymes in HLCs.

A recent mechanistic study revealed that maintenance of mature phenotypes of PHHs and their (glucose) metabolism in vitro, at least partially, depends on blocking epithelial‐mesenchymal transition (EMT) and subsequently identified TGF‐β, Wnt, BMP, Notch, and cAMP signaling as master regulators of this process. However, 5 different chemical compounds, each corresponding to a specific pathway, had to be administered into the culture medium for sufficient EMT inhibition. Building further on the previous knowledge that AKT/mTORC1 signaling is a strong inducer of gluconeogenesis and CYP450 activity, we hypothesized that an upstream pathway and/or common protein(s) could simultaneously activate AKT/mTORC1 and inhibit EMT. Not only would it lead to more mature hepatic phenotypes (and thus better models) in vitro, but it would also broaden the mechanistic understanding underlying hepatic maturation.

Preliminary data suggests that this goal may be accomplished. After targeted inhibition with a small molecule in HepG2 cells, expression patterns were consistent with (i) an upregulation of AKT/mTORC1 as well as estrogen biosynthesis (indicative for strong CYP450 activation), and (ii) a shift in cell metabolism from glucose fueling to β‐oxidation and amino acid consumption with induction of gluconeogenesis (indicative for strong EMT inhibition). Furthermore, the epithelial marker CDH1 (encoding for E‐cadherin) was sharply increased and AKT/cAMP signaling together with typical hepatic TFs that serve as maturation biosensor (HNF4A, FOXO1 and FOXO3) were predicted to be significantly activated. Consistent with this expression data, mitochondrial functionality was confirmed to be improved by quantifying urea production and measuring mitochondrial membrane potentials.

Taken together, gathered preliminary data points towards a novel link between (loss of) the identified target, inhibition of EMT and the enhancement of hepatic maturation TFs as well as CYP450 expression and activation. Generation of knock‐out cells and elaborate bile acid profiling is on‐going and will be applied in further mechanistic studies of DIC.
Formation of Nascent Lipid Droplets at Specialized ER subdomains
Authors: Joshi Amit , Ragusa Joey , Prinz William , Cohen Sarah
Published: May 2022

Lipid droplets (LDs) are neutral lipid containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain–containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain of these proteins is similar to a reticulon homology domain. Like reticulons, MCTPs tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the transmembrane domains (TMD) of MCTP promote LD biogenesis by increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER–LD contact sites. Together, our data indicate that MCTPs form specialized subdomains within ER tubules that regulate LD biogenesis, size, and ER–LD contacts. Interestingly, MCTP punctae are associated with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites.
Identifying Student Misconceptions in Biomolecular Visualization in the Context of the BioMolViz Framework
Authors: Roberts Rebecca , Shor Audrey C. , Jakubowski Henry , Austin Shane , Procko Kristen , Franzen Margaret A.
Published: May 2022

BioMolViz is a community dedicated to improving visual literacy instruction in biology, chemistry, and biochemistry classrooms. To help instructors approach biomolecular visualization instruction, visual literacy concepts are catalogued through the BioMolViz Framework, which presents overarching themes that are subdivided into learning objectives and goals. A main focus of our project is the creation of open‐access assessments associated with each of the Framework learning goals. Through nationwide workshops held over the past four years, faculty teams wrote and revised assessments to probe students’ visual literacy utilizing the BioMolViz Framework. As part of a pre‐workshop activity, we asked faculty to identify student misconceptions in visualization that they observed in their teaching. These were collected, mapped to the framework, and coded for Bloom's taxonomy level. Frequently mentioned misconceptions of broad relevance to biomolecular visualization skills were mapped to the BioMolViz Framework and formatted into a biomolecular visualization literacy misconception survey. We present the process of survey development and invite educators from any higher education institution to contribute to understanding the prevalence of these misconceptions among student populations by participating in our survey. We hope the survey data will allow the community to develop better pedagogical approaches to biomolecular visualization through awareness of student misunderstandings. Our collective contributions can help our students overcome the most common challenges in biomolecular visualization while improving abilities in this foundational skill set.
Teaching Biomolecular Visualization Literacy: Guidelines for Developing Assessments, Images and Rubrics Aligned with the BioMolViz Framework
Authors: Acevedo Roderico , Mertz Pam , Burch Charmita , Austin Shane , Nemmara Venkatesh , Mitton‐Fry Rachel , Hall Bonnie , Fox Kristin , Franzen Margaret A. , Beckham Josh , Procko Kristen
Published: May 2022

