bluuebunny/crossref_metadata_2025 huggingface dataset:
International anatomy partnerships ‐ Results of international student basic sciences internships initiated via the anatomy course
Authors: Wu Anette , Kieff Monica , Noel Geoffroy , Wingate Richard , Kielstein Heike , Sakurai Takeshi , Viranta-Kovanen Suvi , Chien Chung-Liang , Traxler Hannes , Waschke Jens , Vielmuth Franziska , Sagoo Mandeep Gill , Kitahara Shuji , Keay Kevin , Olsen Jorgen , Bernd Paulette
Published: April 2020

Introduction

Anatomists, as scientists, are a great resource for medical students’ scientific research interests. Recently, a new role for the Anatomy course in internationalization of medical education has emerged. Here we report on student experiences from international basic sciences research internships that were initiated via the anatomy course – combining two educational aspects of anatomy: the basic sciences and internationalization of medical education.

Material and Methods

A multi‐directional basic sciences research interchange was initiated as part of an international exchange program that is based on an international partnership of 13 anatomy departments.

200 preclinical medical students initially participated in an online international exchange segment, before embarking on optional basic sciences internships abroad. Based on students’ preferences, anatomy faculty at 13 international medical schools worked together on sourcing and student placement into basic sciences laboratories. Areas of research included a wide range of topics (e.g., immunology, genetics, tissue engineering, diabetes research, etc). At two time points (i.e., before and after travel), a questionnaire with multiple‐choice questions, Likert scales, and open‐ended questions, assessed the students’ learning experience qualitatively.

Results

A total of 82 students participated in international travel during 2019. As of now, 51 have completed the pre travel questionnaire and 26 the post travel questionnaire. Almost all of the partner schools sent and received students. Students improved both their understanding of the basic sciences and their basic sciences research skills, formed networks for future collaboration, and experienced an international cultural interchange. The majority of students felt that the basic sciences experience was important for their careers, and they wished to continue their involvement.

Discussion

Anatomy courses can foster a student’s interest in science and research, and introduce an international component to basic science experiences as study abroad programs. Providing international basic sciences research opportunities expands research options for students, including in areas of interest that are not available at the home institution, provides a baseline for future collaboration and helps students becoming leaders outside of their home institution.
Antibacterial and Antifungal Agent, Triclosan (TCS) Alters Secretion of Interleukin beta and Interleukin 6 from Human Immune Cells
Authors: Wilburn Wendy J.
Published: April 2020

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin (IL)‐6 and IL‐1 beta (IL‐1β) are important pro‐inflammatory cytokines produced by lymphocytes, monocytes, and other cells. Both regulate cell growth, tissue repair, and immune function, increased levels of each have been associated with a number of diseases including rheumatoid arthritis and certain cancers. TCS has been shown to inhibit the lytic function of human natural killer (NK) lymphocytes and to decrease expression of key cell surface proteins on NK cells. Here we examine whether TCS alters the secretion of IL‐1β and IL‐6 from human immune cell preparations. Human peripheral blood mononuclear cells (PBMCs) and monocyte‐depleted (MD)‐PBMCs were exposed to TCS at concentrations of 0–5 μM. Cytokine secretion was measured at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). In this study, both IL‐1β and IL‐6 secretion were altered at one or more concentration of TCS at one or more length of exposure. IL‐1β secretion was increased at 1, 2.5 or 5 μM TCS. Effects of 24 h exposure to 1, 2.5 and 5 μM TCS on IL‐1β secretion from PBMCs treated with selective MAPK pathway component inhibitors were also measured. These results indicate that TCS has the capacity to disrupt secretion of these two important pro‐inflammatory cytokines. Additionally the data show that TCS‐induced increases in IL‐β secretion were dependent on p38 MAPK.

Support or Funding Information

Supported by NIH grant U54CA163066.
RNA Binding Protein FXR1‐miR301a‐3p axis contributes to p21WAF1 degradation in oral cancer
Authors: Majumder Mrinmoyee , Palanisamy Viswanathan
Published: April 2020

RNA‐binding proteins (RBPs) associate with primary, precursor, and mature microRNAs (miRNAs), which in turn control mature miRNA levels, and post‐transcriptional gene regulation (PTR). However, the mechanistic basis of miRNA interaction and stability by RBP remains largely elusive. To understand how RBPs contribute to miRNA stability and PTR, we analyzed miRNAs that are controlled and stabilized by RBP
Fragile
X Mental
Retardation protein‐
1 (FXR1) in head and neck squamous cell carcinoma (HNSCC). We find that FXR1controls the expression of a subset of mature miRNAs, including a highly expressed oncogenic miR301a‐3p in HNSCC cells. We also observed that FXR1 controls the stability of miR301a‐3p by blocking 3′ to 5′ exoribonuclease activity of Polyribonucleotide Nucleotidyltransferase 1 (PNPT1). The miR301a‐3p is degraded by PNPT1 in the absence of FXR1 in oral cancer cells, and the degradation is rescued in co‐knockdown of FXR1 and PNPT1. Also, our in vitro findings show that FXR1 bound miRNA complex protects miR301a‐3p from PNPT1‐mediated miRNA degradation. This work also reveals that FXR1 knockdown destabilizes miR301a‐3p that degrades p21 mRNA, which leads to an increase in p21 accumulation, which explaining why overexpressed FXR1 and miR301a‐3p in HNSCC display p21 downregulation. Thus, our work illustrates the unique role of FXR1 that is critical for the stability of a subset of mature miRNAs or at least miR301a‐3p to target p21, thereby promoting the growth and proliferation of HNSCC.

