bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Low density Lipoprotein Internalization and Transcytosis by HDL; an alternative role for “good” cholesterol
Authors: Fung Karen Yin Yue , Lee Warren , Fairn Greg
Published: April 2020

Atherosclerosis results from the build‐up of low‐density lipoprotein (LDL) cholesterol and immune cells in the arteries leading to their occlusion. High‐density lipoprotein (HDL) is believed to reverse this process by removing the arterial cholesterol from the body. Both LDL and HDL reach beneath the artery by crossing the endothelium through Scavenger receptor B1 (SR‐B1) mediated transcytosis. ApoAI, the protein exclusively found on HDL, mediates the interaction between HDL and SR‐B1. One natural ApoAI variant called Milano (ApoAI‐Mil) is of interest because injections of lipidated ApoAI‐Mil led to enhanced plaque regression. We hypothesize that the natural ability of ApoAI‐Mil to dimerize leads to better interaction with SR‐B1. This could result in better inhibition of LDL transcytosis since both HDL and LDL bind to SR‐B1 for transcytosis and this could also improve HDL transcytosis so that more HDL will reach the target tissue to extract cholesterol. We took an in vitro microscopy approach to quantify internalization of fluorescently labeled LDL in coronary endothelial cells. The ApoAI variants were also lipidated to form fluorescent HDL‐like particles (DiI‐DMPC‐WT or DiI‐DMPC‐Mil) to measure its association with SR‐B1 overexpressing cells. We observed that recombinant ApoAI‐WT can inhibit LDL internalization in coronary endothelial cells. There was also about an 8x‐fold increase of DiI‐DMPC‐WT fluorescence signal in HeLas overexpressing SR‐B1 compared to GFP alone. Furthermore, there was an even higher fold increase (~11x) of DiI‐DMPC‐Mil fluorescence signal compared to GFP alone. Similarly, excess recombinant ApoAI‐Mil led to greater inhibition of LDL internalization compared to ApoAI‐WT. There was no difference in the amount of DiI‐DMPC‐WT or ‐Mil associated with HeLa cells overexpressing Alk1, a LDL‐specific transcytosis receptor. Together this suggests that compared to ApoAI‐WT, ApoAI‐Mil more readily associates with only SR‐B1 which may lead to better inhibition of LDL internalization and to more HDL available to remove arterial cholesterol.

Support or Funding Information

Early Research Award & CIHR
Protective effects of Gladiolus psittascinus bulb Aqueous extract on Nicotine induced Hepatotoxicity in Experimental Rats
Authors: Oyetayo Folake Lucy , Odeniyi Ifeoluwa Adebayo , Olaseigbe Olajumoke Esther , Jegede Funmilayo Olusola
Published: April 2020

Aqueous extract of the bulb of Gladiolus psittascinus was evaluated for protective activities in nicotine induced hepatotoxic rats. Biomarker and antioxidative enzyme activities were analysed using standard analytical procedure. Rats were divided into three groups: Control, Nicotine induced hepatotoxic, nicotine induced but treated Gladiolus psittascinus group. Induction of hepatotoxicity in rats led to significant (P<0.05) reduction of antioxidant enzyme activities. Superoxide dismutase activity (μmol/L) decreased from 18.58± 3.50 in control rats to 9.65± 1.00 in the nicotine induced. This was ameliorated on administration of the bulb extract at 100mg/kg body weight to 17.55± 3.23 in the aqueous Gladiolus psittascinus bulb treated group. Catalase activities (μmol/L) increased significantly from 3.65± 1.43 (Nicotine induced group) to 10.65±1.65 in the bulb extract treated group. Furthermore, plasma aminotransferases activities (U/L) in hepatotoxic rats were significantly reduced to control levels in the bulb extract treated group with Aspartate aminotransferase (AST) showing almost a fourfold reduction (16.51±0.70 (induced rats) to 4.21±0.33(bulb extract treated)) while Alanine aminotransferase (ALT) showed a twofold reduction (5.11±0.70 (induced rats) to 2.50±0.34(bulb extract treated)). Hence, the foregoing shows the potentials of Gladilous psittascinus bulb aqueous extract in enhacing the production of antioxidant enzymes required to scavenge reactive oxygen species. This holds promise for its use as diet therapy in the management and control of hepatotoxicity and oxidative stress related disorders.

Support or Funding Information

Tertiary Education Trust Fund, Nigeria.
Investigating the Role of the Med Protein in Biofilm Formation
Authors: Lara-Garcia Juan , Wacker Sarah
Published: April 2020

