bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Unclumping Assay in Drug Discovery: Sodium Sulfate
Authors: Zem Greg , Crocker Kristel , Deleon Jonie , Telliyan Lucy , Aprelian Kevin , Rosenberg Aryeh , Pouri Nikole , Beltran Gerard , Ramirez Vivian , Kaufman David , Petrosyan Arpineh , Nazarian Deanna , Magistrado Monikajane , Matinian Suren , Hanna Daniel , Eskandari Sera , Atanante Faisal , Nerses Adees , Oppenheimer Steven B.
Published: April 2020

Clumped cells are generally more dangerous than single cells in cancer spread, thrombocytosis and biofilm infectivity. Here a simple 3 component direct kinetic assay is used to examine 2 reagents for anti‐clumping activity using a Prefer (Anatech Ltd., Battle Creek, MI) fixed yeast (Saccharomyces cerevisiae) model that has been recently described by us in detail using other reagents (Amer J Applied Scientific Research 5:28–34 March 2019). In 1212 trials by 17 investigators sodium sulfate and sodium bicarbonate (1–3 mg per ml deionized water) were examined by measuring percentage single cells, number of clumps and number of cells per clump over a 60 min time course, with standard deviations and t‐tests to determine any significant differences between controls and experimentals. Both reagents showed sometimes inconsistent unclumping activity especially in magnitude of effects. When percentage of single cells increased, clump number and/or number of cells per clump generally decreased, helping to validate the assay. An example of these findings in 60 trials at 60 min with 1–3 mg sodium sulfate per ml deionized water: 1 mg 15% increased singles (p<0.01), 29% decreased clumps (p<0.01), 11% decreased cells per clump (p>0.05); 2 mg 12% increased singles (p<0.01), 20% decreased clumps (p<0.01), 30% decreased cells per clump (p<0.01); 3 mg 27% increased singles (p<0.01), 36% decreased clumps (p<0.01), 28 % decreased cells per clump (p<0.02).

Here sodium sulfate showed promise as an anti‐cell‐clumping reagent together with sodium citrate reported previously (Amer J Applied Scientific Research 5: 28–34 March 2019). Sodium citrate is a known human anticoagulant (Platelets 29 (2018) 21–26) independently identified with this assay, helping to validate the assay for drug discovery applications.

Support or Funding Information

Supported by California State University, Northridge, Department of Biology and Center for Cancer and Developmental Biology
Effect of Hypoxia on RAGE Signaling in Pancreatic Cancer
Authors: Taneja Sakshi , Vetter Stefan , Leclerc Estelle
Published: April 2020

Pancreatic cancer (PC) is one of the deadliest cancers owing to its late diagnostic and poor response to chemotherapeutic agents. PC tumors are characterized by large hypoxic regions that contribute to tumor growth, formation of metastasis and resistance to chemotherapeutic agents. Studies have shown that the Receptor for Advanced Glycation End products (RAGE) is upregulated in hypoxic conditions. The goal of this study was to determine the effect of hypoxia on RAGE signaling in two pancreatic cancer cell lines (Panc‐1 and CFPAC). In particular we investigated if hypoxia dependent RAGE upregulation could aftect RAGE signaling and lead to NF‐κB activation. To interrogate RAGE signaling, we first investigated the effect of hypoxia on KRAS upregulation, as well as on ERK and NF‐KB activity levels by Western blot analysis. We use cobalt chloride to create hypoxic‐like conditions by stabilizing HIF‐1α in cells. We confirmed up‐regulation of HIF‐1α and RAGE in cells incubated with cobalt chloride. We also observed increased expression levels of KRAS in hypoxic‐like conditions. Additionally, higher levels of NF‐κB (p65) and of phosphorylated ERK were observed in cells treated with cobalt chloride as compared to untreated cells. In many cell types, RAGE signaling leads to NF‐κB activation resulting in further RAGE upregulation through a feedforward mechanism. Based on these observations, we hypothesize that in hypoxic conditions, stabilization of HIF‐1α leads to RAGE upregulation and signaling, resulting in NF‐κB activation through the activation of KRAS and ERK. We will use a small molecule RAGE inhibitor (FPS‐ZM1) to interrogate the role of RAGE in the proposed KRAS/ERK/NF‐κB pathway.

Support or Funding Information

Department of Pharmaceutical Sciences, College of Health Professions, NDSU, NIH‐COBRE: P20GM109024
Effects of Gold Nanoparticles on Capillary Tube Formation with Special Reference to the Alteration of Pericyte Morphology and Function
Authors: Looprasertkul Sasikarn , Sereemaspun Amornpun , Jindatip Depicha
Published: April 2020