Support or Funding Information

DE025920 and P30 CA138313
Epigenetic Control of Osteoblast Differentiation by Chromobox 3 (Cbx3) Protein 3
Authors: Bagheri Leila , Thaler Roman , Badreldin Amr , Paradise Christopher R. , Khani Farzaneh , Deyle David , Pengfei Zan , Milbrandt Todd , Larson A. Noelle , Dudakovic Amel , van Wijnen Andre
Published: April 2020

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.
The Notion of Cadaver Dissection in the Anatomy Curriculum to Produce Competent Physicians
Authors: Raja Dewan Saiful
Published: April 2020

Many anatomists claim the utmost importance of cadaver dissection to produce competent physicians. Other anatomists say Cadaver Dissection is obsolete. It is a difficult task to prepare and collect embalmed cadaver. Dissection hall maintenance is costly. Exposure to chemicals of a dissection laboratory and contact to human remains is not tolerable to many medical students. Disposal of the dissected cadavers is a cumbersome task too. The number of dissection lab hours and the number of students assigned for one cadaver is variable. Many medical colleges purchased computerized dissection programs. There is a trend of decreasing cadaver dissection hours. Some of the colleges relinquished both the cadaver dissections and prosections. I searched multiple works of literature from one note and PubMed with the necessity of dissection in the anatomy instruction word. Many anatomy instructors have no medical training, and a practicing physician never participates in cadaver dissection. Due to the embalming technique, the normal color and texture of normal human body are altered. A physician learns the relevant clinically oriented anatomy during the residency programs. To get into the residency program, one needs to pass the USMLEs and Clinical Skill examinations. These examinations do not include cadaver dissection nor cadaver structure identification. It is found that a medical student without cadaver dissection assignments had the equivalent scores to the students who spent many hours in cadaver dissections.

Conclusion

It is possible to become a competent physician without the experience of cadaver dissection.

Support or Funding Information

None
Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes
Authors: Smythers Amanda L. , Ford Megan L. , McConnell Evan , Lowery Sarah , Kolling Derrick R.J. , Hicks Leslie M.
Published: April 2020

The target of rapamycin (TOR) kinase is a Ser/Thr kinase and master
regulator, with roles in the regulation of nutritional sensing, protein translation, and autophagy, among others. In Chlamydomonas reinhardtii, a unicellular green algae and model organism for biofuel production and optimization, TOR has been linked to increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target is responsible for the elusive “lipid switch,” the protein or proteins responsible for increasing TAG formation under nutrient limitation and environmental stressors. However, when this lipid switch is activated, overall culture growth is arrested, truncating the alga’s ability to continuously produce TAGs at a level needed for widespread biofuel use.

In order to engineer an effective strain for algal biofuels, the targets and signaling pathways of lipid regulatory networks must be determined. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems. Previous research has used TOR inhibitors to follow phosphorylation pathways and identify potential downstream targets of TOR but little work has uncovered the role of oxidative signaling in regulation. Since TOR inhibition results in an increase in reactive oxygen species analogous to that of nitrogen deprivation, the opportunity for redox control mechanisms is significant.

This study inhibited TOR using AZD8055 and monitored reversible thiol oxidation while simultaneously tracking physiological changes. By pairing these data together, reversible thiol oxidation was related to nearly every major metabolic pathway in the cell, including TAG accumulation, the tricarboxylic acid cycle, and protein translation. Additionally, reversible thiol oxidation corresponded to changes in photosynthetic activity measured through chlorophyll a fluorescence, showing a novel role for TOR regulation of photosynthesis. The delineation of redox‐controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.

