bluuebunny/crossref_metadata_2025 huggingface dataset:
Running Rings Around RNA: The role of Ro RNPs in RNA maturation and decay
Authors: Wolin Sandra L.
Published: June 2021

The Ro 60 kDa protein is a ring‐shaped RNA binding protein that is present in many animal cells and certain prokaryotes. In both animal cells and bacteria, Ro is found complexed with ~100 nt small RNAs called Y RNAs. In vertebrate cells, Ro also binds misfolded RNAs and likely functions in noncoding RNA quality control. Structural and biochemical studies have revealed that Y RNAs bind on the outer surface of the Ro ring. In contrast, the single‐stranded ends of misfolded RNAs insert into the central cavity, while helical portions of these RNAs bind to a large basic platform on the outer surface. Because the binding of Ro to misfolded RNA is largely sequence‐nonspecific, Ro may be able to associate with a variety of structured RNAs that contain both a single‐stranded 3′ end and helices. Consistent with this hypothesis, Ro is required for efficient maturation of 23S rRNA in the eubacterium D. radiodurans. The binding of Y RNAs and other RNAs is mutually exclusive, and studies in bacteria have revealed that one role of Y RNAs is to regulate access of other RNAs to Ro. We are examining the various functions of Ro and the Y RNAs in both mammalian cells and bacteria.
Modulation of the actin cytoskeleton by Salmonella
Authors: Galan Jorge E.
Published: June 2021

Salmonella engages its host cell through the activity of a specialized protein secretion system that direct the translocation of a battery of bacterial effector proteins into the host cells. The bacterial proteins stimulate marked actin cytoskeletal reorganization leading to membrane ruffling and macropinocytosis that ultimately results in the internalization of the bacteria. These cytoskeletal rearrangements are dependent on the function of the small GTP‐binding proteins CDC42, Rac, and RhoG, which through the action of downstream effector proteins, direct the reorganization of the actin cytoskeleton. Two of the bacterial proteins responsible for the stimulation of cellular responses, termed SopE and SopE2, act as exchange factor for Rho GTPases including CDC42, Rac, and RhoG. Other effectors delivered by S. typhimurium into the host cell include the phosphoinositide phosphatase SopB, which stimulates signal transduction pathways that lead to the activation of the RhoG endogenous exchange factor SGEF, which activates RhoG. The cellular responses induced by Salmonella are reversed by SptP, which acts as a GTPase‐activating protein (GAP) for CDC42, Rac, and RhoG.
Connecting development and disease pathways through zebrafish chemical biology
Authors: Peterson Randall
Published: June 2021

The molecular pathways that direct normal embryonic development often resurface as causative factors in adult diseases. For example, bone morphogenetic proteins (BMPs) establish the embryonic dorsal‐ventral axis, but disruption of BMP signaling contributes to the anemia of chronic disease. Similarly, AML1 is a master regulator of embryonic hematopoiesis, but AML1 mutations are causative in a significant proportion of cases of acute myeloid leukemia (AML). This intimate connection between development and disease offers hope that experimentally tractable developmental models may provide new avenues to the study and treatment of adult cancers.

We have developed methodologies that enable high‐throughput small molecule screens to be conducted with whole, living zebrafish. These screens allow us to discover small molecules that modify developmental pathways and also exhibit high specificity, good pharmacological properties, and low toxicity. The small molecules discovered are useful probes for studying embryonic development and can often be developed further to study and treat the cancer associated with the targeted developmental pathway. We are currently exploring the developmental pathways involved in myocardial infarction recovery, AML, and anemia and testing whether our small molecule modifiers will also be helpful for studying or treating these diseases.
Small molecule probes of mycobacterial cell wall assembly
Authors: Kiessling Laura L.
Published: June 2021

Carbohydrates mediate fundamental processes in humans; they also can be essential for pathogen survival. For example, oligosaccharides serve as essential components of the mycobacterial cell wall. Agents that inhibit key biosynthetic steps in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), would be valuable, because TB results in approximately 2 million deaths/year. A key component of the mycobacterial cell wall is the arabinogalactan polysaccharide, which contains galactofuranose (Galf) residues. Because Galf residues are not constituents of human glycoproteins or glycolipids, we have focused on understanding galactofuranose incorporation. To this end, we have discovered a fundamentally new catalytic role for the heterocyclic cofactor flavin (vitamin B2), and we have found that an essential glycosyltransferase mediates oligosaccharide assembly by a processive mechanism. Moreover, we have discovered small molecule inhibitors of galactofuranose incorporation. Small molecules targeting this pathway block mycobacterial growth, a result that highlights the benefits of blocking this glycoconjugate biosynthetic pathway. We anticipate that these inhibitors can illuminate carbohydrate function and lead to the generation of therapeutics effective against strains resistant to current anti‐mycobacterial agents. This research was supported by the National Institute of Allergy and Infectious Diseases.
Roles of non‐coding, Pol IV‐dependent RNAs in megabase‐scale silencing and long‐range heterochromatin interactions in Arabidopsis
Authors: Pikaard Craig Stuart , Preuss Sasha , Costa‐Nunes Pedro , Pontes Olga , Tucker Sarah , Tan Ek Han , Haag Jeremy , Ream Thomas , Wierzbicki Andrzej
Published: June 2021