Pericytes are perivascular cells that wrap around endothelial cells to form wall of capillaries and post‐capillary venules. These cells are primary importance in physiological and pathological processes, especially, involved in angiogenesis. At present, gold nanoparticles (AuNPs) were used for anti‐angiogenesis by inhibiting the activity of endothelial cell proliferation, migration, and tube formation. However, the effects of gold nanoparticles on the alteration of pericyte morphology and theirs function were not reported. Therefore, in this study, 20 nm‐AuNPs were applied to human placenta pericyte monoculture with concentration of 30 ppm. Morphology of pericytes was observed by transmission electron microscopy and cell migration was evaluated by transwell migration assay. Real‐time qRT‐PCR was used to determine Ki‐67, as a proliferation marker, and PDGFR‐β mRNA expression. Moreover, these AuNPs 30 ppm‐treated pericytes were co‐cultured with human umbilical cord endothelial cells on Matrigel® to investigate the 3D tube formation. The results revealed that AuNPs significantly suppressed migration, proliferation, and PDGFR‐β mRNA expression of pericytes. The ultrastructure of pericytes showed AuNPs in several organelles, i.e., swollen or damaged mitochondria, engulfed late endosomes and lysosomes. Moreover, the number of capillary tube formations was essentially reduced and we found that a number of pericytes on these capillary tubes were round shape and did not extend the processes along the tube, which in turn increased incomplete tube formation compared with the control. In conclusion, AuNPs could effect on pericyte biology including proliferation, migration, morphology, and tube formation property. Moreover, the results highlighted the potential application of AuNPs in anti‐angiogenesis by pericyte‐targeted therapy.

Support or Funding Information

The Ratchadaphiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant number RA61/100 and Overseas Academic Presentation Scholarship for Graduate Students from the Graduate School, Chulalongkorn University
Developmental Changes in Total Cardiac mRNA: RNA Complicates Interpretation of Reference Genes
Authors: Jonker Sonnet S , Espinoza Herbert M
Published: April 2020

Finding genes that do not vary with age for use as reference or “housekeeping” can be difficult, as can understanding what experimental gene normalization means in a developmental context. We analyzed expression levels of several potential reference genes in the developing sheep heart, as well as global myocardial mRNA levels compared to total nucleic acid levels. Total nucleic acid, total RNA, messenger RNA (mRNA), and individual gene levels were determined by spectroscopy, dot blot, and PCR. Results were analyzed by ANOVA followed by Tukey’s test, and results reported at P<0.05. Potential reference genes hyaluronidase 2, beta‐2‐microglobulin and ribosomal protein L37a were all found to vary by developmental age. The ribosomal RNA 18s did not vary with age. While total RNA relative to total nucleic acid was constant across development, mRNA was a greater fraction of total RNA in the late‐term fetal heart. These results demonstrate of the difficulty of interpreting normalization of myocardial mRNA expression levels across development.

Support or Funding Information

R01HL142483
Exploring the Physiological Roles of Unsaturated Fatty Acids Using C. elegans
Authors: Kessler Benjamin , Alan Jamie , Lee Kin Sing Stephen
Published: April 2020

Monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), classified broadly as unsaturated fatty acids, are macronutrients that have a wide range of physiological effects. It is known that unsaturated fatty acids play a crucial role in a diet, but it is unclear which specific MUFAs and PUFAs are required and what the physiological consequences of having different amounts of these fatty acids and their metabolites in the body are. Specific omega‐3 and omega‐6 PUFAs have been shown to be beneficial to human diseases, such as cardiovascular disease, inflammatory conditions, and certain cognitive impairments. This study aims to investigate the physiological roles of individual MUFAs and PUFAs. From this study, we will gain the preliminary knowledge for making dietary and treatment suggestions for patients with various disease states and in normal aging. To do this, we will use the model organism C. elegans because of its short lifespan and abundance of genetic tools available (including single and multiple genetic knockouts of fatty acid desaturase enzymes). Additionally, findings from studies in C. elegans reliably translate to human disease. Preliminary data suggests that specific MUFAs and PUFAs are essential for normal lifespan, physiological functions, and neurodevelopment. This study will expand on this by examining every available genetic knockout of fatty acid desaturase enzyme in the worm. We will conduct lifespan assays in order to observe changes in the median and maximum lifespans of different genetic knockouts compared to the wild type as well as thrashing assays, a measure of physical fitness. We hypothesize that certain unsaturated fatty acids play more important physiologic roles than others and the corresponding downstream metabolites are important lipid signaling molecules. Preliminary results from the lab confirm these hypotheses, as the genetic strains tested thus far show variations in lifespan and fitness.

Support or Funding Information

This project was funded by the MSU Startup Fund, NIEHS ‐ R00 ES024806, and NSF ‐DMS‐1761320.
Investigation of the role of GABARAP family proteins in cargo recruitment to the autophagosome
Authors: Amos Shandon , Jensen Liv , Hurley James
Published: April 2020

Autophagy is a conserved cellular process critical for removing cellular waste and recycling nutrients. During autophagy cargo is engulfed by a crescent shaped membrane called the phagophore and subsequently degraded into reusable material. There are two types of autophagy: selective and bulk. Bulk autophagy is a non‐specific process, whereas in selective autophagy, only cargo tagged for degradation is enveloped by the phagophore. A defining characteristic of selective autophagy is the relationship between Atg8‐like membrane proteins and cargo adaptor proteins. A classically studied example of this relationship in mammalian systems is the interaction between cargo adaptor, p62 and Atg8‐like protein, LC3B. It has been shown that LC3B binds to the LC3‐Interacting Region (LIR) in p62. This interaction has been studied extensively, but mammals have two subfamilies of Atg8‐like proteins: LC3 and GABARAP. GABARAP family proteins have significant homology with LC3B and therefore should have binding affinity for proteins with LIR motifs. However, the interaction between GABARAP family proteins and cargo adaptors is not well characterized. The aim of this work is to investigate the ability of GABARAP family proteins to bind to synthetic lipid membranes, and recruit cargo adaptor proteins to these membranes. We used biochemical assays and fluorescence imaging to characterize the activity of GABARAP family proteins in vitro. Our preliminary results show via pulldown that GABARAPL2 has less affinity for cargo adaptor proteins, p62 and NDP52, than LC3B. We also demonstrated using fluorescence microscopy that GABARAPL2 is readily conjugated to lipid vesicles by Atg7, Atg3, and the Atg 5/12/16 complex. Taken together, these results show GABARAPL2 has a different role than LC3B in cargo recruitment to the autophagosome, although the specific role has yet to be elucidated.