Bacillus subtilis is a soil‐dwelling bacterium that serves as a good model organism for the study of different adaptive responses to various environmental conditions. These adaptive responses, including spore formation, cannibalism, and biofilm formation, are controlled by the master regulator Spo0A whose phosphorylation is under the control of five kinases. Initial interest in the protein Med came from its role in the transcription of operons related to cannibalism, and studies show the effect of Med is dependent on one of the five kinases, KinD. These same studies suggest Med may play a role in biofilm formation in a KinD‐dependent manner. The goal of our study of the Med protein is to determine whether it plays a direct role in biofilm formation by directly detecting environmental signals or indirectly through interactions with KinD. To determine whether Med is involved in detecting environmental signals, Δmed and ΔkinD bacteria were compared to wildtype B. subtilis using luciferase reporter assays that quantify the effect of different small molecules on the transcription of biofilm genes. Luciferase assays were compared to biofilm assays under the same conditions to visualize the effect on the biofilm phenotypes. We find that Δmed mutants had a defect in biofilm formation and reduced transcription of biofilm genes compared with wildtype bacteria. However, Δmed mutants were the same as wildtype bacteria in terms of their pattern of stimulation by small molecules. These results indicate that Med is not directly sensing environmental signals and instead is likely to be involved in the biofilm formation pathway indirectly through KinD, which has been shown to bind small molecules through its extracellular CACHE domain. We therefore shifted our focus to investigating interactions between Med and KinD and how Med may affect KinD’s ability to sense small molecules. Med and KinD were purified by tagging them with GST and His tags respectively. Additionally, in the case of KinD we purified constructs of the extracellular and intracellular domains to more precisely determine where the Med protein may be interacting with KinD. Presence of the proteins was determined by SDS‐PAGE and interactions between them was determined by Western blot analysis.

Support or Funding Information

Research was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861.
Investigating mechanisms for acquired resistance to HDAC inhibitors in histiocytic lymphoma U‐937 cells
Authors: Burnett Matthew , Carle Sigrid
Published: April 2020

Xyzidepsin (Xyzi) is an analog of the FDA approved HDAC inhibitor FK228. HDAC inhibitors increase acetylation of histones and other proteins modified by Histone Acetyltransferases (HATs). Through MTT assay analysis we determined the IC50 for Xyzi to be 0.567 μM. Several resistance mechanisms have been suggested for HDAC inhibitors, including the protein ABCB1/MDR1/P‐glycoprotein upregulation, and changes to signal transduction pathways and glycolysis. To further understand mechanisms of resistance a dose escalation protocol with increasing concentration of HDAC inhibitor is used. Autophagy is identified as a resistance mechanism for vorinastat in U‐937 cells. Interestingly, Akt (Protein kinase B, PKB), is an upstream kinase regulatory protein related to glucose metabolism, MDR‐1 expression, autophagy, cell proliferation and apoptosis. Previous studies suggest an association between the regulation of autophagy by Akt through glycolytic pathways, which may indicate a key role of regulator isoenzymes for the glycolysis enzyme 6‐Phosphofructo‐2‐kinase/fructose2,6‐biphosphatase such as, PFKFB3 and PFKB4, in the development of drug resistance. We intend to uncover possible shifts in gene expression which could indicate a combined effect of previously observed resistance mechanisms or alternative mechanism to accommodate an increasing HDAC concentration. The mechanisms for Xyzi and other HDAC inhibitors like as SAHA could then be compared.
Investigation of the Directionality and Kinetics of DNA Transfer by a Bacterial Type IV Secretion System
Authors: Akinleye Olukemi P. , Gemechu Betelhem B. , Ali Sabrina , Berkmen Melanie B.
Published: April 2020

Conjugation is the transfer of DNA from a donor cell to a recipient cell through a Type IV Secretion System (T4SS). The T4SS is encoded by the DNA conjugative element and is assembled in the donor cell membrane. T4SSs can also mediate the mobilization of resident plasmids that do not encode their own conjugation machinery from the donor to a recipient. In addition, in some cases, T4SSs have been shown to mediate retro‐transfer, or reverse mobilization, in which a donor cell containing the conjugative element receives DNA from a recipient cell. Conjugation, mobilization, and reverse mobilization are all important mechanisms of horizontal gene transfer that contribute to bacterial evolution and the spread of antibiotic resistance genes. Previously, it has been shown that the integrative and conjugative element ICEBs1 of Bacillus subtilis exhibits very high frequencies of conjugation and plasmid mobilization. Here, we compare the timing and frequency of ICEBs1‐dependent conjugation, plasmid mobilization, and reverse mobilization. Donor and recipient cells having different antibiotic markers were mixed and placed on solid filters for different periods of time to allow for DNA transfer. After incubation, the cell mixtures were plated on selective media to measure the frequency of DNA transfer. We found that both mating and plasmid mobilization can be detected within 2 minutes and peak after about 1–2 hours. Mating was generally more frequent than mobilization at most time points. Reverse mobilization was not detected until 1–2 hours, and was more than 100‐fold less frequent than mating. Reverse mobilization also required the transfer of T4SS genes to the recipient. Our data are consistent with the T4SS acting unidirectionally; in other words, reverse mobilization requires that a conjugative DNA element be transferred from donor to recipient prior to the recipient making its own T4SS that transfers DNA back into the donor.

Support or Funding Information

This research was supported by Suffolk University and an NSF‐RUI grant to M. Berkmen.
Sema3F Suppresses Tumor Initiation Through Alteration of the Immunological Tumor Microenvironment
Authors: Doçi Colleen L. , Callejas-Valera Juan Luis , Mikelis Constantinos M. , Molinolo Alfredo M. , Gutkind J. Silvio
Published: April 2020