Unlike other eukaryotes, plant genomes encode a fourth class of nuclear DNA‐dependent RNA polymerase, namely RNA polymerase IV (Pol IV). Two non‐redundant forms of Pol IV exist, namely Pol IVa and Pol IVb. Unlike RNA polymerases I, II and III, Pol IVa and b are non‐essential for viability but are required for siRNA‐directed DNA methylation and for the silencing of transposons and other heterochromatic repeats. Both Pol IVa and Pol IVb colocalize with chromosomal loci that give rise to abundant 24 nt siRNAs that are generated by DICER‐LIKE 3 at a distance from the source/target loci, within a nucleolar siRNA processing center. Pol IVa acts early in the pathway and is essential for siRNA production whereas pol IVb act downstream of siRNA production to mediate RNA‐directed DNA methylation. New evidence shows that the Pol IV‐dependent 24 nt siRNA pathway plays a role in dosage control of essential repetitive genes in addition to silencing selfish DNA repeats such as transposons. Other evidence shows that Pol IVb has genome organization functions that are independent of Pol IVa activity or siRNA production, including the higher‐order organization of dispersed heterochromatic repeats into chromocenters. Our progress in understanding the molecular mechanisms responsible for these phenomena will be presented.
Making use of dynamic instability at the microtubule‐kinetochore interface
Authors: Nogales Eva , Wang Hong‐Wei , Ramey Vincent , Barnes Georjana , Drubin David , Westermann Stefan
Published: June 2021

The dynamic behavior of microtubules is essential for their functions. We have obtained the structure of two structural intermediates corresponding to the start and end points in the polymerization cycle that illustrate the conformational consequences of the nucleotide state and how they relate to longitudinal and lateral assembly. We propose that the polymorphism of assembly unique to tubulin reflects an exquisite tuning mechanism for the complex interaction of different microtubule intermediates with cellular factors that need to detect or make direct use of the growing or shortening state of microtubules to play functional roles at the right time and place in the cell. One such cellular component is the yeast Dam1 kinetochore complex. This complex self‐assembles around microtubules into rings and spiral‐like structures, but lacks the classical footprint on the microtubule lattice of motors and structural MAPs, allowing for its passive diffusion on the microtubule. When the microtubule depolymerizes the Dam1 ring uses the conformational strain of GDP tubulin within the lattice as tubulin relaxes into its curved state, to move processively, and without energy consumption of its own. A microtubule‐induced conformational change of the Dam1 complex ensures that ring formation does not precede engagement with a kinetochore microtubule.
Evolutionary Medicine: A Graduate‐Level Seminar Course
Authors: Schiffer Stephen Paul
Published: June 2021

Evolutionary medicine serves as an exciting intersection for the two disciplines of evolution and medicine. It provides for more than just a new perspective on the diseases of man and other animals. From an educational perspective, it makes the subject of evolution both interesting and relevant. With this in mind, a one‐credit graduate‐level seminar course was created at the Georgetown University School of Medicine. The course consisted of weekly sessions that were designed around hypothetical clinical cases prepared by the course director. The clinical cases covered a range of human health problems and while most were written with a current‐day context, a couple cases had an historic context and the final case had a futuristic one. Each student was randomly assigned a case and seminar date during which s/he served as the “Darwinian Doctor of the Day” (DDD). Each session began with the DDD reading the clinical case followed by a roundtable discussion of its evolutionary insights. The DDD then led a detailed presentation on the proximate cause of the disease as well as the evolutionary milestones that helped explain why the disease occurs in humans at all. Each session finished with the DDD handing out an Insight Article (IA) chosen from the scientific or clinical literature that was relevant to the case. The course director then posted a question based on the IA on the course's Blackboard Discussion site and to which each student was required to respond by the next session. The students were graded based upon attendance, participation, their DDD presentation, their timely IA responses, and a mandatory term paper. Student feedback at the end of the course was overwhelmingly supportive indicating that it provided a fascinating perspective on human health and disease.
RNAse One Cloning, Expression and Enzyme Analysis Provides Research Sequence for Biochemistry Laboratory Course
Authors: Bailey Cheryl P
Published: June 2021

Students in a biochemistry laboratory course were posed with the question of how they would design experiments that included a protein of interest. Isolation and characterization of the protein quickly came to the forefront, which directed conversation to why bacterially expressed tagged proteins are so prevalently used in research laboratories. Students utilized BLAST to search for E. coli RNAse One sequence and designed primers PCR to isolate the coding sequence of RNAse One followed by coding sequence for Glutathione‐S‐Transferase (GST). Successful PCR products were cloned into the pGEX™ expression vector. Expressed protein was purified with MagneGST™ beads and protein concentration measured. Total RNA was isolated from Xenopus oocytes and subjected to degradation by purified RNAse One. RNAse One activity was compared to RNAse A and RNAse H with purchased substrates. Literature searches revealed potential further tests with inhibitors. This experience culminated in a poster session with campus faculty invited to participate and ask “how?”, “why?” and “what now?” questions.
Thorax Virtual Anatomy Lab for Pre‐professional Health Sciences Students
Authors: Klinkhachorn Penprapa S. , Walker Elizabeth , Allen Edwin , Altemus Jeffrey , Kraszpulska Barbara
Published: June 2021

