bluuebunny/crossref_metadata_2025 huggingface dataset:
Tooth Replacement in an Anatomical Context: Studies in the Zebrafish
Authors: Huysseune Ann , Crucke Jeroen
Published: June 2021

Most non‐mammalians that possess teeth display lifelong tooth renewal. So far, studies aiming to dissect the mechanisms underlying continuous tooth replacement have focused mostly on the molecular cues that may regulate replacement. However, if we wish to fully understand this process, we also need to consider the cellular environment in which teeth develop.

We have embarked on studies investigating the vasculature and innervation of the zebrafish pharyngeal dentition (there are no oral teeth), and their respective roles in tooth replacement, using light microscopy, 3D reconstructions and immunohistochemistry. First, we have demonstrated that, while the first‐generation teeth develop before the vasculature has reached the last pharyngeal arch on which these teeth develop, functional teeth become surrounded by an elaborate sinusoidal cavity. Capillaries branching off from this sinusoidal vessel supply replacement teeth at an advanced stage in their development, as they enter the pulp cavity only at late cytodifferentiation. Blocking VEGF, a major angiogenic factor, slows down replacement tooth development but does not prevent initiation of new teeth, suggesting that formation of new blood vessels does not trigger the replacement process. Like the more anterior, gill‐bearing arches, the dentigerous pharyngeal arch is innervated by posttrematic rami of the vagal nerve (NX). Sensory fibers enter the pulp cavity at a late stage of cytodifferentiation, shortly before attachment of the tooth. Autonomous fibers have not been identified so far.

Whether nerves follow the vasculature in entering the pulp, as in mammalian teeth, and what the role is of the neurovascular supply in teeth that are renewed anyway, needs further studies.

This study was supported by a grant of the IWT‐Vlaanderen to JC.
Dietary Protein Restriction and FGF21 Influence Bone Morphology
Authors: McNulty Margaret , Laeger Thomas , Albarado Diana , Morrison Christopher
Published: June 2021

Dietary modifications, including protein restriction, affect multiple organs. Previous work shows that protein restriction induces adaptive changes in food intake, growth, and metabolism, and that these effects are lost in FGF21 deficient mice. This indicates FGF21 is required for this response. While previous work has shown FGF21 affects growth plate cartilage, no work has yet been done to identify the impact of these combined conditions on bone growth and remodeling, despite changes in overall size and weight in previous studies. The purpose of the current study was to identify whether dietary protein restriction in wild‐type (WT) or FGF21‐deficient (KO) mice impacts bone. WT or KO mice were placed on a control (NP, 20% casein) or low‐protein (LP, 5% casein) diet (n=39, 9‐10/group) starting at 17.5 weeks of age for 27 weeks. Following sacrifice, cortical and trabecular bone morphology within the femur was evaluated with micro‐CT. No significant differences were identified in bone length, however significant differences were found in trabecular bone volume/total volume (BV/TV; p<0.001). Overall, LP groups had significantly lower values than the NP groups within the same genotype, and KO mice had significantly lower BV/TV values than WT. Cortical area was significantly decreased in the WT‐LP diet compared to WT‐NP (p<0.001), but it was not different in KO mice. However, cortical marrow space area was significantly decreased in KO mice when compared to WTs (p<0.01). These data demonstrate that prolonged dietary protein restriction exerts a marked negative effect on bone health, and that loss of FGF21 signaling alters but doesn't block this negative effect.
Calcitonin Gene‐Related Peptide Elicits Mouse Taste Buds to Release Serotonin
Authors: Huang Anthony , Wu Sandy
Published: June 2021

Gustatory afferent fibers receiving taste signals from corresponding taste bud cells store transmitters such as glutamate and calcitonin gene‐related peptide (CGRP). However, to date, the effect of CGRP on signal processing in taste buds is not well understood.In this study, we characterized metabotropic CGRP receptors that appear to underlie the postulated efferent transmission present on taste cells. Specifically, we reported that Fura 2 loaded mouse vallate taste cells showed Ca2+ mobilization in responses to a low concentration of CGRP (0.1 μM). The findings were that a significant fraction of Presynaptic (Type III) taste bud cells (53%) responded to CGRP, and that only few Receptor (Type II) taste cells (5%) were activated by this transmitter. CGRP8‐37, a CGRP receptor antagonist, reversibly abolished Ca2+ responses and elucidated the actions of CGRP. Removing extracellular Ca2+ had no effect on responses evoked by CGRP stimulation. In marked contrast, applying 1 μM thapsigargin, a SER Ca‐ATPase inhibitor, or 10 μM U73122, a PLC blocker, eliminated Ca2+ responses, suggesting that CGRP stimulation triggered PLC/IP3‐mediated release of Ca2+ from intracellular stores. Using CHO/biosensor cells expressing 5‐HT2C receptors, we also measured taste‐evoked transmitter release from taste buds. Consistent with these results, applying CGRP caused taste buds to secrete 5‐HT, a Presynaptic (Type III) taste cell transmitter. Biosensor responses evoked by stimulating taste buds were reversibly blocked by mianserin, a 5‐HT2C receptor antagonist, verifying that the signals arose from serotonin. We conclude that CGRP as an efferent transmitter may act via serotonergic mechanisms to modulate the final output of taste signaling. Grant Funding Source: Supported by SIUSOM Startup Funds.
Combination of Sub‐effective Doses of NMDA and D1 Dopamine Receptor Antagonists Impairs Executive Function in Rats
Authors: Desai Sagar , Allman Brian , Rajakumar Raj
Published: June 2021