Experts in the life sciences glean critical information from biomolecular visualizations. When teaching, we assume our students grasp the concepts in these depictions of biomolecules in the same way that experts do. However, biomolecular visualization (BMV) skills must be intentionally taught and assessed to gauge student understanding. To identify misperceptions and assess comprehension, BioMolViz developed a framework outlining the required skills for biomolecular visualization literacy. Over the past four years, BioMolViz conducted numerous workshops to engage the broader community in creating assessments aligned with the BMV framework, with the goal of developing a repository of validated assessments that can be used by the broader community. Workshops were shifted to an online format during the COVID‐19 pandemic, and a new working group emerged, the BioMolViz Fellows. This subset of workshop participants continued writing and revising assessments remotely, augmenting the group’s peer review and validation process. Our workshops and the ongoing work of the Fellows generated over 200 assessments at various stages in our validation process, with 80 that underwent at least two rounds of revision, and 27 assessments are pending review by an external expert panel. Here, the Fellows present guidelines for generating excellent images using popular visualization software, such as PyMOL, Chimera, and iCn3d. We also provide tips and tricks for designing or reframing questions and rubrics to help instructors maximize student understanding of important biomolecular concepts. Fellows crafted these guidelines using the BMV framework coupled with the lessons learned through workshops.

If you are interested in joining our growing community of professionals dedicated to improving BMV instruction, then we’d be delighted to connect with you.
A Collapsed Fingers Subdomain is the Basis for DNA Polymerase β I260M Mutator Activity
Authors: Chavira Cristian G. , Fijen Carel , Alnajjar Khadijeh , Sweasy Joann
Published: May 2022

DNA Polymerase β fills single nucleotide gaps as a part of the Base Excision Repair pathway (BER); thus, deficiencies in Pol β can lead to increased mutation frequency in the cell, which can result in cancer. Our lab has previously shown that the I260M germline mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide selectivity in a sequence context dependent manner, incorporating G opposite A. To identify the molecular mechanism of the reduced fidelity of I260M, we studied polymerization using single turnover kinetics, and conformational changes using steady state fluorescence and stopped flow FRET. Our data indicate that the I260M mutation affects the fingers region of Pol β by creating a “collapsed” state in both the opened (in the absence of nucleotide) and closed (prior to chemistry) states. Importantly, we show that the incorrect incoming nucleotide binds more tightly to I260M when compared to the wild‐type by a factor of 3.4x. Based on this data, we found that the collapsed fingers subdomain state of I260M may decrease nucleotide discrimination in I260M, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity. A model is being developed to compare the rate of the “fingers closing” conformational change and the reverse reaction of nucleotide release between I260M and the WT.
Glutamine metabolism co‐ordinates the cell‐cycle with cell fate in stem cells
Authors: Kikani Chinan , Xiao Michael
Published: May 2022

Activation of stem cell proliferation is a critical event in tissue regeneration. The metabolic switch in adult stem cells from the oxidative to the glycolytic mode of carbon utilization is essential for rapid proliferative bursts, but its impact on self‐renewal is unclear. During the glycolytic mode of glucose utilization, glutamine‐derived carbons drive the mitochondrial TCA cycle. While glutamine is required for stem cell proliferation burst, its role in maintaining stem cell self‐renewal property remains unclear. Here, we show that withdrawal or chemical inhibition of mitochondrial glutamine metabolism blunted adult muscle stem cell proliferation, but also reactivated the transcription of self‐renewal‐associated transcripts, such as Pax7, to reduce stem cell heterogeneity and build the self‐renewing stem cell population. Thus, surprisingly, glutamine withdrawal preserved and accentuated the self‐renewing stem cell population. This effect of glutamine is mediated via reductive carboxylation of alpha‐ketoglutarate. Mechanistically, we extensively show that glutamine inhibited cell‐cycle linked self‐renewing network during the G2‐M phase of cell‐cycle to drive the exit from self‐renewal during the terminal mitosis phase before differentiation. Thus, we propose that glutamine metabolism plays an unexpected role in building the progenitor population that is uniquely primed for differentiation during tissue regeneration.
Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative
Authors: Chakraborty Manas P. , Bhattacharyya Sudipta , Roy Souryadip , Bhattacharya Indira , Das Rahul , Mukherjee Arindam
Published: May 2022

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID‐19 patients (Treon et al., Blood, 2020). Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases (Ashleigh et al., Oncotarget,2015). Previous report shows that binding of curcumin to active conformation of DYRK2 at sub nanomolar range makes it an excellent DYRK‐2 inhibitor(Banerjee et al., PNAS,2018), but curcumin's fate is tainted by its instability in the cellular environment and its nonspecific binding nature(Kathryn et al., J. Med. Chem.,2017). Besides, the planar symmetry makes curcumin less druggable toward asymmetric binding pockets such as kinases (Knight et al., Chem. Biol. 2005). small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity (Hanson et al., Chembiol., 2019). Here, we show that functionalization of the 4‐arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor which binds specifically to the inactive conformation of Hck, similar to type‐II kinase inhibitors that are less promiscuous.