The traditional anatomy classrooms for professional students at our university have become too small to accommodate the growing demand for undergraduate anatomy courses. We have offered an online undergraduate human anatomy course since 2002, to serve as a prerequisite for higher programs. We are now addressing the question asked by students “Is there a lab with this course?” Virtual anatomy labs targeted for specific groups of students are now under development. The laboratory text Learning Human Anatomy, by Dr. Julia Guy at Ohio State University, with companion CDROM Anatomy Lab enables the student to work independently before coming to the gross anatomy lab. Virtual and real visits to the anatomy lab will be arranged in addition to the online assignments when possible. The students can choose the following lab modules to fit their needs: Limbs and Back, Thorax, Abdominopelvic Region, and Head and Neck. The combination of the general anatomy course and the Virtual Lab in areas of interest will provide sufficient information for these students to fulfill prerequisite requirements and prepare for higher level anatomy courses. (Supported in part by the Neurobiology and Anatomy Department, West Virginia University School of Medicine, account # 491030050, fund# 12305495)
An Animal Physiology lab project that promotes undergraduate student interest and responsibility
Authors: Onken Horst , Cataldo Carmine S , Coppolo Jaclyn A , Lamb Christina M , LoRe Erica G , Post Amanda K , Zangara Nicole E
Published: June 2021

This lab accompanies a lecture about comparative animal cell and systems physiology. Fiddler crabs (Uca pugnax) are collected during a field trip to mudflats of Arthur Kill between Staten Island and New Jersey. However, other euryhaline animals could replace the fiddler crabs. Students are responsible for the animal care throughout the semester. The crabs are acclimated to waters of 3 different salinities (30, 20 and 10 ‰). Students use simple approaches to determine the osmotic concentrations in the external media and in the animals' hemolymph to reveal the osmotic gradients maintained by the crabs. Passive salt loss is estimated from the conductivity increase of a volume of distilled water after inserting 1‐2 crabs, allowing to estimate the energy needed to maintain the osmotic gradient. Finally, the metabolic rate of the crabs in the different waters is measured and compared to the theoretical expectation on the basis of different assumptions about the metabolism of the crabs. At the end of the course, the animals are returned to their natural habitat.
Neuroendocrine regulation of behavior demonstration in the teaching laboratory
Authors: Cline Mark A
Published: June 2021

Students enrolled in Human Anatomy and Physiology courses, after learning about the neuroendocrine system in lecture, participated in a teaching exercise designed to demonstrate how peptides affects behavior. Young chicks were centrally injected with either α‐melanocyte stimulating hormone, angiotensin II, corticotropin releasing hormone, neuropeptide Y or a control and the students monitored the chick's behaviors to determine which treatment each chick had received. Peptides were selected for their diverse behavioral effects. Prior to coming to lab, students used the primarily literature to research the behavioral effects of these peptides in mammals. In the lab, students were given feed, water and an observation arena and decided on a method to record behaviors. After the observation period, students wrote a research paper in journal form describing their observations. This activity demonstrated peptidergic regulation of behavior, the use of an alternative vertebrate research model, utilization of primarily literature, laboratory animal handling, experimental design, and preparation of a research manuscript. Students reported that the activity was engaging and educationally valuable.
Stem/Progenitor Cell Mobilization for Tissue Regeneration
Authors: Lee Sang Jin , Van Dyke Mark , Atala Anthony , Yoo James
Published: June 2021

Introduction

In this study we explored the possibility of utilizing host stem or progenitor cells for tissue regeneration in vivo. We investigated whether these cells could be mobilized into target specific sites for regenerative medicine applications.

Materials and Methods

Polyglycolide polymers were implanted subcutaneously in mice to recruit host cells. The scaffolds were retrieved at 1, 2, 3 and 4 weeks after implantation for analyses. Cells contained within the retrieved scaffolds were cultured and differentiated into osteogenic, endothelial, myogenic, and adipogenic cell lineages using the respective cell differentiation conditions.

Results and Discussion

The retrieved implants showed a progressive increase in neovascularization and host cell infiltration with time. Tissue infiltrates within the scaffolds showed the evidence of collagen deposition after 2 weeks of implantation and continued to increase over time. The culture of infiltrated cells showed the expression of stem cell markers and these cells were able to differentiate into various cell lineages.

Conclusions

This study demonstrates that stem or progenitor cells can be mobilized and recruited into target specific sites. These findings suggest that host stem cells may be maneuvered to achieve tissue regeneration in vivo using a target specific scaffold system.
Establishment of a stable progranulin deficient cell line: a model of frontotemporal dementia with ubiquitin‐positive inclusions
Authors: Mao Qinwen , Hatanpaa Kimmo J. , Sontag Estelle , Hladik Crista L. , Xia Haibin , White Charles L.
Published: June 2021

Mutations in the gene coding for progranulin (PGRN) are responsible for many cases of familial frontotemporal lobar degeneration with ubiquitin‐positive inclusions (FTLD‐U). Mutations in progranulin create null alleles resulting in decreased progranulin protein or haploinsufficiency, which leads to reduced PGRN‐mediated neuronal survival. In order to elucidate the molecular mechanisms involved in the pathogenesis of FTLD‐U, a cell line stably expressing shRNA against PGRN (shPGRN) under the control of tetracycline was established. HEK 293 cells were transfected with H1‐driven shPGRNs and progranulin‐expressing plasmids and effective shPGRNs were screened by Western blot. Human neuroblastoma SHSY5Y cells transfected with shPGRN expressing vector were found to have decreased cell growth rate and mitosis, and redistribution of TDP‐43 protein to cytoplasm. A line of SHSY5Y cells stably expressing shPGRN under the control of tetracycline (Tet on system) was established. The results indicate that it is feasible to establish a stable progranulin deficient mammalian cell line, which will be a powerful tool to study the pathogenesis of FTLD‐U and explore its potential treatments. The study was supported by NIH ADC grant AG12300, the UT Southwestern Friends of the Alzheimer's Disease Center, the Winspear Family Center for Research on the Neuropathology of Alzheimer Disease, and the McCune Foundation.
Spatial and Temporal Similarities between Laminin‐332 and Keratin‐5 during Wound Repair
Authors: Gerecke Donald R , Gordon Marion K , Svoboda Kathy , Lu Shou‐En , Chang Yokechen
Published: June 2021