Contemporary research in the pathophysiology of cognitive deficits in schizophrenia has provided a multifactorial view, in which the neurotransmitters dopamine, gamma aminobutyric acid (GABA) and glutamate are disturbed in the prefrontal cortex (PFC) of schizophrenic brains. This view is supported by the fact that current antipsychotic drugs, which are primarily dopamine receptor blockers, are not effective in treating the cognitive symptoms of schizophrenia. We hypothesized that abnormality in dopamine, GABA and glutamate neurotransmissions act synergistically to cause certain cognitive symptoms of schizophrenia. We tested the effect of blockade of all three neurotransmitter systems on executive function using an operant conditioning‐based attentional set‐shifting task to assess behavioral flexibility. In a series of dose‐response studies, we determined the effective doses of the specific antagonists for dopamine D1 receptors (SH‐23390), GABA‐A receptors (bicuculline methiodide) and NMDA receptors (MK‐801). Next, sub‐effective doses were administered subcutaneously 25 min before the set‐shifting task. Our results indicate that while the single antagonist treatments did not impair set‐shifting, combination of sub‐effective doses of SH‐23390 and MK‐801 severely affected performance. Present findings will help to elucidate mechanisms underlying cognitive deficits in schizophrenia.

Acknowledgement: Supported by NSERC and the Dept. of Psychiatry, University of Western Ontario.
A Dismantabled Human Brain In Papier‐mâché By Auzoux, Circa 1870: Description and Tri‐dimensionnal Photographical Reconstruction
Authors: LE FLOCH‐PRIGENT PATRICE , GILLOT JEAN‐BERNARD
Published: June 2021

The « dismantabled » brain in papier mâché by Auzoux was made since 1858. This real‐sized model is quite rare. This anatomical model could be dismantabled in 16 asymetrical pièces between the right and left parts. The thickness planes were mainly horizontal cross‐sections. They represented the gross‐anatomical structures without any vessel. This model was conform to the neuro‐anatomical data in the second half of the 19th century which were quite modern ones. By the selection of the horizonal plane, this anatomical model was a pre‐figuration of the actual images of the brain obtained by the CT‐scan and the MRI.
Crushed sciatic nerve treated with latex protein associated with low‐level laser: long term functional study
Authors: Dias Fernando , Watanabe Ii‐sei , Iyomasa Mamie , Joaquim Coutinho‐Netto , Issa Joao P , Fazan Valeria Paula
Published: June 2021

This study evaluated the effects of natural latex angiogenic fraction (F1 protein – 0,1%) associated with low‐level laser (LLL 15J/cm2, 780nm) on the crushed sciatic nerve (15 kgf, axonotmesis). Male Wistar rats (250g) were separated into 6 groups (n=6): C=control; E=exposed nerve; I=injured nerve; L=LLL treated; F1=F1 protein treated and LF1=F1+LLL. The functional analysis included mechanical allodynia, paw pressure withdraw and lower limb grip strength performed at 1, 7, 14, 28 and 56 days after nerve injury. Mechanical allodynia: Groups C and E had smaller initial values in comparison to all others. Groups I, L, F1 and LF1 reduced values with time but only F1 and LF1 reached values similar to controls after 7 and 14 days, respectively. Paw pressure: The uninjured paw data was similar between groups during the experimental time. The injured paw baseline values were higher in I, L, F1, LF1 compared to C and E reaching similar values after 56 days. The F1 and L groups had the largest reductions after 7 days, while LF1 reached control values after 14 days. Grip Strength: All groups increased values over the experimental time but at day 1, C and E groups showed values twice as high compared to the others. LF1 was similar to C in the acute period (7 days) and F1 improved this parameter after 28 days; I and L had lower and similar values during the 56 days period. The functional analyzes showed improvement over time after crush injury. Treatment with the F1 protein, associated or not with LLL accelerated the functional recovery bringing the values closer to controls in a very acute period of time (7 and 28 days after injury). FAPESP 2014/07253‐4
The Role of Histone H1 in S. cerevisiae
Authors: Leicher Rachel , Schilit Samantha , Holmes Scott
Published: June 2021

Linker histone H1 is a fundamental component of chromatin in eukaryotes with unclear functions in chromosome biology. We are utilizing genetic and biochemical approaches to determine the role of histone H1 in chromatin structure and function in budding yeast. First, we are assessing the effects of H1 gene deletions in strains with point mutations in core histones H3 and H4. With changes in silencing as a sensitive reporter for changes in chromatin, we are identifying H1's interaction with the nucleosome and overall function in chromatin. When H1 is deleted, we find a statistical overrepresentation of specific histone region mutations with the same silencing phenotype, such as the H3 tail deletions, suggesting that H1 is interacting with specific regions of the nucleosome. Through a biochemical lens, we are also addressing how histone H1 interacts structurally with DNA. To examine its binding specificity to DNA, we are examining histone H1's association with holiday junctions in vitro. Through the approaches of genetics and structural biology, we will gain insight on the role of histone H1 in chromatin.
Construct of RNAi plasmids and Their Application For the Study of Meiotic Proteins Through RNA Interference
Authors: Hope June , Hillers Ken
Published: June 2021

Sexual reproduction in eukaryotes requires production of haploid gametes from diploid cells. This is accomplished through meiosis, in which two successive divisions of a diploid cell in the germ line results in four haploid gametes. This process is highly regulated, and requires the action of many key proteins in order for chromosomes to segregate properly and meiosis to be successful. In the absence of a protein involved in meiosis, one may expect chromosome missegregation, failure of meiosis, and eventual cell apoptosis. In this study, the function of meiotic proteins are analyzed through the process of RNA interference in Caenorhabditis elegans, a model organism for the study of meiosis. RNA interference (RNAi) reversibly modifies the expression of proteins due to the introduction of double‐stranded RNA (dsRNA), which results in the reversible depletion, or silencing, of the protein encoded by the complementary gene. In previous work, we used proteomics to identify candidate meiosis proteins. In order to evaluate these candidates, we constructed RNAi plasmids that would target the gene under investigation. A successful RNAi construct will result in the decreased expression of the target protein. If the protein is required for proper completion of meiosis, the process will be disrupted, resulting in the reduction in viability of C. elegans progeny. This poster will present data on the construct of RNAi plasmids and their subsequent effects on meiosis in C. elegans, as well as direct analysis of the cytology of meiosis through fluorescence microscopy.
Nucleosome Dynamics Around a DNA Double Stranded Break During Repair by Gene Conversion.
Authors: Tsabar Michael , Hicks Wade , Haber James
Published: June 2021