SEMA3F is an antilymphangiogenic signaling molecule expressed in the proliferating basal layer of normal epithelium. In orthotopic models of head and neck squamous cell carcinoma, loss of SEMA3F or its receptors results in enhanced tumor growth and locoregional metastasis, leading to speculation that SEMA3F may also prevent cancer initiation. To determine whether the loss of SEMA3F enhances susceptibility to cancer development, we employed a two‐step carcinogenesis model in a robustly immunogenic outbred strain of wild‐type (WT) or Sema3F knockout (KO) mice. Animals were treated with a single application of 7,12‐Dimethylbenz(a)anthracene (DMBA) followed by twice‐weekly applications of 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) for 24 weeks. Sema3F KO animals had a modest but statistical decrease in tumor latency compared with WT. Additionally, both the number and size of lesions was significantly decreased in KO animals. Together, this suggested that loss of Sema3F may be influencing cancer development and progression primarily through modulation of the tumor microenvironment. By histochemical staining, KO animals demonstrated much higher percentage of immune infiltrate within the tumors. To investigate whether immunosurveillance may contribute to the diminished cancer progression in KO animals, we analyzed the mRNA expression of inflammatory cytokines from these animals. Analysis of the inflammatory tumor microenvironment in these lesions revealed a strong induction of both IL‐12α and IL‐1β in KO animals. Primarily produced by macrophages, IL‐12α has been shown to suppress the development of skin papillomas and suppress tumor growth and development. Compellingly, macrophages but not leukocytes express Sema3F receptors. Consequently, we propose that expression of Sema3F in the context of chemical carcinogenesis functions as a macrophage chemorepellent and antilymphangiogenic molecule, thus delaying the onset of an adaptive immune response and increasing the latency and tumor burden. Further, Sema3F may be an attractive target for cancer immunotherapies given its autocrine and paracrine signaling roles.

Support or Funding Information

This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research and by Marian University Indianapolis.
Fabrication of a Physiologically Relevant Novel Lung‐on‐a‐Chip for the Study of Human Pulmonary Disease and Drug Toxicity
Authors: Williams Marcus A. C. , Wiens Cooper , Blackwater Elizabeth , Killifer Avery , Vonesh Lily , Vargas Koby , Stalder Madeline , Hamilton Adam , Mancha Sophie , Crawford Jerry , Li Yiyan , Blake David , Jessing Jeff
Published: April 2020

In the scope of biomedical research, many advances have been made in the field of pulmonology with the use of multiple animal models and traditional cell culture experimentation. While these methods are useful when studying single cellular pathways or overarching biological processes, they often falter when studying the meticulous nature of human disease. In this work, we propose the development of a novel Lung‐on‐a‐Chip device that would more accurately mimic the physiological boundaries of human alveoli than animal models or traditional in vitro studies. Utilizing multiple microfabrication techniques, we are developing a microfluidic device with an ultra‐thin (25μm) biodegradable porous silicon membrane. Current data suggests that human pulmonary epithelial and endothelial cells are viable and prolific on the proposed microfabricated silicon membrane in extended studies (14 days). Due to the biodegradability of the fabricated novel silicon membranes, it has been observed in long term studies that cells can remodel and degrade the porous silicon membrane. This degradation allows for physiological cellular contact between membranes mimicking a true blood gas exchange interface as observed in vivo. To further validate this model’s ability to recapitulate human physiology, we have begun to co‐culture with immunological cell types and are monitoring for response to bacterial and tumor antigens. Preliminary data suggests that immunological cells are able to remodel the porous silicon membrane substrate and migrate across cellular boundaries in response to environmental cues as seen in vivo. Broadly, we believe that this model may be used to further characterize and study human pulmonary disease and drug toxicity.

Support or Funding Information

Maximizing Access to Research Careers Undergraduate Student Training in Academic Research Award# T34GM092711NSF PREM for Functional Nanomaterials, Award# 1827847STROBE NSF Science and Technology Center on Real‐Time Functional Imaging, Award# 1548924.
A mitochondrial recruitment assay to characterize a family of DENN‐domain proteins, Rab GDP/GTP exchange factors.
Authors: Kumar Rahul , Laflamme Carl , McPherson Peter S.
Published: April 2020

Intracellular membrane trafficking controls the levels, localization and functional activity of a myriad of proteins and is thus critical for cellular function. Rab GTPases are molecular switches that regulate membrane trafficking by toggling between GTP‐bound (active; membrane‐bound) and GDP‐bound (inactive; cytosolic) states. These GTPases are controlled largely by guanine nucleotide exchange factors (GEFs) that mediate the exchange of GDP for GTP. Mammalian cells possess 61 Rabs but many of their GEFs are unknown. One of the largest family of Rab GEFs contain an evolutionarily conserved protein module called the DENN domain. There are 18 DENN domain proteins but many of their corresponding Rab GTPase partner(s) are either not known or are controversial. This is due to the fact that all studies concerning DENN domains have used biochemical and in‐vitro assays, which do not reflect physiological conditions. Here we describe a cell‐based GEF assay as a method to identify GTPase substrates of DENN domain proteins. By overexpressing DENN domains fused to a mitochondrial targeting sequence we can drive the recruitment of physiological GTPase substrates to the mitochondrial membrane, which can then be detected by immunofluorescence analysis. We validated our assay with a known DENN/Rab pair, DENND1A and Rab35. Using a panel of GFP‐tagged Rabs we have resolved controversies over previously reported DENN/Rab pairs and have identified new Rab substrates for DENN domains. Identifying the full complement of DENN/Rab pairs will provide new insights into membrane trafficking and cellular function.