A mouse ear skin vesicant model was wounded using sulfur mustard and the skin glycoprotein, laminin‐332 examined in a time‐course study. Mouse skin biopsies 6, 12, 24, 72, and 168h after SM exposure demonstrated a direct correlation in skin damage over time. Chain‐specific antibodies showed overlap of laminin‐332 and the basal keratinocyte marker, keratin‐5 in the basal cells of the epidermis and the lining of the sebaceous glands. The mRNA levels of the laminin‐332 chains declined early (within 24h), then increased to a maximum level by 72h post‐treatment. The laminin‐á3 and laminin‐â3 mRNAs returned to normal levels by 168h post‐treatment, however, the laminin‐ã2 chain mRNA levels remained about 10 fold higher than the other two chains at later timepoints (72 and 168h). The higher laminin‐ã2 mRNA level corresponded to increased amount of expressed protein (Western blot analysis). Processed forms of several different sizes for the laminin‐ã2 chain protein also increased over time. This was especially true 72h post‐exposure, when keratinocytes were undergoing hyperproliferation. Confocal microscopy demonstrated an overlap in distribution between keratin‐5 and laminin‐ã2 in the 72h samples and we speculate that the laminin‐ã2 functions to enhance epithelial cell migration.
Proliferation and extracellular matrix protein expression in vascular smooth muscle cells cultured for aortic valve tissue engineering
Authors: Appleton Andrew James Edward , Appleton Christopher Thomas George , Boughner Derek , Rogers Kem
Published: June 2021

OBJECTIVES: Vascular smooth muscle cells are a potential autologous cell source for aortic valve tissue engineering. We hypothesized that combining basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) with transforming growth factor beta‐1 (TGF‐β1) treatment would allow temporal control of rat aortic smooth muscle cell (RASMC) proliferation and ECM production.

METHODS: Growth factor (alone or in combination with TGF‐β1) ability to induce myofibrolast‐like phenotype in RASMCs in monolayer culture was assessed. Cell proliferation was measured by MTT assay following treatment with growth factor combinations. Total RNA was isolated to assess ECM gene expression by real‐time PCR.

RESULTS: Combinations of growth factors which included PDGF showed the greatest increases in proliferation. Immunofluorescence for alpha‐smooth muscle actin (α‐SMA) in conditioned cultures demonstrated an inverse correlation between proliferation and myofibroblast phenotype, with the combination of TGF‐β1+bFGF+EGF showing the greatest α‐SMA expression. Finally, TGF‐β1+EGF+PDGF treatment showed a significant increase in versican, fibronectin and type I collagen mRNA expression.

CONCLUSION: A combination of TGF‐β1+EGF+PDGF, can increase RASMC proliferation and induce ECM gene expression profiles similar to those of native aortic valve interstitial cells.
Interaction between cardiac heme oxygenase and natriuretic peptide system
Authors: Armstrong David William John , Tse M. Yat , Melo Luis G. , Liu Xiaoli , Pang Stephen C.
Published: June 2021

Genetic mouse models have recently shown that cardiac heme oxygenase (HO) and natriuretic peptide system (NPS) are essential for proper tissue dynamics and gene expression in the heart. However, the interaction between these two systems has not yet been investigated. The objective of this study was to determine the effect of ANP disruption on gene expression of the HO system, and to determine the effect of HO‐1 ablation and cardiac‐specific HO‐1 over‐expression on the NPS. Three mouse models were utilized: ANP−/−, HO−/− and HO‐1 cardiac‐specific transgenic (HO‐1 TG), and the respective wild‐type controls. Organs were excised, weighed, and snap‐frozen in liquid N2. Total RNA was extracted and analyzed by real‐time PCR. As expected, ANP−/− mice exhibited significant left ventricular hypertrophy. Surprisingly, HO‐1−/− mice exhibited splenomegaly (almost three fold increase in spleen mass). There was no significant difference in ventricular HO‐2 mRNA expression in ANP−/− mice, however ventricular HO‐1 mRNA expression was significantly lower in ANP−/− mice compared to wild‐type controls. These data support the notion that HO and NPS interact with each other. The significance and the mechanism of splenomegaly in the HO−/− mice remain to be elucidated. Supported by the Heart and Stroke Foundation of Ontario.
Congenital aqueductal stenosis due to downregulation of a dorsalizing gene
Authors: Sarnat Harvey B , Flores‐Sarnat Laura
Published: June 2021