Support or Funding Information

This research was supported by a National Science Foundation CAREER award (MCB‐1552522) awarded to L.M.H and a National Science Foundation award (Cooperative Agreement OIA‐1458,952) to D.R.J.K. A.L.S. was funded by the NASA West Virginia Space Grant Consortium (NNX15AK74A and NNX15AI01H).
Neuroprotective Effects of Lithium in Chemotherapy‐induced Cognitive Impairments
Authors: Nguyen Lien D. , Fischer Tom T. , Ehrlich Barbara E.
Published: April 2020

Chemotherapy, a life‐saving treatment for cancer patients, is often associated with debilitating, sometimes irreversible, side effects. Approximately 40% of cancer survivors suffer from cognitive impairments, with symptoms including memory lapses, learning difficulties, and troubles with focusing, planning, multi‐tasking, and verbal communication. However, essential questions concerning the specific cognitive functions affected, the trajectories of cognitive changes, and the underlying mechanisms remain unclear. We aimed to investigate these questions by developing a mouse model of paclitaxel‐induced cognitive impairments. We also examined the potential neuroprotective effects of lithium, a compound previously shown to be beneficial in multiple neuropsychiatric and neurodegenerative disorders. Mice receiving four intraperitoneal injections of paclitaxel showed impaired short‐term spatial memory acquisition within a week of the last injection, which persisted for at least three weeks. Using Golgi‐Cox staining, we observed reduced dendritic arborization and spine density in the hippocampus and the cortex following paclitaxel injection, suggesting that these regions were particularly vulnerable. Importantly, lithium pretreatment completely rescued the behavioral and cellular deficits observed. Subsequently, we showed that paclitaxel treatment resulted in the upregulation of protein kinase C (PKC) both acutely and chronically, suggesting that dysregulated PKC signaling contributed to the neurological dysfunctions observed. In conclusion, we establish a molecular and cellular basis for paclitaxel‐induced cognitive impairments. Our findings also suggest a role for lithium as a protective agent to prevent cognitive impairments induced by paclitaxel and other chemotherapeutics.
Structural and functional characterization of threonylcarbamoyl adenosine biosynthesis in bacteria
Authors: Paranagama Naduni , Luthra Amit , Bayooz Susan , Jeon Youngmin , Lyumkis Dmitry , Swairjo Manal
Published: April 2020

tRNA, the central molecule of translation, is heavily modified post transcriptionally with noncanonical nucleosides that constitute ~83% of all RNA modifications. Modifications of the anticodon stem‐loop (ASL) of tRNA are required for translational fidelity and codon diversification. The ASL modification N6‐threonylcarbamoyl adenosine (t6A) at position 37 in ANN‐decoding tRNAs (N is any nucleobase) is universally conserved and its deficiency in humans causes renal‐neurological disease. Due to its essentiality in bacteria, the t6A pathway has emerged as a potential antibacterial target. Bacterial t6A biosynthesis begins with synthesis of the intermediate threonylcarbamoyl adenylate (TC‐AMP) from threonine, ATP and bicarbonate by the TsaC enzyme, followed by transfer of the threonylcarbamoyl moiety from TC‐AMP to A37 of substrate tRNA by the theonylcarbamoyl transfer (TCT) complex, comprised of the three proteins TsaB, TsaD and TsaE. We recently showed in T. maritima that while the TsaBD complex is the substrate binding platform responsible for TC transfer, the complex becomes inactivated after each t6A synthesis cycle, and TsaE‐dependent ATP hydrolysis “resets” the platform for multi‐turnover catalysis. However, the nature of the inactivation and reset steps remain unclear. Recent reports of a kinase activity for TsaE prompted us to investigate the role of this activity in the t6A cycle. Using a radiochemical kinase assay and mutagenesis experiments we show that T. maritima TsaE switches between a highly stable multimeric form possessing a tyrosine autokinase activity and a monomeric form lacking kinase activity and functioning as an ATPase in t6A synthesis. Further, to elucidate the structural basis of the specificity of t6A biosynthesis to ANN‐decoding tRNA substrates, we initiated cryo‐EM studies of the tRNA‐bound TCT complex. The preliminary maps reveal tRNA bound on the back surface of the TsaD subunit with its anticodon lodged in the TC‐transfer site and acceptor stem stabilized by the C‐terminal tail of TsaB, consistent with previous biochemical data. Collectively, the results provide insight into the mechanism of TC transfer, and suggest a regulatory role of TsaE that links t6A synthesis to other cellular processes.

Support or Funding Information

Funding: NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).
Simple and Affordable Kinetic Assay of Nucleic Acids by Gel Staining
Authors: Guillen Danielle , Schievelbein Mika , Patel Kushkumar , Jose Davis , Ouellet Jonathan
Published: April 2020

In an effort to circumvent the pitfalls of using radioactive labeling to measure kinetics in undergraduate labs, a gel staining kinetics assay was developed. The IR‐3 enzyme and substrate, a single stranded DNA that cleaves another single stranded DNA only in the presence of Zn++, was used as a simple and inexpensive DNA model. The rate of the IR‐3, or D‐Zyme, was successfully determined by staining PAGE‐Urea gels with SYBR Gold and then viewing them at 302 nm with epi‐illumination. The open source software ImageJ (version 1.52K) was utilized to quantify the relative band intensities, the ratio of cleaved product to the addition of product and substrate, gave percent cleavage. When percent cleavage was plotted against time in Minitab (version 18.1, Minitab, Inc.), the rate of the reaction could then be determined by nonlinear regression curve‐fitting to the single exponential. To certify this technique, it was also tested on two other models, one being a modified version of the D‐Zyme, while the other was a known Hammer Head Ribozyme, trans RzB. It was demonstrated that the use of SYBR Gold post‐migration staining can be used to quantify RNA and DNA bands for cleavage kinetic analysis, without substrate labeling. Although this method is comparatively less sensitive than radioactive and fluorescence labeling, it has several advantages such as: i) ease of use; ii) cost effective; iii) no half‐life decay; iv) absence of substrate labeling; v) precision enabling curve‐fitting; vi) versatility with non‐structured and structured RNAs or DNAs.