Moreover, the lead compound showed no inhibitory effect on two other kinases (DYRK2 and Abl) which can be explained by difference in p‐loop dynamics between Abl and src family kinases. We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signalling pathway in triple‐negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodelling its target affinity and cellular stability.
Role of Histone Chaperone APLF in Mammalian Embryo Development
Authors: Varghese Pallavi C. , Rajam Sruthy M. , Mukherjee Ananda , Dutta Debasree
Published: May 2022

Embryonic development involves orchestrated events regulated by a plethora of factors. Epigenetic regulation is one such key aspect regulating embryonic development. We are investigating the role played by Aprataxin and PNK‐like factor (APLF), a histone chaperone, in regulating cellular transitions in the context of embryonic development. APLF is documented as a DNA damage‐specific histone chaperone and has already been studied in our lab in the context of the generation of induced pluripotent stem cells and breast cancer metastasis as a regulator of epithelial to mesenchymal transition.

We are looking into the expression profile, functional role, and mechanism of action of APLF in embryonic development. The expression study in pre‐and post‐implantation mouse embryos and mouse trophoblast stem cells (TS) through different days of differentiation showed a dynamic expression pattern of APLF, clearly indicating prospective functional significance. The localization of APLF in mouse embryos was enhanced in trophectoderm (TE) and lineages derived from TE, which contributes to the extraembryonic lineage crucial for developing a proper maternal‐fetal connection. Functional studies by sh RNA mediated knockdown (KD) of Aplf in mouse embryos by microinjection induced the hatching of embryos in vitro at E3.75, with a significant increase in Cadherin 1 (Cdh1) and Caudal‐type homeobox 2 (Cdx2) expression. Hatching is a crucial event whereby the embryo breaks out of the protective sheath called zona pellucida and prepares itself to attach itself to the uterine wall by the process of implantation. The Aplf KD embryos failed to implant in vivo. Rescue experiments neutralized the effects of AplfKD both in vitro and in vivo. We used differentiated TS cells as our model system to understand the mechanism behind the observed phenotype in Aplf KD embryos. Reduced expression of Snail Family Transcriptional Repressor 2 (Snai2) and TEA Domain Transcription Factor 4 (Tead4), and the gain in Cdh1 level, marked the differentiation of AplfKD TS cells. Embryo implantation is one of the pioneer developmental stages where EMT comes into action. Significantly high level of epithelial marker Cdh1 and downregulation of mesenchymal marker Snai2 in Aplf KD TS cells would have impaired the crucial EMT process operational during implantation.

Further studies are being conducted to unravel the underlying mechanism completely. Hence, our findings suggest a novel role for APLF during the implantation and post‐implantation development of mouse embryos. As we observed these phenotypes in the mouse embryo, we checked APLF expression in the human placenta, and interestingly, our preliminary studies showed its expression in cytotrophoblast and syncytiotrophoblast cells. As we have seen that a delicate balance of APLF is crucial to achieve implantation and attain proper pregnancy, we anticipate that it would contribute to establishing a felicitous maternal‐fetal connection.
HUNK Phosphorylates Rubicon to Support Autophagy, Promoting Tumorigenesis in HER2+ Breast Cancer
Authors: Dilday Tinslee Y. , Yeh Elizabeth
Published: May 2022