Congenital aqueductal stenosis is a frequent cause of fetal hydrocephalus. We studied 9 human fetuses at midgestation with aqueductal stenosis for clues to etiology. Two had holoprosencephaly. Associated dysgeneses in all 9 included hypoplasia or agenesis of the corpus callosum; hippocampal and cerebellar dysplasias; absence of the dorsal median septum, confirmed by vimentin. The sagittal intercollicular fissure was absent or shallow with collicular continuity across the midline. In 2 cases, only the rostral mesencephalon was involved. These findings suggest downregulation of a gene acting in the dorsoventral gradient of the vertical axis of the neural tube, and also in rostrocaudal and mediolateral gradients.
Increased expression of SOX2 in Ammon's Horn Sclerosis: Further evidence for neurogenesis in epilepsy
Authors: Eschbacher Jennifer Mary , Coons Stephen
Published: June 2021

Neurogenesis persists throughout adult human life in the dentate gyrus and subventricular zone. Recent evidence points to increased neurogenesis in the hippocampus of epileptic animal models. The abnormal migration of new neurons to ectopic locations in the dentate gyrus, in conjunction with aberrant integration of new neurons into the granule cell layer, has been hypothesized to contribute to increased seizure activity. Many studies have also shown an increase in neurogenesis secondary to CNS insults such as stroke and trauma. We therefore hypothesized that we would see increased expression of SOX2, a neural stem cell marker, in hippocampal sclerosis. Using immunohistochemical analysis, we examined the hippocampus from nine patients for SOX2 expression. Seven were from patients with a known history of temporal lobe epilepsy and histologically confirmed Ammon's horn sclerosis and two were from normal autopsy controls. We found many immunoreactive cells throughout the hippocampous, including the subventricular zone, in all seven seizure cases, and no expression of sox2 in the autopsy controls. These results provide further evidence in support of active neurogenesis in epilepsy and suggest that SOX2 plays a role in neurogenesis in hippocampal sclerosis.

This research was funded and supported by Barrow Neurological Institute.
Functional Analysis of an Arabidopsis mitochondrial‐targeted DNA primase/helicase
Authors: Hunt Travis J , Liu Bin , Powell Brady K , Cupp John D , Nielsen Brent L
Published: June 2021

Twinkle primase/helicase is thought to be the single functional mitochondrial DNA (mtDNA) primase in eukaryotes. This protein is be essential for mtDNA maintenance and may be involved in regulation of mtDNA copy number in animal cells. This protein functions both as a DNA primase to initiate DNA replication and as a DNA helicase to unwind the DNA for replication. We have identified a putative mitochondrial‐targeted Twinkle homologue in Arabidopsis thaliana. Western blot analysis using an antibody specific for a unique region of this protein confirms that it is localized only to mitochondria. Bacterial expressed recombinant protein has DNA helicase activity and is being tested for DNA primase activity. Homozygous T‐DNA insertion mutants in the promoter of this gene show significantly reduced growth of plants, which abort before flowering. An insertion in an exon near the middle of the gene results in less effect on plant growth. Current work includes quantitative PCR to examine differences in mtDNA levels in mutant versus wild‐type plants, western blot and reverse transcriptase PCR analysis of expression levels at various stages of growth, and transmission electron microscopy to examine differences in mitochondrial morphology in the mutants. This research is funded by a grant from the NIH and by a BYU Mentoring Environments Grant.
Reduced expression of mitochondrial targeted RecA causes phenotypic differences in Arabidopsis
Authors: Cupp John D , Willis Van , Nielsen Brent L
Published: June 2021

Animals, yeast, and plants have similar numbers and types of genes expressed in their mitochondrial genomes. One difference, however, is that plants, specifically Arabidopsis, contain highly branched linear DNA whereas animals have circular DNA and yeast have both linear and circular mitochondrial DNA (mtDNA). We propose that mitochondrial targeted RecA is essential for mtDNA recombination and is responsible for forming the observed branching of mtDNA. We have observed differences in growth rate and plant morphology in Arabidopsis mutants that contain either a transfer DNA insertion or single base mutation in the nuclear encoded mitochondrial targeted RecA gene. The organization of the mitochondrial cristae is decreased when these mutants are observed by transmission electron microscopy. These morphological differences correspond to a decrease in RecA protein expression observed by western blot. The relative abundance of DNA in the mitochondrion is decreased when examined with quantitative PCR. When these mutant plants are challenged under DNA damaging conditions the differences are magnified. We conclude that mitochondrial RecA is essential for normal plant and mitochondrion morphology and may be involved in mtDNA replication and repair. This research was funded by the NIH.
Characterization and Differential Inhibition of Topoisomerase IB from the Nosomial Multidrug Resistant Pathogen Pseudomonas aeruginosa by Fluoroquinolone Drugs
Authors: Jain Teesta , Grove Anne
Published: June 2021

P. aeruginosa is an opportunistic pathogen responsible for many life‐threatening conditions in immunocompromised patients of neutropenic cancer and bone marrow transplant. P. aeruginosa encodes a putative topoisomerase (PAT) that shares sequence and biochemical characteristics with the vaccinia topoisomerase IB (VT). PAT has been purified and shown to relax supercoiled DNA without ATP but in the presence of MgCl2/MnCl2. The predicted tyrosine at the active site (Tyr292) when mutated to phenylalanine does not perform the relaxation reaction. In addition, the catalytic domain with the truncated amino terminal topo (81–333) performs the same repertoire of reactions as the wild type enzyme.