Support or Funding Information

1) Canada First Research Excellence Fund, awarded to McGill University for Healthy Brains for Healthy Lives. 2) ALS Canada
Regulation of clathrin adaptor protein complex‐1(AP‐1) by a Laa1 protein containing complex
Authors: Duncan Mara Colleen
Published: April 2020

The clathrin adaptor protein complex‐1 (AP‐1) is a critical regulator of membrane traffic at the trans‐Golgi network. Its recruitment depends on the function of the small GTPase‐Arf1, phosphatidyl‐inositides, and members of the HEATR5 family, a poorly characterized family of proteins with members found in all eukaryotes. Although members of the HEATR5 family are known to be important for AP‐1 localization in diverse organisms, how they contributed to AP‐1 recruitment was unclear. We recently identified a conserved co‐factor for the yeast HEATR5 protein, Laa1, that acts as an AP‐1 binding cofactor. This co‐factor, Laa2, binds directly to AP‐1 and this interaction is required for AP‐1 localization, suggesting that direct interaction between the Laa1‐Laa2 protein complex and AP‐1 is essential for AP‐1 localization. Using proteomics and database mining, we have identified additional binding partners for Laa1. Phenotypic analysis suggests these binding partners are required for normal AP‐1 function, similar to Laa1 and Laa2. Here, we report the network of physical interactions between Laa1, Laa2, and two additional binding partners. Moreover, we find many of these proteins are unstable in the absence of their binding partners suggesting they may for a stable complex in vivo. These results suggest that a multi‐subunit complex centered on Laa1 controls AP‐1 functions.

Support or Funding Information

Protein Folding Disease Initiative at the University of Michigan NIGMS R01 GM129255
IroT/MavN is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by In‐vitro Real‐time Transport
Authors: Abeyrathna Sameera Sandaruwan , Abeyrathna Nisansala Sandamali , Thai Nathan , Sarkar Prithwijit , D’Arcy Sheena , Meloni Gabriele
Published: April 2020

Iron plays a vital role in all living organisms due to its key role as an enzyme co‐factor, central to diverse metabolic processes. However, because of its high reactivity, speciation, and insolubility in oxidative environments, organisms have evolved sophisticated networks for intercellular iron acquisition without reaching toxic levels. In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, the causative agent of Legionnaires’ disease, a putative transmembrane protein, IroT/MavN, is inserted onto the surface of the host pathogen‐containing vacuole to facilitate intravacuolar iron acquisition from the host, bypassing the problem of Fe(III) insolubility and mobilization.

In this work, we have developed a platform for purification and the functional reconstitution of IroT/MavN in artificial lipid bilayer vesicles (proteoliposomes) to identify and characterize the IroT/MavN mediated metal transport properties. By encapsulating the fluorescent reporter probe Fluozin‐3, by real‐time metal transport assays, we revealed that IroT/MavN is a high‐affinity and high‐capacity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains and loops important for substrate recognition and rapid‐kinetic translocation. In addition, by encapsulating the pH indicator pyranine, we demonstrated that Fe(II) translocation is coupled to H+ counter‐transport, suggesting that IroT/MavN is a Fe(II)‐H+ antiporter.

This work establishes the substrate transport properties involved in a novel transporter family important for iron acquisition at the host‐pathogen intravacuolar interface and provides chemical tools for comparative investigation of the translocation properties in other iron transporter families.

Support or Funding Information

The work was supported by the Robert A. Welch Foundation (AT‐1935‐20170325 to G.M.) and by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM128704 to G.M.).
Glucocorticoids ameliorate arthritis through epigenetic regulation of CCL17 production
Authors: Achuthan Adrian , Lupancu Tanya , Lee Ming-Chin , Fleetwood Andrew , Cook Andrew , Hamilton John
Published: April 2020

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, which leads to poor quality of life due to the debilitating effect of inflammation. Clinical trials in rheumatoid arthritis targetting the cytokine, granulocyte macrophage‐colony stimulating factor (GM‐CSF) are showing promise although its mode of action remains largely unknown. We have previously shown that that GM‐CSF drives CCL17 production via a new interferon regulatory factor 4 (IRF4)‐dependent pathway in human monocytes and mouse macrophages, as well as in vivo. Importantly, in arthritis and pain models IRF4‐regulated CCL17 formation mediates the proinflammatory and algesic actions of GM‐CSF.

Glucocorticoids (GCs) are potent anti‐inflammatory and immunosuppressive agents broadly used in anti‐inflammatory therapy, albeit with adverse side effects associated with long‐term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action on immune cells.

We report here that GM‐CSF‐induced CCL17 expression is inhibited by GCs in human monocytes and mouse macrophages. Moreover, we provide evidence for the first time that GCs suppress GM‐CSF‐induced IRF4 expression via regulating the expression and activity of JMJD3, which demethylases trimethylated‐H3K27. Further, we demonstrate that GCs suppress the expression of JMJD3, via the recruitment of GC receptor‐alpha to the negative GC response element in the upstream region of JMJD3 gene. Significantly, we measured elevated levels of CCL17 in synovial fluid from patients with RA compared to healthy controls. Using synovial fluid mononuclear cells from RA patients, we provide molecular evidence for the anti‐inflammatory actions of GCs through epigenetic regulation of IRF4 expression and downstream inhibition of CCL17 production. The delineated pathway potentially provides new therapeutic options for the treatment of inflammatory diseases and their associated pain.