Support or Funding Information

Monmouth University Summer Research Program
CYP24A1‐induced vitamin D insufficiency promotes breast cancer growth
Authors: Osanai Makoto , Ono Yusuke , Takasawa Kumi , Takasawa Akira
Published: April 2020

Vitamin D plays a critical role in the maintenance of tissue homeostasis by regulating the expression of genes affecting cell proliferation, differentiation, and apoptosis. The vitamin D 24‐hydroxylase CYP24A1 functions in vitamin D target tissues to degrade the hormonal form of vitamin D. Existing knowledge regarding dysregulated CYP24A1 expression supports its candidacy as a putative oncogene. In our presentation, we demonstrate that the suppression of constitutive CYP24A1 expression by CYP24A1‐specific shRNA conferred target cells with increased susceptibility to apoptosis and consequently inhibited anchorage‐independent growth in breast carcinoma cells. In addition, suppression of vitamin D metabolism following knockdown of CYP24A1 significantly reduced tumor growth in vivo. Since these data provide substantial evidence for a pro‐survival and stimulatory oncogenic effect of CYP24A1 in breast carcinoma cells, we will discuss the potential feasibility of CYP24A1‐inhibitory cancer therapies.
Exploring Structural Determinants and Functional Aspects of Micron‐scale Protein Assemblies in Living Cells
Authors: Levin Tal , Seisdedos Hector Garcia , Empereur-Mot Charly , Levy Emmanuel
Published: April 2020

Proteins are increasingly often seen as forming micron‐scale assemblies in their folded state. Such assemblies include, for example, CTP synthase, Glutamine synthase or Acetyl CoA carboxylase, all of which can form filaments under particular environmental or cellular conditions. Despite the rising prevalence of these micron‐scale structures, their structural basis and functional roles remain poorly understood at a cellular level. In particular, are there general structural principles underlying the formation of these assemblies? Is their accidental formation deleterious to cells? Do they naturally co‐localize with the quality‐control machinery of the cell? Does altering their formation impact cell fitness? To address these questions, we endogenously mutate several yeast proteins to either promote or inhibit their micron‐scale self‐assembly. We will present the results of these experiments, which provide answers to the questions posed above.

Support or Funding Information

European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 819318)
Kinetic and Computational Investigations into the Origin of Metal‐Ion Specificity in Matrix Metalloproteinase‐1
Authors: Grove Laurie E. , Palladini Jacob
Published: April 2020

Matrix Metalloproteinases (MMPs) are a family of Zn‐dependent proteases responsible for cleaving peptide bonds. MMPs have essential roles in cell proliferation, differentiation, immune responses, among many others. Human MMPs have been implicated in a number of diseases including cancer and arthritis and as such have been the focus of studies related to their roles. We are interested in exploring the high specificity for the Zn2+ ion by MMP‐1 using kinetic assays and computational modeling. Human MMP‐1 contains a classical HEXXHXXGXXH Zn‐binding domain. Both the Zn2+ and second sphere Glu219 residue are involved in the first step of catalysis – deprotonating a Zn‐coordinated water. Interestingly, the Volvox species has a proteinase (VMP3) with a modified QEXXHXXGXXH metal‐binding domain that displays enhanced activity with Cu2+ and is much less active with Zn2+. In our study, a variety of metal‐substituted samples of MMP‐1 were prepared using dialysis to remove the Zn2+ ion. Kinetic assays using a FRET substrate demonstrate less than 5% activity in all cases when Cu2+ is substituted for Zn2+. To further explore these results, we developed a series of computational models of the MMP‐1 active site substituting either Zn2+ or Cu2+ into the active site. Density Functional Theory (DFT) as implemented in the ORCA program was used to develop these models, simulating the protein environment using either solvation models or by directly including second sphere residues as part of the quantum mechanics (QM) calculation. Models were validated by comparison to experimental results, including the kinetic data and spectroscopy data in the case of the Cu2+ models. Our work shows two effects at play. First, the first sphere geometry in MMP‐1 is primed to support a tetrahedral coordination environment, which is preferred by Zn2+ whereas Cu2+ prefers a square planar coordination environment. Second, the positioning of the Zn2+‐OH2 unit in the active site positions the water for deprotonation by Glu219. In the Cu2+ models, the distortion of the Cu2+‐OH2 unit due to the less stable coordination species increases the energy associated with proton transfer. This work provides a clear example of how first and second sphere active site residues are finely tuned to maximize enzyme activity.