Support or Funding Information

This investigation was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC.
Tissue‐specific roles of cardiolipin in the control of systemic energy homeostasis
Authors: Gerhart-Hines Zachary
Published: April 2020

The mitochondrial inner membrane is a dynamic interface that supports bioenergetic processes, intracellular communication, and transport of metabolites, lipids, and ions. The phospholipid, cardiolipin, is one of the most enriched components of this membrane and is classically ascribed an indispensable role in mitochondrial structure and function. Disruption of cardiolipin biology is causally linked to numerous pathophysiologies, including diabetes, nonalcoholic fatty liver disease, Parkinson’s disease, and Barth syndrome. Yet how cardiolipin controls metabolic function of individual mammalian tissues remains largely unknown. Therefore, we generated conditional genetic models with cardiolipin synthesis deficiency in tissues with predominantly uncoupled (i.e. brown adipose) or coupled (i.e. skeletal muscle) mitochondria. We found that tissue‐specific loss of cardiolipin had dramatically different outcomes on respiration and systemic energy homeostasis. Cardiolipin deficiency in brown adipose significantly reduced organismal insulin sensitivity and glucose tolerance, whereas loss of cardiolipin in skeletal muscle unexpectedly improved glycemic control. Moreover, in contrast to the dogmatic belief that cardiolipin is ubiquitously required, we found that certain skeletal muscle fiber types retained respiratory capacity in the face of cardiolipin depletion through mitochondrial biogenesis and fiber‐type switching. Our collective findings on tissue‐specific roles of cardiolipin reshape the fundamental understanding of mitochondrial structure and function with potential therapeutic implications for phospholipid disorders.

Support or Funding Information

Novo Nordisk Foundation and the European Research Council (aCROBAT 639382)
An AAA‐ATPase using an airlock‐like translocation mechanism for folded proteins
Authors: Beckmann Roland
Published: April 2020

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly enigmatic. Here, we present the structure of an AAA‐ATPase of the inner mitochondrial membrane, which facilitates the translocation of a protein, which requires folding and incorporation of a Fe2‐S2 cluster before translocation and subsequent integration into a larger complex. Surprisingly, this AAA‐ATPase assembles into exclusively heptameric homo‐oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together, they form two aqueous vestibules, the first accessible from the mitochondrial matrix and the second positioned in the inner membrane, both separated by the seal forming middle domain. Based on this unique architecture, we propose an airlock‐like translocation mechanism employed by this exceptional ATPse.
Rhomboid Proteins in Cell Signaling
Authors: Freeman Matthew
Published: April 2020

Cellular membranes are important biological interfaces. Not only do they compartmentalise cells from the environment and organelles from each other, but they also act as signalling platforms. Membrane proteases mediate many of their functions and are primary triggers of signalling. Rhomboids, which are intramembrane serine proteases, are a relatively recently discovered family of membrane proteases that control both signalling and membrane quality control. Rhomboid proteases are members of a much wider rhomboid‐like superfamily, most of which are pseudoproteases, having lost their proteolytic activity.

The bottleneck in understanding the biological role of rhomboid proteases is identifying their substrates. This has proved to be a difficult problem with all proteases and there is no single definitive approach. We have now successfully developed a combined bioinformatic and experimental pipeline to reveal the substrates if mammalian rhomboids. This has led us to uncover a role for rhomboids in controlling calcium signalling and I shall discuss this novel mechanism of signalling homeostasis, as well as its physiological consequences in human cells.
piRNA Biogenesis and Function in Drosophila
Authors: Siomi Mikiko
Published: April 2020

PIWI‐interacting RNAs (piRNAs) repress transposons to protect the germline genome from their harmful invasion. To this end, piRNAs stoichiometrically associate with members of the PIWI protein family to form piRNA‐induced silencing complexes (piRISCs) and target them to their RNA substrates via Watson‐Crick (RNA‐RNA) base pairing. Drosophila expresses three PIWI members, Piwi, Aubergine (Aub), and Argonaute3 (Ago3); the lack of any of these members causes transposon derepression, leading to severe defects in gonadal development and infertility, showing their functional non‐redundancy. In Drosophila ovarian somatic cells (OSCs), transposon‐repressing piRNAs originate from a soma‐specific piRNA cluster flamenco (flam) on the X chromosome. The flam locus is rich in transposon remnants, whose directions of insertion at the locus are mostly opposite to the direction of flam transcription. Therefore, by their nature, flam‐derived piRNAs are created to act as antisense oligonucleotides to silence their parental transposons. OSCs express Piwi but no other PIWI proteins. The Piwi‐piRISC assembly takes place in the cytoplasm. The complex is then imported into the nucleus and represses transposons at the transcriptional level. In the current model, Armitage (Armi) brings the Piwi‐piRISC precursor (pre‐piRISC) to mitochondria, where Zucchini‐dependent piRISC maturation occurs. We recently found that Armi is necessary for Piwi‐pre‐piRISC formation at Yb bodies and that Armi triggers the exit of Piwi‐pre‐piRISC from Yb bodies prior to translocation to mitochondria. Piwi‐pre‐piRISC resists leaving Yb bodies until Armi binds Piwi‐pre‐piRISC through piRNA precursor. Lack of Armi N‐terminus also blocks the Piwi‐pre‐piRISC exit from Yb bodies. Thus, Armi determines Piwi‐piRISC processing, in a multilayered manner, from precursor formation and quality control to inter‐organelle translocation for maturation.
The role of Organelle Contact During Chlamydia Developmental Cycle
Authors: Derré Isabelle
Published: April 2020