In eukaryotes, double‐stranded break repair (DSB) takes place in the context of chromatin. Saccharomyces cerevisiae mating type (MAT) switching provides an opportunity to investigate nucleosome dynamics around a well‐characterized DSB. MAT switching is initiated by synchronous induction of a DSB by HO endonuclease at the MAT locus following which the DSB ends are resected and the Rad51 recombinase is loaded on the 3‐ended single strand. Rad51 facilitates strand invasion with a homologous donor sequence, which is required for initiation of new DNA synthesis and the resulting repair.

A hurdle in studying nucleosome positioning around the MAT locus has been the weak nucleosome positioning around the MAT locus relative to the heterochromatic donors HML and HMR. To address this hurdle we designed a novel system in which we inserted the Xenopus laevis 5S nucleosome positioning sequence adjacent to the MAT locus. Using this system we have, for the first time, monitored the loss of chromatin protection following resection of the DSB end and the re‐establishment of chromatin structure following repair. We show that nucleosome protection is lost around a DSB in a resection‐dependent manner. Further, we show that nucleosome protection is reestablished following repair of a DSB but not to the level of protection seen prior to induction of a DSB. This reestablishment depends on repair of the DSB and not on passage through the cell cycle. We show that complete protection is reestablished after several cell cycles, suggesting that there is a mechanistic difference between repair‐dependent and replication‐dependent nucleosome positioning.
Untethering Glucocorticoid Receptor‐Mediated Transcriptional Repression of Inflammatory Genes
Authors: Rye Emily , Ortlund Eric
Published: June 2021

The glucocorticoid receptor (GR), a ligand regulated transcription factor, that controls the expression of thousands of genes driving both up‐ and down‐regulation at equal frequencies. To activate transcription, GR cooperatively dimerizes on DNA at glucocorticoid response elements (GREs). However, the mechanism of gene repression by GR is more debated but it thought to occur through DNA‐dependent and DNA‐independent mechanisms. The DNA‐dependent mechanism involves direct binding of GR to the newly discovered negative glucocorticoid response elements (nGREs). The DNA‐independent mechanism involves GRs interaction with activator protein‐1 (AP‐1), which is a central transcription factor in immune systems that drives expression of inflammatory response genes. This mechanism, known as “tethering”, is the prevailing model for GR‐mediated repression at inflammatory genes. However, recent discoveries regarding DNA‐dependent transrepression at nGREs reveal that the mutations used to develop the tethering hypothesis are inadequate. We show using x‐ray crystallography, in vitro, and in cell assays that GR binds directly to nGRE‐like sites, leading us to hypothesize that GR‐mediated repression on inflammatory genes occurs through an alternative mechanism involving direct DNA binding.
Lack of Glucocorticoid Receptor Hypersensitivity‐Related Polymorphisms in an Undergraduate Population
Authors: Leibovitz Evan , Cohen Brian
Published: June 2021

Many phenotypic similarities exist between Cushing's Syndrome (CS) and Metabolic Syndrome (MetS). As the main diagnostic criterion for CS is elevation of the steroid hormone cortisol, a glucocorticoid, we hypothesize that MetS, in some cases, represents a CS like state in the absence of elevated cortisol. One potential mechanism by which this could occur is elevated sensitivity to glucocorticoids as a result of mutation of the glucocorticoid receptor (GR).

Two particular polymorphisms of the GR that correlate with hypersensitivity to cortisol are the asparagine to serine mutation at the 363rd codon of the receptor gene (N363S) as well as a mutation at the BclI site on the second intron of the receptor gene. The objective of this study was to investigate the presence of these two polymorphisms of the human glucocorticoid receptor that are associated with hypersensitivity to cortisol in a population of college students.

Student DNA was isolated using a chelex isolation method from buccal (cheek) cells. A novel set of allele specific primers were designed to detect the two hypersensitivity‐causing polymorphisms. Using polymerase chain reaction (PCR), in a population of white, non‐obese, college aged students neither heterozygotes nor mutant homozygotes of the two GR alleles were detectable.

Our finding of a prevalence of zero is not surprising as our population of non‐obese students is unlikely to have cortisol hypersensitivity. This is evident by their normal range BMIs and lack of MetS and CS like symptoms. However, the population did allow us to test the specificity of the PCR method we developed. Future research will investigate the prevalence of the polymorphisms in morbidly obese adults seeking bariatric surgery and it is expected that the mutant alleles will be detected in this population.
T4 Factor Involved in Phage‐induced Host mRNA Degradation
Authors: Qi Dan , Otsuka Yuichi , Yonesaki Tetsuro
Published: June 2021

Objective

Identification and characterization of T4 factor involved in phage‐induced host mRNA degradation

Results

Bacteriophage T4 causes a shift of gene expression from host Escherichia coli to T4 immediately after infection. In this process, host mRNAs are rapidly degraded, which may facilitate T4 gene expression by providing precursors for transcription and free ribosomes. Recently we found that the rapid degradation of host mRNAs was partially suppressed and a decay intermediate of lpp mRNA was markedly accumulated in Δ(39‐56)6 phage‐infected cells. Since the 4.6‐kb region missing in Δ(39‐56)6 phage contain 8 orfs, we tested the effect of each orf on host mRNA degradation and found that the causal gene is dda.2, a T4 early gene, located immediate downstream of an early promoter.
The average number of phages produced in a cell infected with a deletion mutant of dda.2 (Δdda.2) was one‐third, in comparison to that of wild type, indicating that dda.2 is necessary for efficient propagation of T4. On the other hand, over‐expression of Dda.2 was found to be toxic for cells. With the induction of plasmid‐borne dda.2, cell growth was stopped within 30 min, and cell viability was significantly reduced. In addition, amounts of E. coli mRNAs, such as lpp and ompA were considerably reduced. Furthermore, the decay of ompA and lpp mRNAs was accelerated after induction of plasmid‐borne dda.2.