Human epidermal growth factor receptor 2‐positive (HER2+) breast cancer is one of the three clinical subtypes of breast cancer and is defined by having HER2 gene amplification that coincides with HER2 protein overexpression. HER2 is amplified in 15‐30% of breast cancers and overexpression of this gene is a predictor of survival in breast cancer patients. HER2‐targeted therapies have been successful in treating HER2+ breast cancer; however, over time, HER2+ breast cancer can develop resistance to these therapies. Therefore, there is an urgent need to establish novel targeted agents. One proposed method of resistance to these HER2 agents is dysregulation of autophagy where cancer cells can shift to a cytoprotective autophagy phenotype to avoid cell death. Previous work in our lab established that Hormonally Upregulated Neu‐associated kinase (HUNK) is a Serine/Threonine (S/T) protein kinase that is overexpressed in HER2+ breast cancer and is responsible for promoting autophagy, thereby leading to therapeutic resistance. Our previous work shows that HUNK phosphorylates an autophagy protein, Rubicon, in the N‐terminal domain at S92, promoting autophagy in 293T cells. However, we have yet to establish a role for this phosphorylation site within HER2+ breast cancer cells. Therefore, the objective of this study is to test whether Rubicon S92 phosphorylation plays a role in promoting tumorigenesis in HER2+ breast cancer. We generated multiple scientific tools: a phosphoserine antibody at S92 in Rubicon, a phospho‐mimetic (S92A) mutant, and a phospho‐deficient (S92D) mutant. We have established an autophagy phenotype within a HER2/neu model; mouse mammary tumor virus driven neu(rodent form of HER2) expressing mammary tumor cells (MMTV‐neu) genetically engineered to be HUNK wild type (WT) or HUNK knockout (KO). We performed an endogenous Rubicon immunoprecipitation (IP) and found that KO cells are deficient in S92 phosphorylation alongside with a lack of autophagy response, measured using LC3‐II western blot. These findings confirm that HUNK is required for Rubicon S92 phosphorylation in a genetically tractable model. Utilizing these cell lines alongside the phospho‐mimetic and phospho‐deficient Rubicon mutants will allow us to determine the role that HUNK plays in promoting tumorigenesis within HER2+ breast cancer in‐vitro and in‐vivo.
Impaired Transcription Elongation by RNA Polymerase I Results in Defective Ribosomal RNA Processing
Authors: Huffines Abigail K. , Engel Krysta L. , Schneider David A.
Published: May 2022

RNA polymerase I (Pol I) synthesizes the majority of the ribosomal RNA (rRNA) in a eukaryotic cell, which is then assembled into the mature ribosome with ribosomal proteins. Pol I transcribes a single gene, the 35S gene in yeast, containing three gene elements (18S, 5.8S, and 25S) and four spacer regions. Previous literature demonstrates that processing events occur both co‐ and post‐transcriptionally so that the spacer regions are removed and degraded, resulting in mature 18S, 5.8S, and 25S rRNAs. While it has been established that rRNA processing begins during transcription, the relationship between Pol I transcription elongation properties and processing events have not been well defined. The objective of this project was to determine whether transcription elongation kinetics have a regulatory role in rRNA processing, and we hypothesized that if transcription elongation was impaired, this would result in defects in processing. To test this hypothesis, we generated a yeast strain containing a point mutation in RPA190, the largest subunit of Pol I. We found that there was a modest decrease in transcription elongation rate and an increase in pausing in the mutant compared to wild‐type Pol I in vitro. To analyze elongation effects of the mutant Pol I in vivo, we used native elongating transcript sequencing, which probes for Pol I occupancy on the ribosomal DNA template at single‐nucleotide resolution. Our in vivo results corroborated the in vitro findings, indicating that this mutation introduced an increase in pausing across the template during transcription elongation. Furthermore, Northern blots and polysome profiling indicated that rRNA processing and ribosome assembly were impaired when this mutation was introduced. These three independent in vitroand in vivo experimental strategies converge to demonstrate that transcription elongation and Pol I pausing may play a regulatory role in downstream processing steps.
Investigating the miRNA regulatory landscape of OGT and OGA via the 3’UTR and 5’UTR regions utilizing the miRFluR high‐throughput platform
Authors: Chu Thu
Published: May 2022

The dynamic post‐translational modification of serine or threonine residues by O‐linked N‐acetyl‐beta‐D‐glucosamine (O‐GlcNAc) contributes to diverse cellular processes including epigenetic modifications, transcription, metabolism, and cell signaling that play significant roles in development and normal physiology. O‐GlcNAcylation is catalyzed by O‐GlcNAc transferase and the modification is removed by O‐GlcNAcase (OGA). These genes are highly regulated at multiple levels, but little is known about their regulation by microRNAs (miRs). miRs are small non‐coding RNAs that fine‐tune protein expression through binding to messenger RNA (mRNA). In this work, we built a comprehensive dataset of OGT and OGA regulation via both their 3’UTR and 5’UTRs. Downregulation was almost exclusively mediated through binding to the 3’UTR. We observed independent regulation of OGT and OGA by the majority of regulatory miRs, which did not overlap. However, we did see significant co‐regulation of OGT and OGA by a subset of miRs. This is in keeping with the known transcriptional regulation of these genes.