Elucidating the molecular mechanisms supporting host‐pathogen interactions is a crucial step towards the eradication of infectious diseases. We study the obligate intracellular bacterium Chlamydia trachomatis, which is the leading cause of sexually transmitted infection of bacterial origin in the United States. Upon invading the epithelium of the genital tract, Chlamydia completes its life cycle in a membrane‐bound vacuole, called the inclusion. To convert the inclusion into an intracellular niche favorable for survival and replication, Chlamydia has evolved sophisticated mechanisms to manipulate the eukaryotic cell. Many studies have focused on understanding how Chlamydia, intercept the vesicular trafficking of the host cell. However, our laboratory has shed light on sites of intimate contact, with no fusion occurring, between the Chlamydia inclusion membrane and the endoplasmic reticulum (ER). Based on similarities to inter‐organelles membrane contact sites (MCS) observed in naïve cells, we refer to these structures as ER‐Inclusion MCS. Over the past several years, our lab has identified several molecular components of ER‐Inclusion MCS. More specifically, we have uncovered a role for Inc proteins (a subset of Chlamydia specific proteins that are embedded into the inclusion membrane) in the subversion of the endoplasmic reticulum. The recurrent theme is that, at ER‐Inclusion MCS, distinct Inc proteins mediate the recruitment of and associate with specific host factors that usually associate to distinct MCS in naïve cells. Here, we will discuss our current understanding of how these Inc protein‐host factor complexes contribute to the formation and function of ER‐Inclusion MCS and the establishment of Chlamydia replicative niche.

Support or Funding Information

This work is supported by NIH‐NIAID grants R01 AI101441, R21 AI141841 and R03 AI146649.
Structure of the Alternative Complex III from Flavobacterium johnsoniae in a Supercomplex with Cytochrome c Oxidase
Authors: Gennis Robert , Sun Chang , Benlekbir Samir , Venkatakrishnan Padmaja , Wang Yuhang , Hong Sangjin , Hosler Jonathan , Tajkhorshid Emad , Rubinstein John L.
Published: April 2020

Alternative Complex III (ACIII), like Complex III, catalyzes the oxidation of membrane‐bound quinol and reduction of cytochrome c, but is structurally unrelated to Complex III. We solubilized the ACIII directly from membranes of Flavobacterium johnsoniae using styrene maleic acid (SMA) copolymer in the absence of traditional detergents. The ACIII was isolated as a functional 1:1 supercomplex with an aa3‐type cytochrome c oxidase (cyt aa3) within SMA copolymer nanodiscs. We determined the structure of the ACIII component of the supercomplex to 3.4 Å resolution by cryo‐EM and constructed an atomic model for its six subunits, two of which are anchored to the lipid bilayer with N‐terminal triacylated cysteine residues, resolved here for the first time. The structure also contains a [3Fe‐4S] cluster, a [4Fe‐4S] cluster, and six hemes c along with 11 phospholipid molecules. The ACIII is in direct contact with subunit III of the cyt aa3 component of the supercomplex. The structure revealed that this subunit is structurally modified from the canonical form of subunit III to facilitate association with ACIII, suggesting a specific role of the supercomplex in the respiratory system of this bacterium.

Support or Funding Information

This work was supported by funds from the National Institutes of Health (R01‐HL16101 to R.B.G., P41‐GM104601, U54‐GM087519, and R01‐GM123455 to E.T.) and Canadian Institutes of Health Research (MOP‐81294 to J.L.R.); J.L.R. was supported by the Canada Research Chairs program. Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy, supported by grants from the Simons Foundation (349247), and the National Institute of General Medical Sciences (GM103310, OD019994). Molecular dynamics simulations were performed at Blue Waters (ACI‐1713784 to E.T.) and XSEDE (MCA06N060 to E.T.). Blue Waters is supported by the National Science Foundation (OCI‐0725070 and ACI‐1238993) and the state of Illinois. XSEDE is supported by the National Science Foundation (ACI‐1548562). We thank Mark Mcbride for kindly providing us with the Flavobacterium johnsoniae UW101 strain. We thank Bridget Carragher and Clint Potter for facilitating access to the Titan Krios and Zhening Zhang for collecting data. We also thank Mohammad Mazhab‐Jafari for advice on building atomic models.
The Role of tRNA Derived Small RNAs in Gene Regulation in Normal Tissues and Cancer
Authors: Kay Mark A.
Published: April 2020

The function of tRNAs is not merely to provide the specific amino acids for a growing peptide chain during protein synthesis. Precursor and mature tRNAs can be enzymatically cleaved into various tRNA derived small RNAs. While there are thousands of different RNA species that can be generated, the function of most are not known. I will provide a brief introduction into this field and describe the mechanism of how at least one specific tsRNA derived from the 3′end of a mature tRNA is involved in regulating ribosome biogenesis (Kim et al. Nature 2017). This 3′tsRNA is upregulated in many tumors. Reducing the tsRNA in patient derived hepatocellular carcinomas using antisense oligonucleotides induced apoptosis and tumor shrinkage in mice. This as well as other 3′tsRNAs are being studied as potential targets for cancer therapeutics. I will provide an update on current approaches we are developing to help us identify, quantify, and define the targets of the myriad of various tsRNAs present in various cell types. This will further allow investigation as to their role in maintaining health and disease states.