Conclusions

1. T4 dda.2 is involved in phage‐induced host mRNA degradation, which is thought to be important to facilitate T4 gene expression.

2. T4 dda.2 has a significant role in bacterial physiology probably with a modulation in mRNA metabolism.

Source of the research support: The ministry of education, culture, sports, science and technology, Japan
Modifications Modulate Anticodon Loop Dynamics and Codon Recognition in E. coli tRNAArg1,2
Authors: Sarachan Kathryn , Cantara William , Spears Jessica , DeMirci Hasan , Murphy Frank , Ranganathan Srivathsan , Chen Alan , Agris Paul
Published: June 2021

In E. coli, three of six arginine codons (CGU, CGC and CGA) are decoded by only two tRNAArg isoacceptors. The anticodon‐stem loop (ASL) domains of tRNAArg1 and tRNAArg2 differ only at position 32: tRNAArg1 is posttranscriptionally modified to contain a 2‐thiocytidine (s2C32). Both isoacceptors also contain naturally‐occurring inosine (I34) and 2‐methyladenosine (m2A­­37) modifications. To investigate the functional roles of these modifications, six ASL constructs differing in modification profile were analyzed using binding studies and structural and computational methods. Ribosome filter binding assays showed that while I34 facilitates wobble codon binding, both s2C32 and m2A37 modulate that effect by negating binding to the rare CGA codon. When a non‐naturally occurring s2C32 was introduced for C32 in an unrelated S. cerevisiae tRNA ASLLeu construct also containing I34, the same functional restriction of CGA binding was observed. The NMR and x‐ray crystallographic structures of the ASLs showed only a minor anticodon loop perturbation upon inclusion of s2C32. The mechanism of its effect on codon binding was then investigated by molecular dynamics simulations, which suggested that both s2C32 and m2A37 afford the ASL greater flexibility and conformational accessibility and explained their ability to adopt one structure free in solution and two others when bound to the cognate arginyl‐tRNA synthetase or to codons on the ribosome.
Insights into Spliceosomal Activation from Conformational Dynamics of the U4/U6 di‐snRNA
Authors: Hoskins Aaron , Rodgers Margaret , Brow David
Published: June 2021

Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large‐scale structural rearrangements during splicing. This is exemplified by the U6 snRNA which can exist in isolation in the U6 small nuclear ribonucleoprotein (snRNP); basepaired with U4 in the U4/U6 di‐snRNP, U4/U6.U5 tri‐snRNP, and spliceosomal B complex; or basepaired with the pre‐mRNA intron and the U2 snRNA in the catalytic spliceosome. Through genetic evidence and structure probing from the Brow, Guthrie, and Luhrmann labs, it was previously proposed that the U4/U6 di‐snRNA consisted of a three‐helical junction core domain flanked by single stranded regions. Using single‐molecule Förster resonance energy transfer (smFRET), we discovered a dynamic equilibrium between RNA conformations formed by the flanking single stranded regions. Our smFRET data is consistent with the presence of two mutually exclusive structures: (1) a U4/U6 di‐snRNA containing both basepaired U4/U6 and the U6 telestem and (2) a U4/U6 di‐snRNA with extended basepairing located near U4/U6 stem I. These data show that the U6 telestem persists in the U4/U6 di‐snRNA and that its formation can be regulated by novel basepairing interactions with U4. Furthermore, these dynamics are responsive to the presence of spliceosomal proteins such as the Prp8 RNaseH domain. We propose a model for U4/U6 unwinding that relies on disruption of U4/U6 stem I, and we have tested this model by unwinding U4/U6 with oligonucleotides in the absence of a RNA helicase. We hypothesize that unwinding of stem I by the Brr2 helicase promotes U4 release during spliceosomal activation and that the presence of a RNA helicase may not have been a prerequisite for U4 snRNP evolution.
Investigation Of Copper Exchange Between The Avian Egg Proteins Riboflavin Binding Protein, Phosvitin And Vitellogenin
Authors: Matchynski James , Benore Marilee , Chamoun Ali , Smith Sheila
Published: June 2021

Riboflavin binding protein (RBP) has been recently reported to bind copper in vitro. The exchange of copper between common egg proteins previously determined to bind copper in vivo, phosvitin and its precursor vitellogenin, is examined via FPLC, Atomic Absorption spectroscopy and isothermal titration calorimetry.
Fragment‐based Drug Discovery Targeting KEAP1/Nrf2 Binding
Authors: Zhong Mengqi , Lynch Andrew , Jehle Stefan , Kozakov Dima , Vajda Sandor , Allen Karen , Whitty Adrian
Published: June 2021

The goal of our research is to discover small molecule lead structures for modulation of the protein‐protein interaction between Kelch‐like ECH‐associated protein 1 (Keap1) and Nuclear factor erythroid 2 related factor 2 (Nrf2). We developed a Fluorescent Anisotropy assay for Keap1 inhibitors using FITC‐labeled Nrf2 9‐mer peptide containing the Keap1 binding motif. Using this assay we screened a library of small molecular fragments, and identified a number of hits with diverse chemotypes for further advancement. A number of these fragment hits were crystallized with Keap1, allowing us to compare their binding modes with that of Nrf2 peptide and with known Keap1 inhibitors. We additionally compared the experimental fragment binding modes with the locations of binding energy hot spots identified using the computational FTMap algorithm. The results identify new chemotypes that can bind at the Nrf2 binding site on Keap1, and advance our understanding of the binding properties of this target site.
An Intrinsically Disordered Low Complexity Domain is Required for U1‐70K Self‐association
Authors: Bishof Isaac , Diner Ian , Seyfried Nicholas
Published: June 2021