In summary, this work provides a better understanding of OGT and OGA regulation through miRNA binding via both the 3’UTR and 5’UTR regions.
Identifying Deubiquitinase Regulating the DNA Damage Response Pathway
Authors: Cranley Juliana , Baker James , Pauer Emma , Cheranda Nina , Weng Fangli , Wang Yuqi
Published: May 2022

In response to DNA damage, cells often activate Mec1‐dependent DNA damage checkpoint, resulting in the activation of the effector kinase Rad53 that promotes cell cycle arrest and DNA repair. The checkpoint proteins and the DNA damage response pathway are often regulated by ubiquitination, a reversible post‐translational modification, raising the possibility that checkpoint activation may be regulated by a deubiquitinating enzyme. To test this, we screened a collection of yeast deubiquitinating enzyme deletion mutants and monitored their responses to DNA damage agents. Interestingly, we find that one deubiquitinating enzyme deletion mutant displayed consistently decreased levels of checkpoint activation. Further analysis indicated that this mutant does not affect either the abundance or the stability of checkpoint activators, but it rather has a profound effect on Mec1 itself. This analysis suggests deubiquitinating enzyme as a novel regulator for Mecl1 and DNA damage checkpoint pathway.
Serial crystallography and kinetics reveal how the FimH bacterial lectin tweaks between mono‐ and multivalent binding of high‐mannose N‐glycans
Authors: Bouckaert Julie M. J.
Published: May 2022

Background

The lectin domain of the fimbrial adhesin FimH from Escherichia coli recognizes with the highest affinity and in seemingly monovalent fashion oligomannosides‐3 and ‐5 N‐glycans. This happens with a high affinity (Kd=20 nM) because the alpha1,3‐linked arm remains free from any further alpha1,2‐mannose substitution. The latter causes an at least 10‐fold affinity loss in creating the oligomannose‐6 N‐glycan (1).

Methods

The binding of FimH to pauci‐ and oligomannoside‐containing N‐glycans and similarly glycosylated proteins, as well as its dimannoside endings Manalpha1,2, Manalpha1,3 and Manalpha1,6, have been profiled using the FimH Lectprofile kit (GlycoDiag, Applied Glycomics, France) and a newly designed paucimannose‐containing glycan array. The kinetics of binding, ka and kd, were analysed using surface plasmon resonance and compared with earlier incomplete data (2).

Results

Mono‐ and multivalent binding were found in co‐crystal structures of the FimH lectin domain with alpha1,6‐fucosylated oligomannose‐3 and with oligoannose‐6, respectively. The kinetics of binding indicate that once alpha1,2‐linked mannose comes into play, the complexes are not so stable, as previously also measured using calorimetry (3), and accompanied with a high dissociation constant. Molecular dynamics simulations further support that it is not the total interaction energy that determines the final retained complex (metastable complexes), but the kinetics of interaction with the three possible dimannoside endings.

Conclusions

Kinetics and stability of the complexes drive the most favoured arrangement, via either a unique or multivalent interaction, between the N‐glycan and the FimH protein in the crystal.

References :

(1) Bouckaert, J., Mackenzie, J., de Paz, J. L., Chipwaza, B., Choudhury, D., Zavialov, A., Mannerstedt, K., Anderson, J., Pierard, D., Wyns, L., Seeberger, P. H., Oscarson, S., De Greve, H., Knight, S. D. (2006) The affinity of the FimH fimbrial adhesin is receptor‐driven and quasi‐independent of Escherichia colipathotypes. Molecular Microbiology 61, 1556‐1568

(2) Sauer, M. M., Jakob, R. P., Luber, T., Canonica, F., Navarra, G., Ernst, B., Maier, T., Glockshuber, R. (2019) Binding of the bacterial adhesin FimH to its natural, multivalent high‐mannose type glycan targets. J Am Chem Soc 141, 936‐944

(3) Dumych, T., Bridot, C., Gouin, S. G., Paryzhak, S., Szunerits, S., Bilyy, R., Bouckaert, J., (2018) A novel integrated way for deciphering the glycan code for the FimH lectin. Molecules 23, 2794
Gene Therapy for Brain Tumors: Identification of New Therapeutic Targets Based on RNA Structure
Authors: Sine Laura , DeMarco Victoria , Hintelmann Thomas , Reardon Sean , Hicks Martin J.
Published: May 2022