Support or Funding Information

National Institutes of Health ‐ National Institute of Diabetes and Digestive and Kidney Diseases R01‐DK114483
The Piwi‐piRNA Pathway: a New Paradigm in Gene Regulation
Authors: Lin Haifan
Published: April 2020

Small non‐coding RNAs have been recognized as key players in gene regulation. In 2006, we and others discovered a novel class of small RNAs that interact with Piwi proteins in the germline of diverse organisms. These Piwi‐interacting RNAs (piRNAs), mostly 26–32 nucleotide in length and correspond to all types of genomic sequences, represent a distinct small‐RNA pathway. In my talk, I will report our recent progress that reveal the crucial roles of the Piwi‐piRNA pathway in mediating the regulation of mRNA and lncRNA stability by transposons and pseudogenes, which represent a new paradigm of regulation among the major constituents of the genome.

Support or Funding Information

This work was supported by the NIH grant R37HD42012 and a Mathers Foundation award.
Promoting Mental Wellbeing
Authors: Vanderford Nathan L.
Published: April 2020

Current reports indicate that 30–50% of academic trainees are dealing with some level of mental health issues. This presentation will highlight the breadth and depth of the mental health concerns in the academic trainee population. Methods of promoting and supporting system‐ and department‐level changes to address these issues will be discussed.
A revolution in biochemistry and molecular biology education informed by basic research to meet the demands of 21st century career paths
Authors: Black Paul N.
Published: April 2020

The National Science Foundation estimates that 80% of the jobs available during the next decade will require math and science skills, which requires programs in biochemistry and molecular biology to be transformative using new pedagogical approaches and experiential learning as foundations for careers in industry, research, intellectual property law, education, engineering, health‐care professions and other interdisciplinary fields. These efforts expect a scholarly environment that values the individual student, integrates current trends from the primary literature to provide essential fundamentals in the discipline and experimentally‐directed research, collection and analysis of data and scientific writing. Current trends in the discipline dictate these efforts must necessarily include critical thinking, experimental‐testing, computational‐modeling and inferential logic. In essence, modern biochemistry and molecular biology education must informed by, and integrated with cutting edge research. This environment relies on internal and external research support, commitment by members of the faculty to provide the requisite mentoring, access to instrumentation and state‐of‐the art facilities. New academic environments and resource centers must be provided to establish a culture of pedagogical excellence and faculty engagement leading to innovation in the classroom and laboratory through teamwork and active learning. These efforts must not lose sight of the importance of multidimensional programs that enrich science literacy in all facets of the population, including the general public, students and teachers in K‐12 schools, non‐biochemistry and molecular biology students and other stakeholders. As biochemistry and molecular biology educators, there is an obligation to provide students will the skills that allows them to be innovative and self‐reliant. The next generation biochemistry and molecular biology students must be taught the importance of being scientifically and technologically literate, importance of scientific discourse, and skills required to be problem solvers of the 21st century.
Synthetic NF‐kB: a Building Approach to Study Complex Signaling Behaviors
Authors: Wei Ping
Published: April 2020

Quantitative features of cellular signaling behaviors have drawn much attention due to their capabilities to carry extra information to regulate comprehensive downstream gene expression, and thus determine dramatically different cell fates. Remarkably, such quantitative signaling behaviors were often oscillatory or pulsatile. The particular properties of oscillatory signaling dynamics (e.g., amplitude, frequency) have shown great benefit to coordinate or differentially regulate gene expression in stress signaling and inflammatory response. Due to the complexity and redundancy in natural cells, it remains challenging to completely understand how the complex temporal behaviors are programmed in parameters or structures of the signaling circuits. Here, we took a synthetic approach to study the underlying principles of such complex signaling behaviors, via a reconstituted human nuclear factor κB (NF‐κB) system in S. cerevisiae. This synthetic NF‐κB signaling circuit can robustly oscillate with extensive tunability, which allows us to systematically explore the signaling capabilities by tuning the circuit structures and parameters. Our synthetic NF‐κB represents a general inhibitor‐transcriptional factor model design. The principles revealed in this synthetic system could facilitate our understandings in similar natural signaling systems.
RNA Modification in Cancer
Authors: Chen Jianjun
Published: April 2020