The RNA binding protein U1‐70K aggregates via an unknown mechanism in Alzheimer's disease (AD). Interestingly, AD brain homogenates can induce the aggregation of soluble U1‐70K from control brain or recombinant U1‐70K (rU1‐70K), rendering it detergent‐insoluble. The intrinsically disordered C‐terminus of U1‐70K is necessary for aggregation and harbors two low‐complexity (LC) domains, LC1 and LC2. Similar LC domains have been shown to polymerize into amyloid‐like aggregates in vitro, and the RNA binding proteins TDP‐43 and FUS, which also harbor LC domains, form detergent‐insoluble amyloid‐like aggregates in neurodegenerative disease. Our objective is to determine if the LC domains of U1‐70K are necessary for self‐association. Addressing this question may provide mechanistic insight into the aggregation of U1‐70K in AD. To investigate this question we performed co‐immunoprecipitations of full‐length rU1‐70K and various mutants lacking either the LC1, LC2, or both LC domains. These assays revealed that the LC1 domain (amino acids 231‐308) of rU1‐70K is necessary and sufficient for association with native U1‐70K. Similarly, we utilized bio‐layer interferometry to measure the binding affinity of the LC1 domain with a synthetic peptide corresponding to amino acids 252‐270 of U1‐70K. These data provide evidence for a direct self‐association between regions of the LC1 domain. Finally, by immunocytochemistry we show that rU1‐70K mutants lacking the LC1 domain have an impaired ability to form nuclear granule structures. Together these data indicate that the disordered LC1 domain is necessary for U1‐70K self‐association and subnuclear compartmentalization.

Sources of Research Support:

NIRG‐12‐242297 (NTS) and T32‐NS007480 (IB)
Hsp90, Hsp60 And HSF‐1 Genes Expression in Muscle, Heart and Brain of Thermally Manipulated Broiler Chicken
Authors: Al‐Zghoul Mohammad
Published: June 2021

The effect of thermal manipulation (TM) was investigated during embryonic days (ED) 12‐18 on the hatchability, body temperature (Tb) and on thyroid hormones (T3 & T4) and on mRNA levels of heat shock proteins (Hsp90, Hsp60 and HSF‐1) in muscle, heart and brain tissues and during thermal challenge (TC) at days 10 and 28 post‐hatching. One thousand five hundred fertile chicken eggs were randomly allocated into four groups: Control group (37.8°C), TM1 (39°C for 9h), TM2 (39°C for 12h) and TM3 (39°C for 18h). Chicks from each treatment group were then randomly sub‐divided into 2 treatment groups (Naive and TC). At days 10 and 28 of age, chicks in TC groups were subjected to thermal challenge (TC) at 43.0°C for 6h while naïve chicks were kept under regular conditions. Chick's response to TC was evaluated by determination of body temperature (Tb), plasma T3 and T4 levels and mRNA levels of Hsp90, Hsp60 and HSF‐1 in muscle, heart and brain tissues. TM adversely affected chick's hatchability rate. The Tb of TM chicks decreased compared to the control group during the first 2 weeks of age. Interestingly, during TC at days 10 and 28 of age, the Tb of TM chicks was significantly lower than those of the controls. When compared to the control, TM resulted in a significant increase in mRNA levels of Hsp90, Hsp60 and HSF‐1in muscle, heart and brain tissues during embryogenesis and during TC at days 10 and 28 post‐hatching. Results of this study indicated a long‐term enhancement of Hsp90, Hsp60 and HSF‐1 gene expressions associated with improved thermotolerance acquisition in thermally challenged chicks without adversely affecting body performance parameters.
A Low Affinity Cavb4a/Rem2 Interaction is Required for Inhibition of Cav2.1 Ca2+ Currents
Authors: Xu Xingfu , Zhang Fangxiong , Zamponi Gerald , Horne William
Published: June 2021

RGK (Rem, Rad, Kir/Gem proteins) proteins, including Rem2, cause profound inhibition of high‐voltage activated Ca2+channels containing intracellular regulatory b subunits. All RGK proteins bind to Cavb subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies solution state nuclear magnetic resonance (NMR) and calorimetric techniques to map the binding site for Rem2 on human Cavb4a and to measure the binding affinity. Our experiments revealed two binding surfaces on the b4 GK domain contributing to a 156 μM Kd interaction: 1) a hydrophobic pocket surrounded by four residues, mutation of any of which completely disrupted binding; and 2) a nearby surface containing three residues that when individually mutated decreased affinity. Cav2.1 Ca2+currents were completely inhibited by Rem2 when co‐expressed with wild‐type Cavb4a, but were unaffected by Rem2 when co‐expressed with a Cavb4a site 1 mutant. These results provide direct evidence for a low affinity Rem2/Cavb4 interaction and its essential role in Cav2.1 inhibition by Rem2.
Expression and Purification of Rickettsial Outer Membrane Protein B
Authors: Choi Bok‐Eum , Abeykoon A , Chao C C , Wang G. , Gucek M. , Ching W M. , Chock P B. , Buchanan S. , Yang D C H.
Published: June 2021