Support or Funding Information

Wentworth Institute of Technology
Cross Regulation of Phospholipid and Unsaturated Fatty Acid Biosynthesis
Authors: Dible Sarah , McDonough Virginia
Published: April 2020

As most cellular phospholipids require unsaturated fatty acids (UFAs), we investigated the potential cross regulation of phospholipid and UFA biosynthesis. The Δ‐9 desaturase, encoded by OLE1, is the sole source of endogenous UFAs in Saccharomyces cerevisiae. Inspection of the OLE1 promoter revealed several sequences that are close matches to the regulatory sequence UASINO found in many phospholipid biosynthetic genes, which is the target of regulation by Ino2p, Ino4p and Opi1p. We examined if these proteins also play a role in regulation of OLE1 expression. Reporter gene analysis showed that when uracil prototrophy was placed under control of the OLE1 promoter, poor expression was observed in the opi1 and ino4 mutants. When lacZ was placed under control of the OLE1 promoter, reporter assays confirmed that expression was poor in the tested mutants. qPCR analysis demonstrates that the down regulation of OLE1 occurs normally in opi1, ino2 and ino4 mutants, however, its expression under growth conditions without exogenous UFAs is poor compared to wild type cells. Fatty acid analysis revealed that normal regulation of OLE1 occurred in response to unsaturated fatty acid (UFA) supplement in the opi1 mutant, indicating that post‐transcriptional regulation is still functional. Finally, the 2‐hybrid assay was used to examine potential interactions between Ole1p and Opi1p, Ino2p, Ino4p and known OLE1 regulators Mga2p and Spt23p, however no unexpected interactions were detected. This preliminary work indicates that phospholipid regulators Opi1p, Ino2p, Ino4p play a role in OLE1 expression, but not through regulation in response to UFA supplements or through Mga2p and Spt23p.

Support or Funding Information

Research reported was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM132853
Contacts and Confirmations that Regulate Hexameric Helicases at the Replication Fork
Authors: Trakselis Michael
Published: April 2020

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. The hexameric helicase is at the leading front of the replisome and is a nexus of control for both the initiation and elongation phases of DNA replication. These proteins are comprised of a N‐terminal DNA binding domain and a C‐terminal motor domain that assemble into a toroidal hexameric structure. Two main superfamilies of hexameric helicases exist: the RecA family, which contain helicases from phage and bacteria, and the AAA+ family, which contain helicases from archaea and eukaryotes. Interestingly, the measured translocation polarity of 5′‐3′ or 3′‐5′, respectively, dictates the orientation of these helicases at the replication fork.

Using site specific DNA cleavage experiments and presteady‐state FRET, we have unequivocally shown the then N‐terminal domain of the archaeal MCM helicase leads the way for unwinding (N‐first). The MCM hexamer itself can bind model DNA substrates in multiple conformations; however, the correct ‘N‐first’ conformation is dictated by 1) the presence of an excluded strand, 2) the ssDNA polarity (3′‐5′), and 3) the C‐terminal winged‐helix (WH). This orientation is opposite to that of the bacterial DnaB helicase which proceeds C‐first in the 5′‐3′ direction.

To determine the cellular impact of the excluded strand interaction in the steric exclusion and unwinding model (SEW), we created and tested several CRISPR/Cas9 genetic mutations of E. coli dnaB that correlate with mutations on the exterior surface. These DnaB mutations alter the conformation of the hexamer creating a spectrum of constricted states that all result in faster DNA unwinding. Genetic dnaB mutations lead to decoupling within the replisome, buildup of ssDNA, activation of the SOS response, and loss of genomic integrity. Therefore, the SEW interaction and the central channel diameter is adaptive and dynamic to properly maintain the DNA unwinding within the confines of the replisome to regulate speed of DNA replication and prevent chromosomal instability.

Support or Funding Information

National Science Foundation Division of Molecular and Cellular Biosciences (NSF1613534)
Activity‐Dependent and Compartment‐Specific Neuronal Redox Dynamics
Authors: Radhakrishnan Saranya , Tantama Mathew
Published: April 2020

Reactive oxygen species (ROS) are integral components of redox signaling in cells and mediate physiologically essential signaling across compartments. Under pathological conditions, excessive presence of ROS can lead to oxidative stress that damages cellular components and leads to cellular dysfunction and death. To understand dynamic redox changes and identify the mechanisms that disrupt redox homeostasis, we need high‐precision tools that can be used to define redox processes in live cells under physiological and pathophysiological conditions.