N6‐methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNAs, has been shown to play essential roles in various normal bioprocesses. We show recently that histone H3 trimethylation at Lys36 (H3K36me3) guided m6A deposition co‐transcriptionally. Evidence is emerging that m6A modification and the associated regulatory proteins also play critical roles in cancers. We have revealed the important functions of m6A regulatory proteins (e.g., FTO, METTL14 and IGF2BP1/2/3) in the development and drug response of cancer, especially acute myeloid leukemia (AML), and in the self‐renewal of leukemia stem cells (LSCs). We found that FTO is highly expressed in certain subtypes of AMLs including AMLs carrying t(11q23), t(15;17), NPM1 mutation, and/or FLT3‐ITD, in which FTO plays an essential oncogenic role in promoting leukemogenesis and in affecting drug response through post‐transcriptionally regulating expression of its critical target RNAs (such as ASB2 and RARA) in an m6A‐dependent manner. Subsequently, we found that FTO is also a direct target of R‐2‐hydroxyglutarate (R‐2HG), which is produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes and has been reported as an oncometabolite. We show that by targeting the FTO/m6A/MYC‐CEBPA axis, R‐2HG exhibits a broad and intrinsic anti‐tumor effect in vitro and in vivo. We also show that METTL14 is overexpressed in AML and plays a critical role in the development and drug response of AML, and is also required for the self‐renewal of leukemia stem cells, by post‐transcriptionally regulating of expression of a set of essential oncogenes such as MYC and MYB. Very recently, we have developed highly selective and effective FTO inhibitor compounds that shows potent therapeutic effects in treating human AML and solid tumors in preclinical animal models, demonstrating that as an mRNA demethylase, FTO is a druggable target for cancer treatment. Collectively, our studies highlight the critical roles and therapeutic implication of m6A modification and its regulatory proteins in cancers.

Support or Funding Information

This work is supported in part by the National Institutes of Health (NIH) Grants R01 CA214965 (J.C.), R01 CA236399 (J.C.), R01 CA211614 (J.C.). J.C. is a Leukemia & Lymphoma Society (LLS) Scholar.
Control of Macrophage Activation by Coenzyme A
Authors: Divakaruni Ajit S.
Published: April 2020

Macrophages are innate immune cells responsible for processes as varied as killing pathogens, initiating wound‐healing programs, and presenting antigens to T cells. To achieve this broad range of functions, they acquire pro‐ or anti‐inflammatory programs (or ‘polarize’) by integrating intrinsic, extrinsic, and environmental signals. It is now well established that metabolic changes are critical for this process, though it remains unclear precisely which metabolic alterations are indispensable for (rather than merely being associated with) activation.

Pro‐inflammatory macrophage polarization with lipopolysaccharide (LPS) collapses mitochondrial ATP production. This profound decrease in oxidative phosphorylation is associated with increases in glycolysis, signaling metabolites from the TCA cycle, and mitochondrially derived redox signals. Conversely, anti‐inflammatory activation with interleukin‐4 (IL‐4) is associated with substantial increases in oxidative phosphorylation and fatty acid oxidation (FAO). Previous work in our laboratory has shown that although FAO and mitochondrial ATP production are strongly associated with the IL‐4 response, these are surprisingly dispensable, whereas coenzyme A homeostasis is obligatory [Divakaruni et al. (2018)
Cell Metabolism]. This presentation will cover our current understanding of the essential metabolic requirements of macrophage activation, with a focus on the potential mechanisms by which coenzyme A may control the IL‐4 response.
Using narrative in STEM education
Authors: Lansiquot Reneta
Published: April 2020

Interdisciplinary pedagogy, especially team‐taught courses, can advance STEM success for all students. This presentation focuses on three pedagogical exemplars: learning communities, co‐teaching, and team teaching. It explores my use of narrative, both written and in virtual worlds, in interdisciplinary courses over the past decade. I will also illustrate how to apply the insights of interdisciplinary pedagogy to STEM education.
The Role of VPS13 and Related Proteins in lipid transport at membrane contact sites
Authors: Reinisch Karin
Published: April 2020

VPS13 proteins, whose dysfunction in humans underlies neurological disorders including Huntington‐like and early onset Parkinson’s diseases, are lipid transport protein active at membrane contact sites. VPS13 and proteins related by the presence of a chorein_N sequence, such as the autophagy protein ATG2, constitute a novel class of lipid transporters that solubilize tens of lipids within a wide hydrophobic channel. An intriguing possibility is that these proteins are bridges across which glycerolipids traverse the cytosol between membranes.
Tuning receptor signaling through ligand engineering
Authors: Garcia Chris
Published: April 2020

I will present the results of studies in our laboratory investigating the role of extracellular ligand‐receptor complex structure on the nature of signaling outputs in a range of systems. Most cell surface receptors are activated by some degree of ligand‐mediated clustering or oligomerization, but it is less clear to what degree the nature of the downstream signal induced by ligand can be ‘tuned’ or manipulated by natural or engineered ligands that engage the receptors with alternative geometries or binding parameters. Typically, ligands that elicit distinct pharmacologies through a single receptor have been observed GPCRs, but we have engineered ligands for T cell and cytokine receptors that exhibit partial agonism and inverse agonism. As one example, we have modulated cytokine receptor signaling by altering the orientation and proximity of a cytokine receptor dimer using diabodies and other synthetic designed scaffolds as surrogate ligands. We directly correlated the intact receptor dimer structures, determined crystallographically, with functional outcome and we find that indeed such systems are ‘tunable’ by remodeling dimer topology and distance. Thus, induced alterations in receptor dimer geometry can ‘tune’ signaling and extracellular ligands that enforce large receptor inter‐subunit distances can counteract intracellular oncogenic ligand‐independent receptor activation. The approach of modulating signaling output through receptors is important to overcoming the therapeutic limitations of cytokine pleiotropy. In my talk I will discuss recent results bearing on this topic.