Support or Funding Information

NHMRC Project Grant and Seeding Ideas Grant from the University of Melbourne
Determining the Biosynthetic Pathway of Rhodoquinone in C. elegans
Authors: Reinl Samantha R. , Drexelius Julia , Salinas Gustavo , Shepherd Jennifer
Published: April 2020

Parasitic helminths infect more than 1.5 billion people, with third world countries being the most affected. Parasites are transmitted through soil and contaminated water and they feed on host tissue in order to grow and reproduce. Infections cause anemia, malabsorption of nutrients, and loss of appetite. While inside a host, parasites anaerobically respire by using an alternative electron transport chain that uses rhodoquinone (RQ) as an electron carrier instead of ubiquinone (Q), which is used in aerobic respiration. Mammalian hosts produce and use Q for aerobic respiration, but they do not make or require RQ. An ideal anti‐parasitic drug target would be an enzyme that is used only for the biosynthesis of RQ ‐ not the biosynthesis of Q. Caenorhabditis elegans have been used as a model system for parasitic helminths in order to investigate RQ biosynthesis. RNAi feeding experiments were performed to identify genes required for several steps in the RQ biosynthetic pathway in C. elegans. Several genes were identified that are common to both Q and RQ biosynthetic pathways (coq‐3, coq‐5, and coq‐6). However, knockdown of the kynu‐1 gene from the kynurenine pathway affected only RQ production. Additional experiments involving knock‐outs of other C. elegans genes in the kynurenine pathway confirmed the requirement of kynu‐1 and the corresponding arylamine products for the biosynthesis of RQ. The roles ofcoq‐2 and coq‐3 MV genes in the biosynthesis of RQ in C. elegans were further investigated in this study using RNAi knockdowns and characterization was performed with RT‐qPCR and LC‐MS.

Support or Funding Information

Gonzaga Science Research Program and Agencia Nacional para la Innovación y la Investigación ANII FCE_1_2014_104366
Making the Connection: Linking Lecture and Lab Using Drosophila melanogaster in an Introductory Molecular Biology Course
Authors: Rogers Ryan Patricia
Published: April 2020

A common challenge for introductory lab courses is bridging the gap between lecture content and lab experiments. Given that the margin between these areas is dramatically decreasing with the introduction of flipped classroom and experiential learning, I have developed a project that uses in silico and in vivo methods to bridge the two components in an introductory biology course. Over the course of 6 weeks students are challenged to use open access databases to identify an unknown protein and characterize its function and tissue expression patterns. Once the protein is identified, the project shifts from computational to technical by using the genomic DNA sequence to design primers and amplify the corresponding gene from dissected Drosophila tissue. By introducing the project immediately following the module on gene expression students have a basic understanding of transcription, translation, and protein folding, thus they are able to visualize relevant relationships between each concept. As the lecture focus shifts toward cellular processes such as transport and communication, they are able to enhance their understanding of protein structure as it relates to cellular function. Further, they gain an understanding of isoforms and tissue specific expression patterns, thereby enhancing their understanding of gene expression as a whole. Each phase of the project is accompanied by guided analysis questions to help direct students to the appropriate resources and to ensure that they are able to successfully connect each concept. The project culminates in both an oral presentation and a written research paper, which gives students two opportunities to communicate their findings. Both methods emphasize data presentation and analysis in addition to scientific accuracy. Students that successfully complete the project leave the course with an enhanced understanding of transcription, translation, protein folding, and protein function, which are all learning outcomes for the course. Moreover, students develop computational and molecular lab techniques accompanied by enhanced abilities to synthesize related concepts into a larger narrative.

Support or Funding Information

This work was supported by funds from the Department of Sciences at Wentworth Institute of Technology
Kelch‐like Proteins Have A Sweet Spot: Site‐specific Glycosylation Influences Metabolic Regulation and Protein Homeostasis
Authors: Huynh Duc T. , Chen Po-Han , Hu Jimin , Smith Timothy J. , Chi Jen-Tsan , Boyce Michael S.
Published: April 2020

The Kelch‐like (KLHL) family of proteins, a conserved group of 42 human members, binds to E3 ubiquitin ligase complexes to target protein substrates for proteasomal degradation. Although mutations in KLHL genes cause various human diseases—from cancer to neurodegeneration—their functional regulation remains understudied. Post‐translational modifications in general influence protein fates, yet how they modulate KLHL proteins is not fully explored. In this context, our laboratory has examined O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc), a nutrient‐sensing, intracellular glycosylation governed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) that decorates serines (S) or threonine (T) residues in response to cellular stimuli. Using global transcriptional responses to OGT or OGA inhibition and chemical biology approaches, we previously reported that O‐GlcNAcylation of KEAP1, a KLHL protein, is required for the ubiquitination and regulation of the transcription factor NRF2 and the cellular response to oxidative stress. Given the functional conservation of the KLHL family, we hypothesized similar roles for O‐GlcNAcylation in other KLHL proteins, such as gigaxonin that promotes the degradation of intermediate filament (IF) proteins. Human loss‐of‐function gigaxonin mutations are known to cause IF accumulation in neurons, resulting in neuronal swelling and ultimately giant axonal neuropathy, a fatal neurodegenerative disease. By mass spectrometry, immunofluorescence and co‐immunoprecipitation assays, we showed that gigaxonin glycosylation sites at residues S272 and T277 are important for clearing IF aggregates in gigaxonin−/− cells. Taken together, our findings implicate O‐GlcNAc modification of KLHL proteins in governing protein homeostasis of E3 ligase substrates, suggest O‐GlcNAcylation as a general mode of regulation for other KLHL proteins, and inspire future tool developments to detect site‐specific glycosylation changes in human health and disease.

