bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThis 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 InformationMaximizing 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 Information1) 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 InformationProtein 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 InformationThe 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 InformationNHMRC 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 InformationGonzaga 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 InformationThis 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 InformationResearch on this project in the Boyce Lab is supported by NIH grants R01GM118847 and R01NS111588 and a gift from the Hannah’s Hope Foundation