This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, for the treatment of glioblastoma multiforme (GBM). Currently, individuals diagnosed with GBM have a short life expectancy of 12‐14 months. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of cancer‐driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. We have examined the ‘pre‐mRNA structurome’ of EGFR to evaluate the accessibility of targetable regions. To advance our therapeutic strategy, we have analyzed the secondary structure of the EGFR transcript using selective 2’ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE‐MaP). SHAPE‐MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5’ splice site and hidden elements of a cryptic polyadenylation signal. Based on these structural profiles, we generated RNA therapies that interact with structural elements to unravel the hidden polyadenylation signal with the potential to activate expression of the short therapeutic isoform. In this project, we cloned these therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre‐mRNA transcript.
Codon Optimization of saCas9 Gene Enhances Protein Expression in Human Kidney Cells to Compensate for Difficulties in Delivery Vectors to Boost CRISPR‐Cas9 in vivo Therapeutic Application
Authors: Cheng Benjamin , Cooper Eli , Krishnan Anoushka , Groshong Tyler , Madejski Irene , Sundaresan Avirath , Holmes Grace , Gupta Ishir , Leschly Emma , Hauser Paul , De Luke
Published: May 2022

The novel gene editing technology CRISPR‐Cas9 shows great promise in revolutionizing treatments of genetic disorders; however, currently the only practical treatments are ex vivo, limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process. The large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We hypothesize that a Cas9 gene codon optimized for human cells will support significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies.

To investigate our hypothesis, we selected a commonly used parent saCas9 Addgene’s public plasmid database. We engineered a novel, codon‐optimized gene sequence from the parent using GenScript’s OptimumGene™ algorithm. We will compare protein expression via ELISA assay and Western Blot analysis. In this poster we will present the data gleaned from these assays.
Hypoxia Re‐Oxygenation Modelling Using Cancer Cells Expressing Cell Cycle and Cell Death Probes to Understand the Dynamics of Resistance Mechanisms
Authors: Tiwari Shivanshu Kumar , Kumar T. R. Santhosh
Published: May 2022

Background

Most solid tumors are difficult to treat with conventional cancer drugs. Non‐responsiveness to drugs and the recurrences even after successful treatment are serious problems. Although, extensive studies have been performed to understand the mechanisms of resistance and tumor recurrences, the complex and variable nature of microenvironmental factors that contribute this remains as a challenging topic.

Hypoxia is also one of the key factors that play a crucial role in tumor progression. Hypoxia‐Induced Factor 1 α (HIF 1‐α) are involved in the resistance of hypoxic tumors to radiation therapy as well as conventional drugs. However, the growing tumor is often exposed to hypoxia and re‐oxygenation because of the tumor driven hypoxia and occasional angiogenic response that primarily affect the cell cycle and cell death.

Hypothesis

We hypothesize that the defined models of cell cycle and cell death in the setting of hypoxia‐reoxygenation will help us to understand the hypoxia‐mediated cell signaling and resistance mechanisms.

Methodology

We have employed cancer cells expressing cell cycle probe FUCCI to study the dynamics of cell cycle progression during hypoxia and reoxygenation using real‐time imaging. Similarly, cancer cells were expressed with real‐time probes for cell death, FRET‐based caspase sensor to track their long‐time fate. These innovative strategies allowed us to visualize both cell cycle and cell death events in hypoxic and re‐oxygenation condition and to track the long‐time fate of cancer cells.

Results & Conclusion

We have shown that the hypoxic exposures alter the cells’ ability to undergo apoptosis dynamically after its recovery under normoxia. Initially, the cells adapt to hypoxia through mitophagy dependent manner that alters its cell cycle progression both in normoxia and hypoxia. Despite adaptive mitophagy, hypoxic cells remain sensitive to re‐oxygenation and also to cancer drugs owing to the oxidation damage to organelles.

However, rare cells survive during these two events progresses towards expansion with a highly variable mitochondrial density that governs differing functional traits of cell cycle, cell death response, ability to invade and migrate. This functional heterogeneity appears to play a key role in temporally regulated tumor resistance and recurrences.
From Flat to Bulb ‐ Novel Insights in Caveolae Membrane Curvature
Authors: Matthaeus Claudia , Sochacki Kem , Puchkov Dmytro , Haucke Volker , Lehmann Martin , Taraska Justin
Published: May 2022