Support or Funding Information

Research on this project in the Boyce Lab is supported by NIH grants R01GM118847 and R01NS111588 and a gift from the Hannah’s Hope Foundation
Isolation of Intricate α‐Synuclein Assemblies Across the Dynamic Landscape
Authors: Lucas Heather R.
Published: April 2020

Cardinal features of Parkinson’s disease (PD) include the development of granular inclusions known as Lewy bodies that are enriched with aggregates of α‐synuclein (αS). αS has long been established as an intrinsically disordered protein, and the array of its physiologically relevant oligomeric conformations is ever‐expanding. In recent years, a higher order conformation was identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant. The monomer and tetramer reside in a dynamic coexistence within cells, yet the canonical mechanism by which the tetramer assembles remains elusive due to the various folding patterns and low energy barriers involved in αS structural transitions. A shift from metastable tetramers to the monomeric form likely serves as a mechanism for disease initiation. The underlying pathology causing this shift remains unknown, but the importance of understanding tetramer stability and disassembly has therapeutic potential that cannot be overemphasized. Hence, a platform for investigating analytical approaches to stabilize tetrameric αS is warranted, as are methods to discriminate αS conformational states. Isolation of tetrameric αS is complicated by its dynamic nature, thus thorough bioanalytical studies on this conformer have been hampered by accessibility issues. We recently developed a robust recombinant expression platform that enables purification of native tetrameric αS without any detergents, crosslinkers, or other structure‐modifying additives by the sequential combination of anion exchange and size exclusion chromatography steps. The successful isolation of this intricate αS conformer is confirmed through various analytical methods, including advanced light scattering techniques, mass spectrometry, immunoblotting, and circular dichroism spectroscopy for secondary structural analyses. The development of multifaceted and innovative approaches that are necessary to tease apart the dynamic landscape of αS quaternary structure will be presented. Moreover, novel methodology to facilitate localized surface plasmon resonance analysis of this intricate conformer will be highlighted as well as the effects of PD‐relevant structural modifiers present in cellular milieu, including metals and/or liposomes, strengthening the application of this platform to advance tetrameric αS as a therapeutic target. The talk will conclude with new insights on strategies to shift the conformational equilibrium towards tetramer in mixed populations as a means to thwart αS misfolding and aggregation.

Support or Funding Information

Supported by NIH R01 GM134015, NCATS UL1TR002649 & CCTR Endowment Fund, VCoA ARDRAF Award No. 18‐7, and NIA L‐13046.
Identification of Proteins that Interact with Otoferlin to build a ‘Otoferlin Interactome’
Authors: Manchanda Aayushi , Bonventre Josephine , Johnson Colin
Published: April 2020

Otoferlin is a C2‐domain transmembrane vesicular protein that has been associated with congenital deafness. It is majorly responsible for facilitating synaptic vesicle exocytosis in cochlear hair cells. Synaptotagmin and Otoferlin have previously been compared to one another for their calcium sensor functions, but hair cells exocytosis is not as well‐understood as neuronal exocytosis. In case of hair cell synapses, we still lack complete knowledge of the SNARE proteins or their equivalent that aid in the fusion of vesicle membranes to the presynaptic membranes, thereby leading to exocytosis. Currently, a major bottleneck in our understanding of Otoferlin’s role in hearing and deafness can be attributed to the lack of knowledge about its mechanism of functioning. Using brain extracts from mouse, we conducted coimmunoprecipitation experiments to find interaction partners for Otoferlin so as to build a Otoferlin interactome, that can shed some light on the mechanism by which hair cell exocytosis actually occurs. The proteins interacting with Otoferlin were identified by Mass spectroscopy and consisted largely of neuronal proteins that share functions with SNARE proteins or had other neurotransmission‐related functions. We use Human Embryonic Kidney (HEK) cells and a zebrafish model to validate the direct interactors of Otoferlin and conduct further assays to determine their role in hair cell exocytosis and/or endocytosis. This knowledge of Otoferlin interactome leads us to a better understanding of the mechanism whereby neurotransmission is carried out in the inner hair cells.

Support or Funding Information

1R01DC014588 (C.P. Johnson) 4/2016‐3/2021 NIDCD (NIH). In vivo and in vitro studies of the deafness associated protein otoferlin
Analytic Comparison of Talc in Commercially Available Baby Powder and in Pelvic Tissues Resected from Ovarian Carcinoma Patients Shows that the Most Frequent Size Class of Baby Powder Particles is the Same at the Most Frequent Size Class in Resections
Authors: Johnson Kurt
Published: April 2020