Genetically encoded fluorescent sensors that measure cellular redox status can be targeted to specific subcellular compartments to study the redox changes in intracellular compartments during live‐cell imaging. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution. To address this challenge, we developed red fluorescent redox sensors that can be multiplexed with reduction‐oxidation‐sensitive green fluorescent proteins (roGFP) to image dual compartments within the same cell. Our approach utilized Förster‐type resonance energy transfer (FRET) in a spectral relay strategy that extends the fluorescence emission of roGFP into red emission wavelengths. The construct was then characterized both in solution and through dual compartment imaging in cell lines.

ROS increases are ancillary to pathological processes like glutamate excitotoxicity and mitochondrial dysfunction that can propagate neuronal damage. Mitochondria help regulate the metabolic burden through the detoxification of neurotransmitters and the utilization of metabolic substrates, thereby regulating extracellular levels of glutamate. Following preliminary characterization of the sensor in cell lines, we targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents as well as to neuronal stimulation. Glutamate stimulation of neurons results in an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pharmacology indicates that the compartment‐specific redox responses are coupled and involve mitochondrial electron transport. Thus, using our sensors we have observed novel redox phenomena in primary neurons.
An autoinhibitory mechanism in regulating m6A‐methylation of 28S rRNA
Authors: Song Jikui , Ren Wendan , Lu Jiuwei
Published: April 2020

N6‐methyladenosine (m6A) modification represents an evolutionarily conserved epitranscriptomic mechanism that critically regulates RNA metabolism and function. m6A is widely present in mRNA and long noncoding RNA, as well as in ribosomal RNA (rRNA) and small nuclear RNA, mediated by an array of distinct writer enzymes. However, how m6A writers attain substrate specificities remains unclear. CCHC zinc finger‐containing protein ZCCHC4 is a newly identified RNA methyltransferase that specifically mediates the m6A modification on the site 4220 of 28S rRNA, which in turns impacts translation, cell proliferation and tumor growth. To provide mechanistic insights into the ZCCHC4‐mediated m6A RNA methylation, we performed structural characterization of the enzyme‐cofactor complex of human ZCCHC4. Our work reveals that the regulatory domains and the methyltransferase (MTase) domain pack into an integrated fold, thereby creating a multi‐domain RNA‐binding platform. Importantly, the MTase domain adopts an autoinhibitory conformation, in which the active site is occluded from RNA binding by an inter‐domain loop segment. Mutational and enzymatic analyses further indicate that this loop segment directly engages substrate recognition, providing an autoinhihitory mechanism by which the activity of ZCCHC4 is coupled with specific substrate binding. In addition, analysis of the substrate sequence and structure suggests a role of the stem‐loop structure within substrate in governing the substrate specificity. Together, our study uncovers an autoinhibitory mechanism that ensures specific interplay between ZCCHC4 and the RNA substrate, reminiscent of what was seen with the DNA methylation writers.

Support or Funding Information

NIH 1R35GM119721
ApoE genetic variation is associated with differential susceptibility to dyslipidaemia and type 2 diabetes in black Africans
Authors: Tanyanyiwa Donald Moshen , Buthelezi Philani Ernest , Dandara Collet , Bhana Sandeep Amrat
Published: April 2020

Background

Dyslipidaemia in type 2 diabetes mellitus (T2DM) is characterized by a biochemical lipid triad of low HDL‐cholesterol, raised triglycerides, and small, dense low‐density lipoprotein (LDL) particles. Studies on ApoE genetic variation influence in the development of dyslipidaemia in African diabetic patients are few.

Aims and Objectives

This study aimed to evaluate the role of genetic variation in ApoE coding gene on dyslipidaemia in South African diabetic patients.

Methods

Two hundred and forty‐four (n=244) participants were retrospectively recruited from the Baragwanath diabetic clinic. These comprised of two groups: (i) dyslipidaemic, and (ii) non‐dyslipidaemic (controls). The dyslipidaemic group was further divided into three groups according to; i) high cholesterol only; ii) high triglycerides only; and, iii) both high cholesterol and high triglycerides (referred to as the mixed group). Ethical clearance was obtained from the University of Cape Town and Baragwanath/Wits University. Genetic characterisation of ApoE was accomplished using polymerase chain reaction/restriction fragment length polymorphism (RFLP), confirmed by Sanger sequencing.

Results

Of the 244 participants, 165 were dyslipidaemic while 79 were not. A significant (p <0.0001) finding in this study is that ApoE2/3 dyslipidaemic participants had a distribution of only 5.3%, compared to 20.6% among the non‐dyslipidaemic participants. There was a low frequency distribution, 1.8% of among ApoE2/4 among the diabetic participants. There a significant association between HbA1c and age (p=<0.008). TC (p=0.00092), LDL (p=0.0184) and triglycerides (p=0.0175) were strongly associated with poor glycaemic control. Both LDL (p=0.0174 and HDL (p=0.0072) were associated with age.