Fluoroquinolones are a subset of quinolones that are a group of antibacterials. The quinolones inhibit the type II topoisomerase exemplified by DNA gyrase. Sparfloxacin is a synthetic fluoroquinolone, which belongs to the same class as ofloxacin and norfloxacin. Although the PAT is not inhibited by norfloxacin, lomefloxacin, enrofloxacin, moxifloxacin and camptothecin, it is inhibited by sparfloxacin at higher concentrations. Like VT, PAT is resistant to camptothecin and the specific fluoroquinolones which inhibit the viral enzyme.
An interaction between Mex67p and Upf1p at the terminal step of mRNA export facilitates mRNA translation
Authors: Agosto Abby Joyce , Cajigas Iván , Guzzardo Paloma , Krebber Heike , González Carlos
Published: June 2021

Gene expression relies on the communication between the processes of transcription, mRNA processing, export and translation. Once synthesized in the nucleus, an mRNA must become competent for its translocation across the nuclear pore. Formation of an mRNA particle (mRNP) suitable for export is accomplished by the binding of soluble export factors to the nascent mRNA. Following transport, mRNPs must undergo structural rearrangements in the cytoplasm in order to be translated. We have gathered information to support this mRNP remodeling step. Specifically, we have found that Mex67p interacts with Upf1p. We provide evidence to support the notion that the Mex67p‐Upf1p interaction is neither required for the activation of the NMD pathway or for poly‐(A)+ mRNA export. Instead, Upf1p and Mex67p are critical to facilitate the conversion from an export‐competent mRNP to a translation‐competent mRNP, since cells lacking both proteins demonstrate severe defects in protein synthesis. Specifically, our data demonstrate that these cells are unable to efficiently initiate mRNA translation and recognize translation termination codons. We propose that in the cytoplasm, Upf1p communicates the Mex67p‐bound mRNAs with the protein synthesis machinery to facilitate translational competency and couple the terminal step of mRNA export to mRNA translation.

Supported by RISE Award 2R25GM61151, NIH U54 Award CA96297‐03, NIH‐NIGMS SCoRE Award GM008102‐305, and P20 RR 016174 from NCRR‐NIH.
SRp20 and hnRNP A1 binding to IR exon 11 modulate its alternative splicing
Authors: sen supriya , Talukdar Indrani , Webster Nicholas J.G
Published: June 2021

Alternative splicing of the insulin receptor (IR) transcripts generates two isoforms: isoform A (IR‐A), which lacks 36 bp exon 11, and isoform B (IR‐B), which includes exon 11. The IR‐B and IR‐A isoforms show a cell type specific distribution with their relative proportion varying during development, aging and different disease states. Normally, IR‐B predominates in insulin‐sensitive tissues such as liver, muscle, adipocytes and kidney whereas, various pathological conditions mostly express IR‐A isoform. Also, IR‐B has greater insulin responsiveness than does IR‐A. Recent studies demonstrate that IR gene also exhibits metabolic and hormonal regulation of splicing, although the regulatory factors are mostly unknown. Our lab has previously demonstrated that both exon 11 and intron 10 contain regulatory sequences that affect IR alternative splicing both positively and negatively. Here, we focus on the role of specific cis acting regulatory elements within exon 11 of the IR gene. Fine mapping of cis acting elements within exon has been carried out by six linker scanning mutations (LS1‐LS6) throughout the exon 11. Results indicate the presence of an exonic splicing enhancer (ESE) in the first 12 nucleotides and an exonic splicing silencer (ESS) within the central 12 nucleotides of exon 11. Most naturally occurring ESEs have been shown to bind specific SR proteins, whereas the best characterized ESSs are bound by particular hnRNP proteins. Sequence analysis of ESE in IR exon 11 revealed that it contains a (CUCUUC) consensus binding sequence for SR protein, SRp20. The ESS contains a sequence (CAGGCA) that partially matches the previously identified high‐affinity binding sequence for hnRNP A1 (UAGGGA). Co‐transfection in HepG2 cells of IR minigene B and several SR and hnRNP protein expression vectors reveals that hnRNP A1 is a negative regulator, whereas SRp20 is a positive regulator for IR exon 11 inclusion. Co‐transfection of the deletion and linker scanning mutants of exon 11 with SRp20 or hnRNP A1 confirmed the binding location of these proteins in IR exon 11. To establish in‐vitro binding of SRp20 and hnRNP A1 in ESE and ESS respectively, an RNA affinity assay was performed using RNA templates corresponding to wild type and linker scanning mutants in exon 11, coupled to adipic acid beads and incubated with HeLa cell nuclear extract. By western blot analysis of the eluted proteins, we have established that ESE binds SRp20 whereas the ESS binds hnRNP A1. Co‐transfection of minigene B with siRNA against hnRNP A1 increased whereas siRNA against SRp20 decreased the inclusion of exon 11. Purification of these regulatory proteins for in‐vitro splicing assay as well as UV cross linked competition assay using radio‐labeled probes is currently underway. These results demonstrate for the first time that SRp20 and hnRNP A1 play an antagonistic regulatory role in IR alternative splicing via binding to juxtaposed exonic elements.
COX‐2 mRNA stability in Monocytes by Ligation of RAGE. Novel roles of hnRNPK and miR‐16
Authors: Shanmugam Narkunarajaa
Published: June 2021