Support or Funding Information

1Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA. 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA.
Sugary Coats: Synthesis and Secretion of Extracellular Polysaccharides
Authors: Zimmer Jochen
Published: April 2020

Complex carbohydrates are frequently deposited on the cell surface to form cell walls, capsules, or 3‐dimensional matrices called biofilms. Often, the high molecular weight polymers are synthesized intracellularly and transported across at least one biological membrane by dedicated translocation machineries. We are interested in how polysaccharides are assembled and deposited on the cell surface. In particular, bacterial cellulose is a major biofilm component of many enterobacteria and secreted during its synthesis by the membrane‐embedded cellulose synthase, a processive glycosyltransferase. Using the tools of structural and molecular biology, we delineated the mechanism by which the enzyme elongates a cellulose polymer and translocates it across the membrane through a pore formed by its own transmembrane region.

We have also examined O antigens, which constitute the variable region of lipopolysaccharides in the outer membrane of Gram‐negative bacteria. The polymers are synthesized inside the cell on a lipid anchor and transported to the periplasm by the WzmWzt ABC transporter. Crystal structures of the transporter in nucleotide‐free and ATP‐bound states suggest a processive translocation mechanism through a channel formed by the transporter. Molecular dynamics simulations suggest that membrane lipids are a fundamental part of the transporter’s function by sealing its channel in a resting state.
Malva pseudolavatera leaf extract promotes ROS induction leading to apoptosis in Acute Myeloid Leukemia cells in vitro
Authors: Rizk Sandra , El Khoury Marianne , Haykal Tony , Hodroj Mohammad Hassan , Najem Sonia Abou , Taleb Robin I.
Published: April 2020

Malva pseudolavatera Webb & Berthel is a plant from the Malvaceae family that has long been included in the human diet due to its various curative effects. Many plant leaf extracts from the various species of Malva genus have been reported to possess anti‐cancer properties, however, studies on M. pseudolavatera Webb & Berthel leaves have documented anti‐inflammatory and anti‐oxidant effects with no emphasis on their possible anti‐cancer potential. The present study explores the anti‐cancer properties of Malva pseudolavatera Webb & Berthel leaf extract on Acute Myeloid Leukemia (AML) cell lines in vitro and deciphers the underlying molecular mechanism. Treatment of AML cell lines with M. pseudolavatera methanolic leaf extract showed a dose and time dependent inhibition of proliferation and a dose‐dependent increase in apoptotic hallmarks such as an increase in phosphatidylserine on the outer membrane leaflet, membrane leakage in addition to DNA fragmentation. The pro‐apoptotic effect was induced by ROS induction as well as an upregulation of cleaved PARP, an increase in Bax/Bcl‐2 ratio, and the release of cytochrome‐c from the mitochondria. Major compounds of the extract included methyl linolenate, phytol, γ‐sitosterol and stigmasterol as revealed by Gas Chromatography coupled to Mass Spectrometry.

Support or Funding Information

School Research and Development Council, Lebanese American University
Effects of fermented rooibos on sperm from infertile men in vitro
Authors: Opuwari Chinyerum Sylvia , Takalani Ndivhuho Beauty , Adefolaju Gbenga Anthony , Henkel Ralf
Published: April 2020

Rooibos (Aspalathus linearis) is indigenous to the Cederberg region in the Western Cape Province of South Africa. It is produced as unfermented and fermented rooibos tea. Previous studies demonstrated improved sperm parameters in male rats owing to its antioxidant properties. This study aims to investigate the effects of fermented rooibos in proven infertile men in vitro.

Semen samples were collected after 3–5 days of abstinence from 50 patients attending the infertility clinics of Tygerberg and Vincent Palotti Hospitals, South Africa. After liquefaction, semen was washed with HTF‐BSA (1:5), centrifuged for 10 minutes at 500 ×g and exposed to aqueous extract of fermented rooibos (0, 0.10, 1, 10 and 100 μg/ml) for 1h at 37°C. Thereafter, sperm parameters such as motility, vitality, mitochondrial membrane potential, ROS production, capacitation and acrosome reaction were analyzed.

Our results showed no effect of the aqueous extracts at any concentration used on the respective sperm parameters. In conclusion, fermented rooibos has no direct effect on the human sperm in vitro. Hence, the positive effect observed in previous studies may rather be due to the systemic antioxidant effect on the male reproductive system in vivo.