Conclusions

The study showed the effects of ApoE genetic variation on the dyslipidaemia seen in black South African diabetic participants. The study showed that diabetic dyslipidaemia is genetically influenced by Apo E. ApoE2/3 DM patients had a reduced risk of developing dyslipidaemia while ApoE2/4 was associated with reduced risk for T2DM. Therefore, exploration of possible underlying genetic predisposition in dyslipidaemic T2DM patients who respond poorly to standard therapy is recommended.

Support or Funding Information

Departmental support
Fluorescence Localization of the Conjugation Machinery of Bacillus subtilis
Authors: Chen Sirui , Dame Haley B. , Berkmen Melanie B.
Published: April 2020

Conjugation, or mating, is the transfer of DNA from a donor to a recipient cell through conjugation machinery. Conjugation contributes to bacterial genome innovation and the spread of virulence and antibiotic resistance genes between bacteria. DNA is transferred during conjugation through a multi‐protein DNA translocation channel, classified as a Type IV secretion system (T4SS), embedded in the membrane. Our work focuses on characterizing the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Previously, we identified several protein components of the ICEBs1 conjugation machinery, including the bitopic membrane protein ConB and the peripheral membrane ATPase ConE. We have used fluorescence microscopy to observe the locations of ConB and ConE inside cells. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane, predominantly at the cell poles. Here, we show that a fusion of ConB to the red fluorescent protein mCHERRY localizes similarly to ConE‐GFP. Consistent with ConE lacking predicted transmembrane segments, ConE‐GFP’s localization to the membrane requires ConB. In contrast, localization of ConB‐mCHERRY to the membrane does not depend on other conjugation proteins. Current studies are focused on which domains of ConB are required for its own localization as well as the localization of ConE.

Support or Funding Information

This research was funded by Suffolk University and an NSF‐RUI grant to M. Berkmen.
Molecular signatures of neuroinflammation induced by α‐synuclein aggregates in microglial cells
Authors: Sarkar Souvarish , Dammer Eric , Malovic Emir , Seyfried Nicholas , Tansey Malu , Kanthasamy Anumantha , Rangaraju Srikant
Published: April 2020

Alpha‐synuclein aggregates (α‐synAgg) are pathological hallmarks of Parkinson’s disease (PD) and other synucleinopathies that induce microglial activation and immune‐mediated neurotoxicity, but the molecular mechanisms of α‐synAgg‐induced immune activation are poorly defined. We performed quantitative proteomics by mass spectrometry coupled with PCR, immunohistochemical and functional validations studies to define the molecular characteristics of alpha synuclein mediated microglial activation. In mouse microglia, α‐synAgg induced robust pro‐inflammatory activation (increased expression of 864 genes including Irg1, Ifit1 and Pyhin) and increased nuclear proteins involved in RNA synthesis, splicing and anti‐viral defense mechanisms. Conversely, α‐synAgg decreased expression several proteins (including Cdc123, Sod1 and Grn), which were predominantly cytosolic and involved in metabolic, proteosomal and lysosomal mechanisms. Pathway analyses and confirmatory in vitro studies suggested that α‐synAgg partly mediates its effects via Stat3 activation. As predicted by our proteomic findings, we verified that α‐synAgg induces mitochondrial dysfunction in microglia. 26 proteins differentially expressed by α‐synAgg were also identified as PD risk genes in genome‐wide association studies (upregulated: Brd2, Clk1, Siglec1; down‐regulated: Memo1, Arhgap18, Fyn and Pgrn/Grn). We then validated progranulin (PGRN) as a lysosomal PD‐associated protein that, relative to the vast upregulation of activated microglia, is decreased in nigral microglia in post‐mortem PD brain compared to non‐disease controls, congruent with our in vitro findings.

Conclusion

Together, proteomics approach both reveals novel molecular insights into α‐syn‐mediated neuroinflammation in PD and other synucleinopathies.

Support or Funding Information

Work supported by Emory Alzheimer’s Disease Research Center Grant P50 AG025688, Alzheimer’s Association (SR #37102), NINDS (K08‐NS099474‐1) and Emory Neuroscience NINDS Core facilities (P30 NS055077) to S.R.; NINDS (NS100090) and NIEHS (ES026892) to A.G.K
Investigation of the DNA strand separation step by DNA methyltransferase Caulobacter Crescentus
Authors: Konttinen Olivia Rae , Reich Norbert O. , Carmody Jason , Kurnik Martin
Published: April 2020