Outer membrane protein B is an autotransporter protein present in all rickettsial species. Methylation of OmpB has been implicated in bacterial virulence. Two distinct types of lysine methyltransferases (PKMT1 and PKMT2) have been characterized using rOmpB as substrates. Structural studies of OmpB‐MTase complexes are necessary to better understand the mechanistic action of these unique MTases, but have been hampered by poor expression, purification and solubility of OmpB. Using codon‐optimized rOmpB fragments remarkably improved expression in E. coli. Denaturation with 6M guanidinium chloride appreciably elevated the yield of purified 6xHis‐tagged fragments using Ni‐NTA. Renaturation with decreasing concentrations of urea invariably produced insoluble aggregates. Partially refolded rOmpB fragments were then subjected to refolding in the second Ni‐NTA column. Our results indicate that the two‐step Ni‐NTA process produced highly pure and soluble rOmpB. The presence of multiple methylation sites and the circular dichroism spectra of the purified rOmpB fragments indicate that the Ni‐NTA resin facilitates surface‐mediated refolding of rOmpB. These expression, purification and refolding procedures allow us to overcome the obstacles to obtain a large quantity of rOmpB for crystallographic studies. The results also implicate a potential role of in vivo surface‐mediated OMP protein folding.
Hypoxia Reduces Mature HERG Channels through Calpain Up‐regulation and Activation
Authors: Lamothe Shawn , Zhang Shetuan
Published: June 2021

Human ether‐à‐go‐go related gene (hERG) encodes the pore‐forming subunit of the IKr channel, which is important for cardiac repolarization. Loss of function of the hERG channel causes long QT syndrome, a cardiac disorder with high risk of cardiac arrhythmias and sudden death. Patients with medical conditions, such as cardiac ischemia, are associated with prolonged QT intervals and arrhythmias. Since proteases, such as calpain, are upregulated and activated during cardiac ischemia and hypoxia, we investigated the role of calpain in hypoxia‐mediated hERG reduction. We demonstrate that hypoxic (0.5% O2) culture of hERG‐expressing HEK cells and neonatal rat cardiomyocytes led to a significant reduction of mature hERG expression and a concomitant increase in calpain expression. Transfection of hERG‐HEK cells with calpain‐1 reduced mature hERG expression and was accompanied by a significant calpain expression in the cell culture media. Treatment of cells with media from calpain‐1 transfected cells reduced mature hERG channel expression and function. The calpain‐1‐mediated hERG reduction was completely prevented by the membrane‐impermeable calpain inhibitor peptide B27. Replacing the S5‐S6 pore linker of hERG with that of EAG completely eliminated the calpain‐1 and hypoxic culture‐induced hERG reduction. Application of a peptide (BeKm‐1), which binds specifically to hERG's extracellular S5‐pore linker, prevented calpain‐1 and hypoxic culture‐induced hERG reduction. We conclude that hypoxia decreases mature hERG expression by activating calpain, which cleaves the channel protein at the extracellular S5‐S6 pore linker leading to subsequent channel degradation.

Supported by the Canadian Institutes of Health Research & the Heart and Stroke Foundation of Ontario
Distinct Roles of Apolipoprotein A1 and Lecithin‐Cholesterol Acyltransferase in Lipopolysaccharide‐Induced Inflammation
Authors: Petropoulou P.I. , Berbée J. , Hatziri A , Karagiannides I. , Kypreos K.
Published: June 2021

Aim

We studied lipopolysaccharide (LPS)‐induced inflammation under conditions of Apoa1‐ or Lcat‐ deficiency.

Methods

RAW 264.7 macrophages, whole blood and peritoneal macrophages from C57BL/6 (WT), Lcat‐deficient (Lcat‐/‐) and Apoa1‐deficient (Apoa1‐/‐) mice were challenged with LPS and plasma TNFα was measured. WT, Lcat‐/‐ and Apoa1‐/‐ mice were challenged intravenously with LPS and plasma cytokines were monitored. WT, Lcat‐/‐ and Apoa1‐/‐ leukocytes were phenotyped using FACS.

Results

Serum and HDL from Lcat‐/‐ and Apoa1‐/‐ mice display reduced capacity to attenuate the LPS‐induced TNFα response, in vitro. Blood from Lcat‐/‐ and Apoa1‐/‐ mice show an increased TNFα response when stimulated with LPS ex vivo, with more pronounced effect for Lcat‐/‐ blood. Lcat‐/‐ and Apoa1‐/‐ mice show a markedly enhanced and prolonged LPS‐induced inflammatory response in vivo. LCAT‐deficiency is associated with leukocytosis, increased CD11b+/Ly‐6Cmed monocyte and CD4+ lymphocyte levels, but low CD11b+/Ly‐6Chi monocyte levels and CD11b expression. Similarly, peritoneal macrophages from Lcat‐/‐ mice show reduced LPS‐induced TΝFα response. In contrast, APOA1 deficiency is associated with decreased leukocyte, CD11b+/Ly‐6Clo/med/hi monocyte and CD4+, CD8+ lymphocyte levels.

Conclusions

LCAT deficiency renders leukocytes less responsive to LPS, but increases their number in circulation, while APOA1 deficiency results in fewer but more responsive leukocytes.
Lipin‐1 Links Lipid Signaling with Inflammation
Authors: Balboa María , Meana Clara , Lordén Gema , Balsinde Jesús
Published: June 2021

Lipins are Mg2+‐dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Although they regulate cellular levels of important signaling lipids, little is known about the role of lipins in the immune response. Using lipin‐1‐deficient animals, we show in this work that this phosphatase acts as a proinflammatory modulator during TLR signaling and also in the development

of in vivo inflammatory processes. After stimulation with lipopolysaccharide (LPS), lipin‐1 deficient macrophages had a decreased production of DAG, activation of MAPKs and phosphorylation of the AP‐1 transcription factor c‐jun. Consequently, the generation of cytokines like IL‐6, IL12, IL23, or proteins like iNOS and COX‐2 was reduced. In the same line, responses to Salmonella were diminished, although no differences in phagocytosis rates were found. During the development of chemically‐induced colitis these animals exhibited reduced inflammatory responses and were protected against tissue damage and disease severity. These effects correlated with a lower generation of gut pathogenic factors like IL‐17 and IFNg and increased production of the gut protective factor TGFa. These findings demonstrate an unanticipated role for lipin‐1 as a mediator of inflammatory‐based pathological states and support a critical link between lipid signaling and inflammatory responses.