Using polarized light microscopy (PLM) and scanning electron microscopy (SEM), we measured the size and shape of 1,000 talc particles in samples of talc‐containing baby powder (TCBP) and 200 talc particles in surgically resected pelvic tissues (hysterectomies) from talc‐exposed patients with ovarian carcinoma. The most frequent class of particles in TCBP (aspect ratio 1–3.9, area 1–400 μm2)(79.4% of total) can be unequivocally identified as talc, using both polarized light microscopy, where talc particles are birefringent; and, scanning electron microscopy with energy dispersive X‐ray analysis (SEM/EDX), where talc particles show a spectrum with magnesium, silicon, and oxygen in the correct amounts. The talc particles found in resected tissues from ovarian carcinoma patients are similar in size and shape (aspect ratio 1–3.9, area 1–400 μm2)(83.5% of total) to the most abundant morphological class of particles in TCBP, are birefringent in PLM, and show SEM/EDX signals typical of talc. This finding, combined with previous epidemiological literature and tissue‐based analytical studies provides further evidence that the small, isodiametric particles that dominate TCBP can migrate from the perineum and become lodged in distal structures in the female reproductive tract, where they may lead to an increased risk of developing ovarian carcinoma.
Modulation of TRPC store‐operated channels by Orai and STIM proteins
Authors: Kolesnikov Dmitrii , Glushankova Liubov , Skopin Anton , Gusev Konstantin , Shuyskiy Leonid , Sozonova Svetlana , Kaznacheyeva Elena , Shalygin Alexey
Published: April 2020

Store‐operated calcium entry is significant pathway of calcium concentration rise in cytoplasm. The main players in this process are Orai store‐operated channels, which are activated directly by STIM calcium sensors. Data on participation of TRPC channels in store‐operated calcium entry is controversial.

Study was focused on interaction between STIM and Orai proteins with endogenous TRPC and its role in pathology. We investigated activity of TRPC channels in a model object — HEK293 cells and in the podocytes of the rat model of diabetic nephropathy, using single channel recording patch camp technique.

To eliminate Orai channels activity in HEK293 cells we used several experimental approaches: overexpression of dominant‐negative Orai mutant, Orai genes knockout and knockdown via CRISPR‐Cas 9 system and shRNA. Endogenous TRPC1 channels remain active in cells without functional Orai proteins and become insensitive to store depletion.

In experiments with different extracellular calcium concentration we confirmed that calcium entry is needed for TRPC1 activation by store depletion.

In experiments with calcium, barium or sodium as a charge carrier we observed similarities in dependence of open channel probability versus membrane potential between TRPC1 and Orai channels.

The role of STIM calcium sensors in TRPC1 channels activation was investigated in cells with different expression level of STIM1 and STIM2 proteins.

The findings allow us to propose that STIM proteins don’t activate the endogenous TRPC channels directly. TRPC channels are activated downstream to calcium entry through the Orai channels.

Support or Funding Information

The study was supported by grant of Russian science foundation № 19‐14‐00114 (Glushankova L., Skopin A., Kaznacheyeva E.), and by grant of Russian foundation for basic research № 19‐315‐90065 (Shalygin A., Kolesnikov D.).
Decitabine Reduces Kidney Growth in an ARPKD Mouse Model
Authors: Ginsparg Ari Benjamin , Shan Dan , Kim Harrison , Mrug Michal
Published: April 2020

Autosomal recessive polycystic kidney disease (ARPKD) is a progressive disorder affecting especially kidneys and liver. Using the Cys1cpk/cpk mouse, a commonly used ARPKD phenocopy, we observed an increased association between the rate of renal disease progression and kidney DNA methylation. To test the functional relevance of this association in vivo, among FDA‐approved demethylating drugs we prioritized decitabine based on its cystogenesis‐inhibiting effects and lowest toxicity in our cell‐based model. We treated time‐bred Cys1cpk/cpk mice from postnatal day 7 (P7) to P14 either with a vehicle (DMSO) or decitabine (1mg/kg intraperitoneally one, four, or six days per week). All doses of decitabine reduced MRI‐derived total kidney volume (TKV)‐based indices of cystic disease severity. However, four‐ and six‐day per week dosing was associated with reduced body weight gain. Together, our studies point to decitabine, dosed once per week, as a promising renal cystogenesis‐inhibiting strategy.

Support or Funding Information

The project was funded under NIH R25 DK 112731, NIH/NIDDK 1R01DK097423, NIH/NIDDK P30 DK074038, and VA I01BX002298
Heat Waves Produce Ecologically Relevant Expression Profiles for Temperature‐responsive Genes
Authors: Marroquin-Flores Rosario A. , Mortimer Nathan T. , Paitz Ryan T. , Bowden Rachel M.
Published: April 2020

The red‐eared slider turtle (Trachemys scripta elegans), exhibits temperature‐dependent sex determination (TSD), where sex‐specific genetic profiles are produced in response to incubation temperature. Despite over 50 years of study on this topic, we still do not fully understand how temperature affects the molecular mechanisms underlying TSD, and prior studies aimed at characterizing genes in the sex‐determining pathway have primarily used constant incubation temperatures. Such studies fail to capture the thermal variability that developing embryos naturally experience, but have been used to identify potential candidate genes in the sex‐determining pathway. One candidate, cold‐inducible RNA‐binding protein (Cirp), has sex‐specific expression in several species of turtles and other reptiles under constant incubation conditions. We characterized Cirp expression in the gonads of T. s. elegans embryos exposed to temperatures that are a better mimic of natural nest conditions. All eggs were incubated for the first 24 days at male‐producing temperatures (MPT; 25.0 ± 3°C) before a subset were shifted to female‐producing temperatures (FPT; 29.5 ± 3°C) to induce ovarian development via a simulated heatwave. Embryonic gonads were sampled on day 24, then sampled from both FPT and MPT for five consecutive days. In contrast to results from constant temperature studies, we found that Cirp expression did not differ in embryos at MPT and FPT. We then submitted a subset of our samples for RNAseq to identify genes exhibiting differential expression (DE) in the gonads of embryos at FPT and MPT. A preliminary analysis found 74 genes to be upregulated and 102 genes to be downregulated by day 5 of the heatwave. Similar work using constant temperature incubation found 1887 DE genes in the gonads of embryos at the same stage of development. Only 25 DE genes were shared between the constant and fluctuating temperature datasets. Our initial comparison of gene expression under constant and fluctuating conditions suggests that while constant incubation conditions result in many more DE genes, many of these genes may not be differentially expressed under more natural incubation conditions. Further, these findings highlight the importance of using ecologically relevant conditions to identify genes expressed during early gonadogenesis in TSD species.