Support or Funding Information

Thuthuka fundinding instrument, National Research Foundation, South Africa (TTK170426228930)
The Role of the Fibrotic Scar in Repair Following Neuroinflammation
Authors: Dorrier Cayce , Aran Dvir , Haenelt Ezekiel , Lizama Carlos , Cautivo Kelly , Sheehy Ryan , Weiner Geoffrey , Arnold Thomas , Daneman Richard
Published: April 2020

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) in which the body’s immune system attacks the myelin sheath that surrounds and insulates axons. In many cases this myelin is not repaired by myelinating oligodendrocytes, which decreases the efficiency of action potential conduction and leads to neural dysfunction. We hypothesized that a barrier preventing oligodendrocyte lineage cells from repairing damaged myelin is a fibrotic scar. Following CNS injury, a scar consisting of an outer glial scar made up of reactive astrocytes and an inner fibrotic scar made of proteins such as collagen I forms around the site of trauma. In MS the glial scar has also been characterized, but the presence of a fibrotic scar has not been investigated. I have shown that following induction of experimental autoimmune encephalomyelitis (EAE) in mice, which is used as a model of MS, an extensive fibrotic scar forms in the lesioned tissue. Scar‐forming cells were visualized in this tissue using a Col1a1GFP mouse model. The number of these cells increased rapidly in the lesion site following symptom onset and remained high throughout the course of the disease. Lineage tracing and single cell RNA sequencing determined that these cells arise from the proliferation of fibroblasts and not other cells such as pericytes turning on the production of collagen. The objective of this project is to determine the role of the fibrotic scar in tissue repair and to determine mechanisms of scar formation in the CNS. To determine the role of the fibrotic scar following EAE I used the herpes thymidine kinase system to ablate proliferating fibroblasts. Using this paradigm I was able to reduce scar formation by 60% in EAE and found that this reduction resulted in a decrease in motor symptoms in the later stages of disease concurrent with an increase in oligodendrocyte lineage cells in the inflammatory lesions. To understand the signaling pathways that play a role in CNS scar formation, I used FACS to purify Col1a1GFP+ fibroblasts from spinal cords of healthy mice and mice with EAE and analyzed their transcriptome by RNA sequencing. I found that these cells upregulate inflammatory signaling pathways in disease such as the interferon gamma pathway. Deleting the interferon gamma receptor in CNS fibroblasts resulted in a decrease in scar formation following EAE and may be a potential therapeutic target for CNS disorders with fibrotic scarring. In conclusion I identified a fibrotic scar forms following neuroinflammation that arises from the proliferation of CNS fibroblasts, plays a role in disease recovery and forms in part through interferon gamma signaling.

Support or Funding Information

This work was funded by a NIH T32 Pharmacological Sciences Training Grant
Fold‐Switching Sets the Stage for Cooperativity and Competition in the Cyanobacterial Circadian Clock
Authors: Partch Carrie , Swan Jeffrey , Heisler Joel , LiWang Andy
Published: April 2020

Circadian rhythms are an ancient evolutionary adaptation found across the domains of life that synchronize behavior and physiology to the daily solar cycle for optimal fitness. In cyanobacteria, timekeeping is generated by three Kai proteins (KaiA, B and C) that form distinct protein complexes throughout the day, comprising a molecular clock that measures time in ~24‐hour increments. We recently showed that KaiB undergoes a rare transition from a tetrameric ground state to a monomeric, fold‐switched signaling state that binds to KaiC and plays a central organizing role in the evening phase of the clock. Using a version of KaiB locked into its active, fold‐switched state, we determined the structures of several complexes that illustrate how KaiB docks on the KaiC hexamer to recruit KaiA and the output protein CikA. A new structure of the output protein SasA bound to KaiC suggested that structural mimicry of the KaiB fold‐switched state is utilized to transition through distinct phases of the clock. Biochemical studies demonstrate that structural mimicry is used at low concentrations of SasA to cooperatively recruit KaiB and enhance oscillator function, while it leads to competition for KaiC at high concentrations. Collectively, this work reveals how mutually exclusive binding interfaces are used to regulate day/night transitions as well as both input and output signaling in the cyanobacterial circadian clock.

Support or Funding Information

National Institutes of Health R01 grants GM121507 (to C.L.P.) and GM107521 (to A.L.).
The biological activity of extracellular matrix mimetic elastin‐like polypeptide‐laminin α1 peptide
Authors: Truong Anh Tan , Hamada Keisuke , Yamada Yuji , Guo Hao , Kikkawa Yamato , Okamoto Curtis Toshio , MacKay J. Andrew , Nomizu Motoyoshi
Published: April 2020

The extracellular matrix (ECM) is comprised of a large network of proteins essential for providing a physical scaffold as well as biochemical and biomechanical signals for tissue development and repair. Laminins are a major component of the ECM and have a diverse set of biological roles such as cell adhesion, migration, proliferation and differentiation. Laminin α1 chain bioactive RGD containing peptide, A99 (AGTFALRGDNPQG), which was previously identified to promote strong cell attachment has demonstrated utility for cell adhesion applications. A wide array of biomaterials has been created to be used as scaffolds for these types of bioactive peptides, but many are not suited for various reasons such as scalability, convenient reaction conditions or lack of homogeneity. Here, we are the first to describe methods for preparing chemically conjugated and recombinant A99 to elastin‐like polypeptides (ELPs) as the scaffold and characterize their behavior as well as evaluate the coating efficiency, cell attachment and spreading activity. These data suggest that the A99‐ELP fusion has great potential for use as a biomaterial in cell culture applications as they greatly promote cell adhesion and spreading. ELPs are biocompatible protein‐polymers that are thermo‐responsive and self‐assemble in response to heat. Below the lower critical solution temperature (LCST), they are highly soluble but above the LCST, ELPs phase separate, forming coacervates. They provide an excellent structural framework for bioactive peptides and their intrinsic biophysical characteristics allow for simple production with high yields and purity making them an ideal biomaterial for cell adhesion applications.

Support or Funding Information

This research is sponsored by the Japan Society for the Promotion of Science (JSPS), PE18045.