Supported by Grant SAF2013‐48201‐R from the Spanish Ministry of Economy and Competitiveness.
Ceramide‐enriched secreted microvesicles as a mediator of cytotoxic synergy between TNF‐α and IFN‐γ in oligodendrocyte cell death
Authors: Podbielska Maria , Szulc Zdzislaw , Hogan Edward , Bielawska Alicja , Bhat Narayan
Published: June 2021

A combination of TNFα and IFNγ induces cell death in several cell types including oligodendrocytes but the mechanism of their synergistic action is unclear. In this study, using a human oligodendroglioma cell line, HOG as a model, we show that secreted microvesicles (MVs) act as a paratropic mediator of synergistic cytotoxicity. Thus, TNFα and IFNγ are marginally cytotoxic individually but together induce marked cell death. MVs obtained from TNFα‐ but not IFNγ‐treated HOG cells, while mildly toxic to fresh cultures, induced extensive cell death in a fashion resembling the effect of cytokine combination when added to IFNγ‐primed target cultures. Since sphingolipids (SLs) including ceramide (Cer) form an integral part of secreted MVs, we determined the basic SL profiles of the secreted MVs as well as cytokine‐treated 'source’ cells by HPLC‐MS/MS. The treatment with the pro‐apoptotic cytokines time‐dependently increased Cer, dhCer & SM expression, in particular, of C24‐ and C24:1‐Cer species; C16‐, C24‐ and C24:1‐dhCer species; and C16‐, C24‐ and C24:1‐SM species in both intra‐ and extracellular compartments over a course of 48 hrs. That MV‐associated Cer contributes to the cytotoxic activity was supported by the observation that exogenous C6‐Cer replicated, albeit partially, the cytotoxic effect of Cer‐enriched MVs. We conclude that Cer‐laden MVs released from TNFα‐exposed cells collaterally 'spread’ the death signal to IFNγ‐primed target cells to elicit a robust synergistic cell death response.
Spatiotemporal Activation of Yeast Lipin Pah1 and Phospholipid Remodelling during Lipid Droplet Formation
Authors: Barbosa Antonio , Sembongi Hiroshi , Han Gil‐Soo , Su Wen‐Min , Carman George , Siniossoglou Symeon
Published: June 2021

Lipid droplets (LD) are central players in several biological functions but many aspects of LD biology are far from being understood. LD expansion requires the synthesis of the neutral lipids triacylglycerol (TAG) and steryl‐esters. In this work, we used the yeast Saccharomyces cerevisiae to unveil the regulation of enzymes involved in TAG formation, which requires Pah1‐driven synthesis of diacylglycerol and an acyl‐CoA donor. In yeast, LD biogenesis is induced in stationary phase.

Pah1 lacks a transmembrane domain, suggesting recruitment onto membranes under specific conditions. We observed recruitment of Pah1 to a domain of the nuclear envelope before the translocation to lipid droplets. This recruitment is independent of the glucose repression machinery but can be mimicked by physiological cues that govern entry into stationary phase, providing a mechanism for Pah1 activation.

We have shown that phospholipases B and the transacylase Lro1 generate, from phospholipids, the majority of the fatty acids required for TAG synthesis in stationary phase. Under these conditions, Pah1 and the phospholipase associated with LD are in contact with the ER. The conversion of phospholipids into storage lipids prevents aberrant expansion of membranes in stationary phase.

Our data show a nutrient‐dependent recruitment of Pah1 to specific membranes and a rewiring of lipid metabolism towards storage at the expense of phospholipids.

Work supported by grants from the Medical Research Council and the National Institutes of Health.
The Lipid Kinase PIKfyve Controls Lysosome Gene Transcription
Authors: Choy Christopher , Gray Matthew , Li Yang , Botelho Roberto
Published: June 2021

Lysosomes are degradative organelles responsible for molecular degradation, eliminating pathogens, digesting cellular debris and salvaging energy during autophagy. Upscaling of lysosome activity has been observed in autophagy where the transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is stimulated to enhance lysosomal gene expression. The most well characterized regulator of TFEB is the mammalian target of rapamycin complex one (mTORC1). Under nutrient sufficiency, mTORC1 is active and phosphorylates TFEB, resulting in its cytosolic retention. During starvation, mTORC1 is inactivated, TFEB becomes dephosphorylated and it enters the nucleus, upregulating lysosomal gene transcription. Recently, mTORC1 was shown to be recruited by the lysosomal lipid phosphatidylinositol 3,5‐bisphosphate [PtdIns(3,5)P2]. Therefore, we postulated that PtdIns(3,5)P2 may help repress TFEB function by maintaining an active mTORC1. To do this, we used apilimod to inhibit PIKfyve, the enzyme that synthesizes PtdIns(3,5)P2. Depletion of PtdIns(3,5)P2 caused TFEB‐GFP to translocate from the cytosol to the nucleus within 40 minutes and enhanced expression of various lysosomal genes, including cathepsin D. Additionally, apilimod treatment appeared to cause TFEB dephosphorylation similar to mTORC1 inhibition, judged by the faster TFEB gel mobility. However, and strikingly, mTORC1 remained active in apilimod‐treated cells as reported by phosphorylation of two downstream effectors, S6K and ULK. Thus, PtdIns(3,5)P2 appears to control TFEB‐mediated lysosome gene expression, independently of mTORC1 activity. Currently, we are testing whether PtdIns(3,5)P2 binds to and presents TFEB to mTORC1 on lysosomes. This work was funded by Canadian Research Chairs.
Antitumor 1‐nitroacridine, C‐1748, Decreases Pro‐survival Autophagy and Induces Accumulation of Lipid Droplets Resulting in Apoptosis of Panc‐1 Pancreatic Cancer Cells
Authors: Borowa‐Mazgaj Barbara , Skwarska Anna , Augustin Ewa , Pyrek Sebastian , Mazerska Zofia , Radominska‐Pandya Anna
Published: June 2021