Caveolae are 60‐100 nm sized bulb‐shaped plasma membrane invaginations found in various cell types including adipocytes, endothelial cells and myocytes. Their cellular functions comprise endocytosis and signaling processes, cellular lipid uptake, as well as sensing and regulation of membrane tension. Thereby, it was shown previously that caveolae can change their membrane shape from invaginated bulbs to flat domains and vice versa. However it is currently not clear which proteins are responsible for the membrane curvature changes. Here, we report the first detailed structural investigation of caveolae membrane shapes in vivo. To do so, Platinum replica EM images of the plasma membrane in mouse embryonic fibroblasts and endothelial cells were correlated to super‐resolution (STED) fluorescence intensities of proteins associated with caveolae domains. This correlative imaging approach (CLEM) allowed us to precisely localize caveolae related proteins to the underlying membrane curvature. The major structural caveolae proteins such as Caveolins and Cavins were found in all caveolae membrane domains independent of their curvature. Surprisingly, EHD2, a caveolar neck protein, was not only detected at bulb‐shaped invaginations but also at flat caveolae. Contrarily, Pacsin/Syndapin2 and EHBP1 were predominantly surrounding flat caveolae domains indicating that both proteins promote the neck formation. Because dynamin was previously associated with caveolae endocytosis and mobility we analyzed its spatial distribution to caveolae. However, our detailed CLEM analysis failed to determine dynamin to caveolae membrane domains in several cell types. Additionally, dynamin lacking cells did not show severe morphological changes of caveolae suggesting that dynamin is not involved in caveolae membrane curvature changes. Taken together, this study gives novel insights in caveolae structure and their formation from flat to invaginated membrane domains.
Asymmetric RNA Egress Site in Expanded RNA Virus by Cryo‐EM and HDXMS
Authors: Braet Sean , Venkatakrishnan Varun , Ramesh Ranita , Raghuvamsi Palur , Anand Ganesh
Published: May 2022

RNA viruses are metastable macromolecular assemblies that sense their environment through dynamic breathing motions. While surfaces of viral particles have been well characterized by cryo‐EM, dynamic genome‐capsid cores are poorly understood. To address this gap in understanding, we have applied hydrogen deuterium exchange mass spectrometry (HDXMS) together with cryo‐EM to Turnip Crinkle Virus (TCV) as a model system to study RNA genome‐capsid interactions and their importance in environmental sensing and programmed viral disassembly in favorable host environments. TCV undergoes expansion upon abstraction of Ca2+ ions, associated with entry into a plant host cell. We have mapped changes upon expansion in the viral capsid. HDXMS analysis reveals two conformations of the coat protein R‐domain regions in the native state: a minor (~5%) RNA‐bound and a major (~95%) unbound R‐domain. A larger fraction of the R‐domain is bound (~15%) more tightly to RNA in the expanded state, indicating expansion is accompanied by changes in conformation of the RNA‐coat protein core. Cryo‐EM of expanded TCV reveals that the newly formed strong interactions between the capsid proteins and the RNA genome correspond to the asymmetric ribonucleoprotein core packing observed near a unique 5‐fold vertex in the expanded TCV particle. This 5‐fold vertex is the specific point of egress for the genomic RNA into the host. Far from being passively package contents, our results highlight an active role of the genomic RNA‐protein core as an environmental sensor which controls capsid uncoating and RNA release.
A Framework to Assess Liquid‐Liquid‐Phase‐Separation in Bacterial Cells
Authors: Hoang Y. , Azaldegui Christopher , Biteen Julie , Vecchiarelli Anthony
Published: May 2022

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.
Activation of Gαq sequesters specific transcripts into Ago2 particles
Authors: Scarlata Suzanne
Published: May 2022

Hormones and neurotransmitters can activate the Gαq/phospholipase Cβ1 (PLCβ1) signaling system eliciting cellular calcium responses. PLCβ1 also prevents the aggregation of ribosomal and RNA proteins into stress granules, which are halted translation complexes that form in response to cellular stress. Activation of Gαq promotes PLCβ1association releasing bound proteins and promoting the formation of stress granules. However, the cellular impact of stress granules formed from routine Gαq protein signaling is unknown. Here, we have characterized Ago2 stress granules formed in response to Gαq activation in a neuronal‐like cell line. We find these stress granule have a distinct protein composition, and unlike stress granules formed under heat stress, contain only two mRNA transcripts, chromogranin B, which is involved in secretory function, and ATP synthase 5f1b, which is required for ATP synthesis. Our studies show an unexpected pathway where Gαq/PLCβ regulates the translation of specific proteins.
Conformational Plasticity of initiator caspases
Authors: Karadi Giridhar Mithun Nag
Published: May 2022