Advanced glycation end products (AGEs) act via their receptor, RAGE, and play major roles in diabetic complications. We recently showed that AGEs and a RAGE ligand, S100b, can induce the inflammatory cycoloxygenase‐2 (COX‐2) gene in monocytes by both mRNA stability and transcription mechanisms. In this study we examined the mRNA stabilizing mechanisms. S100b treatment of THP‐1 monocytes led to significant accumulation of COX‐2 mRNA at 2 hr. This was mainly due to RNA stability as also confirmed by S100b‐induced increase in luciferase activity of a COX‐2‐3’UTR‐Luc construct. Heterogenous nuclear ribonucleoprotein‐K (hnRNP K) promotes cross‐talk among multiple nuclear factors. RNA immunoprecipitation assays showed that S100b increased binding of hnRNP K to COX‐2 UTR RNA in THP‐1 cells. This was further confirmed by RNA‐EMSA and supershift with hnRNP K antibody. siRNAs to hnRNPK blocked S100b‐induced COX‐2 mRNA stability. Furthermore, micro RNA(miR)‐16 was also involved in S100b induced COX‐2 stability. In untreated cells, siRNA to miR‐16 enhanced COX‐2 3’UTR‐Luc activity and mRNA stability suggesting that miR‐16 can destabilize COX‐2 mRNA. Upon S100b treatment, miR16 levels (QPCR and N.blots) were reduced, while COX‐2 stability was enhanced. These new findings show that diabetic stimuli like AGEs can stabilize inflammatory genes such as COX‐2 by novel RNA regulatory mechanims involving hnRNP K and miR16.
Paralog specificity of mammalian SENPs for SUMO‐1 and SUMO‐2
Authors: Kolli Nagamalleswari , Mikolajczyk Jowita , Mukhopadhyay Debaditya , Dasso Mary , Salvesen Guy , Wilkinson Keith D
Published: June 2021

There are four SUMO paralogs; SUMO‐2, ‐3 and ‐4 are closely related and about 40% identical to SUMO‐1. SENPs are SUMO specific proteases responsible for deSUMOylation. Using a panel of SUMO‐1 and ‐2 derived substrates and inhibitors we examined the distribution and paralog specificity of SENPs. The total deSUMOylation activity in tissues and mammalian cell lysates examined is 15‐fold higher using SUMO‐2 than SUMO‐1 aminomethylcoumarin (AMC). Mammalian cell lysates showed more reactivity with SUMO‐2 vinyl sulfone (VS) than with SUMO‐1VS. Only SENP1 reacts significantly with SUMO‐1VS. To confirm these observations we examined the paralog specificity of the recombinant catalytic domains (cSENPs). The cSENPs acted on SUMO‐2 with Km and Ki values from 50–200nM. cSENP1 and cSENP2 showed a 2–4 fold preference for SUMO‐1. All other cSENPs strongly preferred SUMO‐2. Because cSENP2 reacted with SUMO‐2 derivatives while full length SENP2 in lysates did not, we asked whether specificity was dependent on the N‐terminal non‐catalytic domain. Full length recombinant his6‐SENP1 showed the expected paralog specificity. In contrast the catalytic domain alone, his6‐SENP2 cleaved SUMO‐2‐AMC much faster than SUMO‐1‐AMC. Thus the non‐catalytic N‐terminal domain of SENP2 participates in determining paralog specificity. In summary, SUMO‐2/‐3 modification in vivo may play a more significant role than previously appreciated.
Proteomic Analysis of SUMO Mediated Stress Responses in Yeast
Authors: Ro Kevin , Wohlschlegel James
Published: June 2021

Cells are constantly exposed to a wide variety of environmental stresses and their ability to maintain the integrity of their genome is essential to their survival. One cellular pathway that plays a key role in this cellular response is the SUMO modification pathway. SUMO is a member of the ubiquitin family of small protein modifiers and its covalent attachment to cellular targets is a critical regulatory step for a diverse range of cellular processes that includes transcriptional regulation, cell cycle progression, and cellular DNA damage response. In this study we are investigating how patterns of targets modified by SUMO changes in response to various stresses such as hydrogen peroxide, ethanol, nocodazole, and doxorubicin. This approach involves the use of a quantitative proteomics platform established in our laboratory and is built around a recently developed linear ion trap–orbitrap hybrid mass spectrometer. Ultimately, this work will enable us to understand how cells use SUMO pathways to protect themselves from genotoxic damage and offer insight into how the deregulation of this pathway, a common feature in cancers, may lead to genetic instability. These experiments are currently underway and the results from this work will be presented. Importantly, the SUMO pathway is highly conserved between yeast and humans so that important findings generated by our analysis will likely be directly applicable to humans.
Utilization of a Deoxynucleoside Diphosphate Substrate by HIV‐1 Reverse Transcriptase
Authors: Garforth Scott J , Prasad Vinayaka R
Published: June 2021

The substrates for polymerization by DNA polymerases are, with few exceptions, deoxynucleoside triphosphates (dNTPs); the DNA polymerase from phage RB69 and several non‐template dependent polymerases can utilize a deoxynucleoside diphosphate (dNDP) substrate. In HIV reverse transcriptase (RT) the K65 residue has an electrostatic interaction with the γ‐phosphate of the incoming dNTP. We have previously shown that a K65A mutant of HIV RT retains activity, demonstrating that the interaction between RT and the γ‐phosphate is nonessential. Therefore, we investigated whether the γ‐phosphate of the incoming dNTP was required for polymerization, and found that HIV RT can utilize a dNDP substrate. Further, we demonstrate that RT can catalyze the corresponding reverse reaction, the inorganic phosphate‐dependent excision of primer 3′ terminal nucleotide. Phosphorolytic excision of a chain terminating nucleotide by RT is the major mechanism for AZT resistance in HIV, and has previously been shown to occur via pyrophosphorolysis or ATP‐dependent phosphorolysis. We show that HIV RT is also capable of excising AZT through inorganic phosphate dependent phosphorolysis. This previously undocumented activity of HIV RT has the potential to be an additional route by which HIV acquires AZT resistance. Funding: Public Health Service research grant to V.P.
Absence of intestinal gluconeogenesis in type 1 diabetic rats: Arteriovenous and carbon 13 NMR measurements.
Authors: Baverel Gabriel , Gadet Rudy , Nazaret Rémy , Martin Mireille , Boghossian Michelle , Martin Guy , Ferrier Bernard , Conjard Agnès
Published: June 2021