Support or Funding Information

American Physiological Society William Townsend Porter Pre‐Doctoral Fellowship, Society for Integrative and Comparative Biology Grants‐in‐Aid‐of‐Research, Beta Lambda Phi Sigma Biological Honors Society Weigel Grant
3D Printed Microfluidics for Studying Sleep Pathways in Caenorhabditis elegans
Authors: Honer Madison , Fingerut Jonathan , Nelson Matthew D.
Published: April 2020

Despite sleep being essential and highly conserved at the genetic level, a thorough understanding of the mechanisms of sleep regulation and its core function are lacking. Caenorhabditis elegans, a genetically tractable nematode worm, is a powerful model system for addressing these questions. Conserved neurochemistry and a compact 302‐celled nervous system allow for the identification and rapid characterization of novel sleep‐regulating genes of relevance to more complex animals. Orcokinin neuropeptides, predominantly found in ecdysozoan animals, have been implicated during the regulation of circadian rhythms and molting, however, a role during sleep regulation has not been described. In C. elegans the genes nlp‐14 and ‐15 code for peptides predicted to be orcokinin orthologs. We sought to determine if these peptides control a behavior called stress‐induced sleep (SIS), which occurs following incidents of noxious stimuli that damages their cells. Using a combination of loss‐of‐function and gain‐of‐function approaches, we find that nlp‐14 and nlp‐15 play a central role during SIS, regulating unique aspects of behavioral quiescence. To identify an orcokinin receptor, we have been conducting forward genetic screens to isolate mutants that suppress a strong gain‐of‐function sleep phenotype of nlp‐14. In the past, we employed a manual screening protocol that required an abundance of time and resulted in a high false‐positive rate. To improve upon this, we fabricated screening devices using 3D printing, which allows for higher throughput and selectivity. These microfluidic devices use gravity‐based sorting chambers which are both cheap and easily employed. In these chambers we place mutagenized animals, induced to fall asleep by nlp‐14 over‐expression, in a lower sorting chamber. Sleeping animals are trapped in the lower chamber, while suppressor mutants display gravitaxis behavior, the ability to swim against gravity, thus, swimming to an upper collection chamber. These rare suppressor mutants are then isolated for whole‐genome sequencing in order to reveal the essential genes involved in these sleep‐regulating pathways.

Support or Funding Information

NSF Award #1845020 (PI Nelson)
AKR1B1‐Induced epithelial‐mesenchymal transision Mediated by RAGE‐ Oxidative Stress in the Lens of DM Cataracts
Authors: Cheng Pei-Wen , Chen Ying-Ying , Wu Tsung-Tien
Published: April 2020

Background and purpose

Cataracts in patients with diabetes are a major cause of blindness in developed and developing countries. It’s been known that lens epithelial cells (LECs) would undergo epithelial‐mesenchymal transision (EMT) that formed to cataract. Great deals of studies have demonstrated that overproduction of AKR1B1 and RAGE played a central role in the pathogenesis of diabetic cataract. The aim of this study was to examine whether EMT formation in the DM cataract through an AKR1B1 enhanced AGE and ROS generation in LECs from DM patients.

Methods

Fluorescence was used to estimate the AKR1B1, RAGE, AMPK and EMT biomarkers in the human lens epithelial sections from DM or non‐DM cataracts. All data were expressed as the mean ± SEM (n=12 for each group). A one‐way analysis of variance (ANOVA) with Scheffe’s post hoc comparison was employed to compare the group differences.

Results

Immunohistochemistry staining founding that the pathologic phases and N‐cadherin expression levels were significantly enhanced in the human lens epithelial sections from DM (+) than DM (−) cataract patients. Immunofluorescent staining also demonstrated that AKR1B1 and RAGE were significantly enhanced in the human lens epithelial sections from DM (+) than DM (−) cataract patients. Interestingly, AcSOD2 levels were significantly higher in the LECs of the patients with DM, not in non‐DM cataracts, however, the phosphorylation level of AMPKT172 was has reverse effect. These results indicated that AMPKT172 may be required for reduced superoxide generation and the pathogenesis of diabetic cataract formation.

Conclusions

Collectively, our results revealed that AKR1B1 overexpression would abolish AMPK activation, thereby increasing AcSOD2 and RAGE‐induced EMT formation in the LECs of diabetic cataracts as potentially important elements in the pathogenesis of diabetic cataract.

Support or Funding Information

108‐2320‐B‐075B‐002 ‐