Caspases are an important family of enzymes that are key players in the Apoptotic pathway. In the extrinsic apoptotic pathway, Initiator Caspases: 8, 10 and cFLIP are monomers which need an activation platform to dimerize and subsequent cleavage to produce the active enzyme; Executioner caspases however exist as dimers under physiological conditions and have to be processed at specific regions for them to be activated. We have studied the equilibrium unfolding of inactive (active site mutant) monomeric initiator caspase 8 by urea denaturation studies and obtained thermodynamic parameters. Initiator caspases have a total free energy of 7kcal/mole whereas executioner caspases have a total free energy of 25.8kcal/mole, dimerization contributes to 18.8kcal/mole and the monomeric step contributes to 7kcal/mole which is comparable to the initiators. These results indicate a conservation of the monomeric fold in all caspases with dimers having mutations at sites that modulate networks to stabilize the dimeric fold. The free energy decreases as the pH decreases below pH 7 and rises above pH 8.5 which corresponds to titration of Histidine and Cysteine residues that are important for stabilizing the tertiary structure. Initiator caspases play an important role in a variety of other pathways like necroptosis, cell differentiation and motility which are less well understood compared to its role in apoptosis. These studies are vital to understand allosteric regulation and the conformational plasticity of initiator caspases which could help us design drugs to divert these enzymes in desired pathways to effect the fate of a cell.
Toxic photochemistry and signaling repercussions of blue light in cells exposed to retinal
Authors: Karunarathne Ajith , Thotamune Waruna , Ratnayake Kasun , Wong Kwoon
Published: May 2022

Age‐related visual damage by blue light and its adverse effects on quality of life for many people >55 yrs have been well documented. Eliminating blue wavelengths has been shown to reduce age‐related visual impairment and facilitate vision preservation in the elderly. The blue region of the visible spectrum is abundant in sunlight and common electronic light emitters. Many adults receive increased ultraviolet and blue light exposure upon retirement by moving closer to the tropics and spending more time outdoors. The cornea and lens of the eye are largely opaque to ultraviolet, while blue light readily permeates into the retina. We propose that all‐trans‐retinal (ATR), the light‐sensitive chromophore released from photoexcited photopigments in the retina, could induce age‐related vision impairment. Our data demonstrate that exposing cells incubated with ATR and exposed to blue light or sunlight permanently distorted the chemical properties of the crucial signaling lipid PIP2 and disrupted its subcellular location and signaling. Similar conditions permanently displaced G proteins in the phototransduction cascade and Ras family G proteins from the plasma membrane to the cell interior. Chemical evidence shows the oxidation of their farnesyl lipid anchor during this perturbation. Upon ATR and blue light, the percent positivity for TUNEL stain, indicating DNA damage, is significantly higher in primary cells isolated from mouse retina. We also show α‐Tocopherol, a lipid‐soluble antioxidant, inhibits the above cellular photodamage. Therefore, we now ask the critical question: could resident ATR concentrations in photoreceptor cells cause retina neurodegeneration upon physiologically relevant light exposures?
Reference chart for transverse cerebellar diameter in developing fetuses according to their gestational age in Indian population
Authors: GHATAK SURAJIT , MISHRA SANJAY , AGARWAL DUSHYANT , SINGH PRATIBHA , GARG PAVAN K.
Published: May 2022

Objectives

The objective of the study was to develop reference chart and to evaluate the efficacy of transverse cerebellar diameter (TCD) as one of the fetal biometric parameters used in ultrasonography to determine gestational age in the Indian population. Also, in this study we compared our findings with similar studies done on different ethnic populations to check effect of different ethnicity, environmental and nutritional factors in development of TCD.

Design

A retrospective cross‐sectional study.

Method

In this study routine antenatal check‐ups of 300 singleton pregnant women were done after obtaining ethical clearance from Institute Ethical Committee. Ultrasonographic measurements of various fetal biometric parameters were taken which included, biparietal diameter (cm), head circumference (cm), abdominal circumference (cm), femur length (cm) and transverse cerebellar diameter (cm). Reference chart with mean TCD for corresponding gestational age (GA) in weeks was developed and compared with results of similar other studies.

Results

Statistically significant correlation found between TCD and gestational age (R2=0.92, p=0.0006). Gradually increasing linear pattern obtained between the development of TCD and corresponding gestational age of the fetus. Therefore, it is more reliable parameter to be used in regression formulae used in USG machine to predict accurate gestational age of fetus. When reference charts for TCD is compared with findings in other studies in different ethnic population, it is found that there is statistically significant difference exists.

Conclusions

In normally developing fetuses the TCD has linear correlation with advancing gestational age. Therefore, along with other fetal biometric parameters measurement TCD can be used in determination of gestational age for more accuracy. Thus by preventing over or under estimation of gestational age will help in reducing no judicious use of health resources and medical interventions. A separate reference chart is required for every different population because ethnicity, nutrition and environmental factors can have impact on normal TCD values. This will help to avoid misinterpretation of data to determine gestational age in various ethnic populations.