Recent reports have indicated that type 1 diabetes induces gluconeogenesis in the small intestine of adult Sprague Dawley rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and (ii) have major consequences in the diabetes fields, we have thoroughly re‐examined this question. For this, metabolically viable small intestinal segments from fed and 36h‐fasted streptozotocin‐diabetic Sprague Dawley rats were incubated with [3–13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR methods. Although the segments utilized 13C‐glutamine and accumulated 13C‐labeled products at high rates for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re‐utilization of 13C‐glucose initially synthesized. Arteriovenous metabolite concentration difference measurements across the portal vein‐drained viscera of 36h‐fasted streptozotocin‐diabetic Sprague‐Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the type 1 diabetic rat is a gluconeogenic organ.

Supported by the INSERM.
Popcorn‐Like Conformational Transition of Bio‐Nanoparticle Complex: Analysis and Application
Authors: Lee Tzu‐Cheng , Chou Ching‐Chung , Cheng Pei‐Hsin , Chang Chia‐Ching
Published: June 2021

Nanometer sized diamond (ND), in addition to its noncytotoxicity, intrinsic fluorescence and easiness of surface functionalization, contains ‐C‐N=O functional group inside its structure. The ‐C‐N=O group can be photo‐cleaved by UV light and then releases NO molecule. Here, we use nanosecond pulse laser to trigger photo‐cleavage process based on the multi‐photon absorption. Comparing the Raman spectra before and after laser irradiation reveals the structure of ND changes conformation from sp3 diamond structure to aromatic graphite‐like sp2 bonded structure. Also, the AFM & SEM analyses show that 5‐nm NDs were expanded to ∼100 nm in diameter. Additionally, by conjugating ND with growth hormone(rEaGH), it can recognize cancer cell (A549) which frequently contains high copy number of growth factor receptors. Causing a “Popcorn‐like” conformational transition changes the cell's morphology and makes the cell apoptosis in consequence. Combining with those photophysical and biochemical properties mentioned above, we suggest that ND is potentially a good candidate in cancer therapies and other biomedical applications.
trans‐[Ru(NO)(NH3)4(py)](BF4)3 encapsulated in PLGA nano and microparticles for delivery of nitric oxide to neoplasic cells
Authors: Gomes Anderson J. , Barbougli Paula A. , Espreafico Enilza M. , Tfouni Elia
Published: June 2021

The NO donor trans‐[Ru(NO)(NH3)4(py)](BF4)3 was loaded into poly‐lactic‐co‐glycolic acid (PLGA) particles (pyMP) and the ability of pyMP to deliver NO was tested on the cultured B16‐F10, melan‐a and WM1552 melanoma cells. Scanning electron microscopy and dynamic light scattering revealed that the particles are spherical, have a diameter of 200 to 1200 nm. The complex in solution, in the absence of irradiation, is toxic at higher concentrations (1×10−3 mol.L−1) with cell death attributed to NO release following reduction of the complex in the cell environment. There was no cell death with pyMP, due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. Upon light irradiation, pyMP is able to release sufficient NO that can diffuse out of the matrix, reach the adjacent cell membranes, and kill the tumor cells. The phototoxicity strongly suggests that cell death is due to NO release from trans‐[Ru(NO)(NH3)4(py)]3+. Supported by FAPESP (05/58170‐2), CNPq (151406/2007‐3).
Preparation of immobilized bovine deoxyhypusine hydroxylase and its applications
Authors: Soustek Meghan S , Murtaugh Megan L , Wang Yinglu , Smith Joshua L , Huang Jenq‐Kuen , Wen Lisa
Published: June 2021

Deoxyhypusine hydroxylase (DOHH) catalyzes the second and the final step of eukaryotic initiation factor 5A (eIF5A) maturation which is essential for eukaryotic cell proliferation and survival. Modification of eIF5A has been suggested to be a potential target for anticancer and antiviral therapy. A detailed study of the structure and properties of DOHH is essential. We have previously partially purified DOHH from bovine testis. Our results suggested possible presence of multiple forms of DOHH. However, the native DOHH protein has never been fully purified and characterized from any tissues. Antibody probes specifically recognize DOHH would be a value tool for detection and purification of native DOHH protein. We have elicited polyclonal antibodies against recombinant bDOHH. Here, we report the preparation of immobilized recombinant bDOHH by coupling the protein to CNBr‐activated agarose. The bDOHH‐agarose has been used as an affinity column to purify antibodies from the immune serum. The affinity purified antibodies have been used in detecting recombinant bDOHH in a Western blot. These antibodies are being used to study native DOHH proteins in bovine tissues by Western blotting of bovine tissue extracts after separation by 1D or 2D gel electrophoresis.

Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.