Given poor prognosis of pancreatic cancer, there is a strong need for the development of novel therapeutic agents and discovery of new molecular targets. Changes in lipid metabolism can affect numerous cellular processes, including cancer cell growth, proliferation or differentiation. Here, we show for the first time that DNA‐damaging antitumor agent, C‐1748, affects lipid content in pancreatic cancer Panc‐1 cells which may contribute to its antiproliferative effect. Cytotoxic activity of C‐1748 against Panc‐1 cells was similar to clinically used gemcitabine. Compared with unexposed cells, C‐1748 reduced level of pro‐survival autophagy as shown by decrease in Acidic Vesicular Organelles. Staining with Nile Red showed that inhibition of autophagy was accompanied by accumulation of cytosolic lipid droplets (LDs). The pool of LDs following C‐1748 treatment increased in a dose‐ and time‐dependent manner. Importantly, along with decreased autophagy and development of LDs, C‐1748 progressively triggered mitochondrial dysfunction, caspase‐3 activation indicating on induction of apoptotic cell death. Recent studies indicate that autophagy allows cancer cells to maintain energy supply during stress conditions, and inhibition of autophagy increases triglyceride storage in LDs, what prevents them from being used as energy source. Our study suggests that C‐1748 blocks autophagy in pancreatic cancer cells, which disrupts pro‐survival lipid metabolism and leads to cell death.
Warfarin Metabolite Profiles Reveal the Importance of Factors on Patient Dose‐Responses to Anticoagulant Therapy
Authors: Pouncey Dakota , Hartman Jessica , Boysen Gunnar , Smith Eugene , Miller Grover
Published: June 2021

Coumadin (R/S‐warfarin) anticoagulation therapy remains challenging due to a narrow therapeutic range and high inter‐individual variations in response. We hypothesize that plasma metabolite profiles capture clinically relevant variations in warfarin metabolism as a resource to improve therapeutic strategies to minimize risk. We profiled R‐ and S‐warfarin and ten oxidized metabolites from fifty‐nine patient plasma samples using a novel LC‐MS method. Profiles included R‐ and S‐6‐, 7‐, and 4′‐hydroxywarfarin plus four 10‐hydroxywarfarin diastereomers, yet neither R‐ nor S‐8‐hydroxywarfarin was detected. Profiles differed significantly from those generated by R‐ and S‐warfarin using human liver microsomes and thus indicate variations in secondary metabolism and/or transport contributing to metabolite levels and their impact on dose‐responses. Multi‐linear regression analysis of metabolite levels normalized to parent drugs identified the relative importance and factors influencing their contributions to R‐ and S‐warfarin metabolism as evidence of the ability of metabolite profiles to capture the metabolic phenotype. These seminal findings provide a promising foundation to identify metabolite patterns as predictors of patient dose‐response to warfarin during anticoagulant therapy. Work was supported by National Institutes of Health (5R25HL108825‐02), National Institutes of Health and UAMS Translational Research Institute (UL1RR029884), and American Heart Association (13GRNT1690043).
Role of Cardiac mir‐208a in Nebivolol‐Mediated Signaling
Authors: Mahmood Abuzar , Gul Rukhsana , Luck Christian , Pulakat Lakshmi
Published: June 2021

The myomiR miR‐208a is implicated in cardiac hypertrophy, fibrosis, induction of obesity, and up‐regulation of β‐myosin heavy chain (β‐MHC) and GATA binding protein 4 (GATA4) expression. Nebivolol (Neb), a 3rd generation β1 adrenergic receptor blocker, suppresses cardiac hypertrophy and fibrosis. We reported that Neb treatment attenuated weight gain in obese, leptin resistant rats and suppressed Mammalian Target of Rapmycin Complex 1 (mTORC1) (PMID: 22969779). This study tested the hypothesis that Neb regulates miR‐208a and its effectors (β‐MHC, GATA4) in cardiomyocytes via mTORC1. Treatment of mouse atrial cardiomyocyte HL‐1 cells with Neb (1μM: 12 hrs) significantly suppressed β‐MHC and GATA4 as determined by immunostaining with anti‐β‐MHC and anti‐GATA4 antibodies and nuclear stain DAPI (n=3 experiments; analysis of > 40 cells for each group; P<0.05). Neb also attenuated increases in miR‐208a expression in HL‐1 cells induced by treatment with Angiotensin II (Ang II: 100nM: 12 hrs) as determined by real‐time RT‐PCR (P<0.05; n=5). Losartan (1μM), an AT1R blocker, and Rapamycin (10nM), an mTORC1 blocker, attenuated Ang II‐induced miR‐208a expression, but AT2R blocker PD123319(1μM), NADPH oxidase inhibitor apocynin (500μM) or Phospholipase C inhibitor U73122 (10μM) did not. Rap and Neb also suppressed Ang II‐induced phosphorylation (p) of mTOR (pSer2448), S6K1 (pThr389) and RPS6 (pSer235/236) (P<0.05).

Conclusion

Neb suppresses miR‐208a and its effectors in cardiomyocytes by inhibiting mTORC1. Such suppression of cardiac miR‐208a may underlie Neb‐induced body weight loss in leptin resistant rats.

Research support: LSME, UM‐Columbia (LP) and NIH 1R01HL118376‐01(LP)