Epigenetic DNA methylation by CcrM C. crescentus is an important regulator of gene expression. CcrM methylates adenine bases on hemimethylated double stranded DNA substrates containing 5′‐GANTC‐3′ sites to regulate gene expression. The recent cocrystal structure shows that CcrM uses a unique DNA recognition mechanism by which four of the five basepairs are completely disrupted. The target strand interacts in base‐specific ways whereas the non‐target strand is largely unperturbed, and does not involve any base‐specific interactions. The dimeric enzyme’s methyltransferase domain and c‐terminal domain interact with the DNA target strand and non‐target strand respectively. Here we present a novel assay that tracks the DNA strand separation step by using a fluorescent probe (Pyrollo‐dC). Placement of Pyrollo‐dC within double stranded DNA at the N position of the CcrM recognition site results in a dramatic increase in fluorescence when CcrM binds; this only occurs when the analog is positioned within the target strand, with much smaller effects involving the non‐target strand. Serine 315 and Histidine 317, which reside in the highly conserved 80‐residue C‐terminal tail, interact with phosphates within the non‐target strand and mutation to alanine results in significant losses of activity and a loss of strand separation, as determine by the Pyrollo‐dC assay. Residue G305A does not interact with the phosphate backbone of the non‐target strand and does show fluorescent signal, suggesting that this mutant is able to strand separate. This mechanism appears to be widespread since other enzymes (e.g., HinfI and an ortholog of CcrM, BabI) exhibit fluorescent signals similar to CcrM.

Support or Funding Information

NIH Grant
Expression and purification of human Neuronal PAS domain protein 2 (hNPAS2) for structural and functional studies
Authors: Chen Shawnan , Moreno Brenda , Moriel Karla , Xiao Chuan
Published: April 2020

The circadian rhythm, a roughly 24‐hour biological clock, is found in most living organisms. The oscillation of the rhythm is controlled by a molecular mechanism called the transcriptional‐translational feedback loop (TTFL). In mammals, the circadian rhythm is responsible for a variety of biological and physiological processes such as hormone regulation, digestion, and body temperature. Human neuronal PAS domain protein 2 (hNPAS2) has been proven to work as a transcription factor important in regulating the circadian rhythm. This project aims to determine the structure of hNPAS2 in order to elucidate its function in the circadian rhythm. In this study, full length hNPAS2 gene was cloned into a bacterial expression vector. To obtain high yields of the soluble protein, the expression was optimized. Recombinant hNPAS2 proteins were purified by affinity and size exclusion chromatography. The high concentration of purified hNPAS2 protein will be used in structural and functional studies. The results will deepen our knowledge about hNPAS2 and its role in the circadian rhythm.

Support or Funding Information

Mentored Experiences in Research, Inquiry, and Creativity for Undergraduate Scholors (MERITUS), Campus Office of Undergraduate Research Initiatives (COURI), Office of the Provost and Vice President for Student Affairs, BUILDing SCHOLARS, National Research of General Medical Sciences of NIH
Growing and Sustaining a Nationwide CURE: Genomics Education Partnership Enhances Research Opportunities for Students and Faculty at Diverse Institutions
Authors: Goodman Anya L. , Van Stry Melanie , Lopatto David , Leung Wilson , Reed Laura K. , Community GEP Faculty
Published: April 2020

The Genomics Education Partnership (GEP) is a community of practice focused on the integration of genomics research experiences into undergraduate curriculum and supporting faculty in this endeavor. Founded in 2006, GEP has grown to 100+ participating institutions and introduced thousands of students to eukaryotic gene structure, comparative genomics, and genome evolution. Recently, GEP has transitioned to a collaborative leadership structure. GEP members contribute their expertise and enthusiasm to the community through four committees: science and IT, curriculum development, assessment, and professional development and mentoring. Digital communication tools facilitate collaboration in this geographically distributed community and allow faculty with similar interests to work together. We seek to grow our network of faculty and to increase the diversity of our community by making a variety of training opportunities available, including online mentoring via the QUBES platform and regional node workshops. We are also expanding our repertoire of scientific projects. GEP has partnered with Galaxy to develop G‐OnRamp, an open‐source platform for constructing UCSC Assembly Hubs and JBrowse/Apollo genome browsers to facilitate collaborative annotation of eukaryotic genomes. These tools allow us to expand GEP projects, for example, to include investigations of the evolution of venom in parasitoid wasps and an investigation of the evolution of insulin pathway genes across 27 Drosophila genomes. Our education research is focused on student experiences, attitudes, and learning gains in the Course‐based Undergraduate Research Experience (CURE) setting. An important dimension of CUREs that distinguishes them from the traditional laboratory classroom is iteration: students have opportunities to make mistakes and redo (Auchincloss et al, 2014). The scaling of undergraduate research from an apprentice model to the classroom, however, poses a major challenge for faculty, creating the need to provide feedback to a large number of inexperienced student researchers. We find that custom bioinformatics tools provide rapid feedback to GEP students and allow for iterative revisions of student projects. Student surveys and focus groups reveal that bioinformatics CUREs foster “formative frustration,” whereby students can safely fail in their original analysis, adjust, recover, and succeed; students value this experience. We have created a supportive community of faculty and welcome new colleagues (no experience necessary!) eager for the challenge of bringing genomics research to all undergraduate students (freshmen through seniors). Faculty interested in joining the GEP can contact us at http://gep.wustl.edu/contact_us.

Support or Funding Information

Supported by NSF IUSE‐1915544 and NIH IPERT‐1R25GM130517‐01 to LKR.