bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomical study of the “Three in one” pectoral block. A new analgesia method for breast surgery
Authors: LABROUSSE Marc , BOZIER Eric , ROBERT Jordan , RENARD Yohann , AVISSE Claude , LUDOT Hugues
Published: June 2021

Breast cancer surgery is associated with severe postoperative pain. Novel techniques improving postoperative analgesia have been recently described such as pectoral nerves blocks (Pecs types I and II). The purpose of this study is to describe a new pectoral block targeting the whole breast region nerves and spaces.

Ten women fresh cadavers were dissected. An unique puncture was realized at the medioclavicular line level, 3 cm under the clavicle. The orientation of the needle was medio‐lateral and oblique, tangential to costal plane. Under ultrasound control the first injection was at the serrato‐costal plane level between anterior serratus deep fascia and exothoracic fascia. The second injection was in the serrato‐pectoral plane, localized between anterior serratus superficial fascia and clavipectoral deep fascia. The third injection was more superficial in the interfascial plane between pectoralis major and minor muscles. A last subcutaneous injection is performed at the puncture point.

After dissection of the two sides of each cadaver, serrato‐costal plane injections blocked thoracic intercostal nerves anterior divisions, serrato‐pectoral plane injections targeted long thoracic and thoracic intercostal nerves lateral division, and interpectoral plane dissections blocked lateral and medial pectoral nerves. The supraclavicular nerve is blocked with the subcutaneous injection at the puncture point.

This « three in one » pectoral block realized the analgesia of whole breast region nerves. To our knowledge it's the first time that such a block with an unique puncture is described.
Neuroinflammation and Brain Antioxidant Levels in Ethanol Leaf Extract of Nauclea latifolia and Artemisinin‐combination Therapy in Swiss mice Infected with Plasmodium berghei ANKA
Authors: Edagha Innocent , Ekandem Gabriel , Ekanemesang Udoudo , Etuknwa Bassey , Aquaisua Aquaisua
Published: June 2021

We investigated the histomorphological effects of Nauclea latifolia and Coartem® on the antioxidant levels and hippocampus in Plasmodium berghei ANKA infected mice. Forty‐two mice, 6 per group, between 6‐8weeks, of 22‐24 g, were infected and 5 days p.i., treated for 5 days; Group 1 served 10 ml/kg of (0.9% NaCl). Groups 2 and 3 received 500 and 1000 mg/kg of the extract, groups 4, 5 and 6 received n‐hexane, chloroform and butanol fractions at 1000 mg/kg, group 7 received Coartem® ‐ artemether/lumefantrine 5 mg/kg. Parameters evaluated were parasitemia, organosomatic index, superoxide dismutase, catalase, lipid peroxidation, protein, H&E and GFAP staining. Results showed no significant changes in final body weight and organosomatic index in treated groups compared with control, except in group 5 which was increased (p<0.05) compared to the control, there was significantly (p<0.05) decreased parasitemia in treated groups compared to control, with highest % chemosuppression in Coartem® group. Antioxidant and protein levels were not significantly changed (p>0.05) in the treated groups compared to control, except in group 5 which had significantly reduced Catalase. Hippocampal sections showed mild to severe morphological changes with neuronal loss, patechial hemorrhage, neuronal shrinkage, hydropic vacuolations, neuronal polymorphism, and pyknosis. Reactive astrogliosis showed moderate to severe neuroinflammation in treated groups compared to control, except in Coartem group® which may be associated with JAK2‐STAT3 activated signaling pathway in astrocytes, GFAP up regulation, and inositol 1,4,5‐trisphosphate (IP3)‐dependent Ca2+ signaling in astrocytes. In conclusion, curative therapy with ethanolic leaf extract of Nauclea latifolia and Coartem® against Plasmodium berghei ANKA infection in mice, significantly reduced parasitemia, the ethanolic extract did not reverse the parasite induced astrogliosis, which may influence neuronal survival and death, but neuronal shrinking, neuronal loss and pyknosis were mild to severe and this was dose/fraction dependent, however Artemisinin‐combination therapy phenotypically reduced reactive astrogliosis in the hippocampus, but had numerous hydropic vacuolations. The result may validate the use of Coartem® in severe malaria or cerebral malaria.
Regulation of Mitochondrial Biogenesis and Activity by Histone Acetylation
Authors: Rogerson Daniella , Galdieri Luciano , Zhang Tiatian , Lleshi Rron , Vancura Ales
Published: June 2021

Acetyl‐CoA is a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Concentration of acetyl‐CoA affects histone acetylation and links intermediary metabolism and transcriptional regulation. Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. Here we show that genes required for mitochondrial biogenesis are repressed by histone acetylation. During exponential growth of budding yeast cells in rich, glucose‐based medium, mitochondrial biogenesis is repressed and cells metabolize glucose predominantly by glycolysis. Under these conditions, the cellular level of acetyl‐CoA is high and global histone acetylation is also high. When glucose becomes limiting, the cells enter diauxic shift, upregulate mitochondrial activity and switch metabolism from glycolysis to oxidative phosphorylation. This metabolic transition is associated with a decreased cellular level of acetyl‐CoA. To probe the role of global histone acetylation in regulation of mitochondrial biogenesis and activity, we characterized yeast mutants that express nonacetylatable versions of histones H3 and H4. These mutants displayed increased mitochondrial biogenesis and activity. Taken together, our results suggest a model in which decreased global histone acetylation signals decreased glucose availability and results in a metabolic switch from fermentation to oxidative phosphorylation. This work is supported by a grant from the NIH (GM106324).
Genetic Effects of Low‐Dose Ionizing Radiation on the Chaffinch (Fringilla coelebs) in Chernobyl
Authors: Owens Michael , Bonisoli‐Alquati Andrea , Møller Anders , Mousseau Timothy
Published: June 2021

The meltdown of the Chernobyl Nuclear Power Plant in 1986 resulted in numerous deleterious ecological effects on the area's wildlife. Currently, a 30‐km radius area around the disaster site (known as the Chernobyl Exclusion Zone, CEZ) remains contaminated with low‐dose ionizing radiation at a level that is orders of magnitude higher than surrounding areas. Studies have indicated that the avian populations of the CEZ have higher incidence of partial albinism, abnormal sperm motility, and decreased brain size, among other effects.

Previous investigations have probed the genetic effects of the Chernobyl disaster using gene expression microarrays. These studies (conducted using the common chaffinch, Fringilla coelebs) identified a large number of genes whose expression levels differed between contaminated and clean individuals. To investigate the cause of these abnormal expression levels, we tested for copy number variation in a number of chaffinch genes using quantitative real‐time PCR, using DNA samples from birds within and outside the CEZ. Two gene pairs were selected, along with another single gene, and copy numbers were compared to β‐actin. Each pair consisted of one gene whose expression levels were raised and one gene whose expression levels were decreased. Pairs were selected based on proposed interactions between the two genes in question, as found during a literature review. Preliminary findings suggest that the expression patters of these genes are not correlated with their copy number. This indicates that the molecular effects of the Chernobyl disaster are not mediated at the pre‐transcriptional level for these genes.
Thymine DNA Glycosylase (TDG): The excised base is not retained in the enzyme‐product complex
Authors: Malik Shuja , Pozharisky Edwin , Varney Kristen , Drohat Alexander
Published: June 2021

Thymine DNA Glycosylase (TDG) is an enzyme that initiates base excision repair (BER) pathway and has important functions in maintaining genetic integrity and modulating epigenetic regulation. TDG was discovered as a repair enzyme that removes T from G:T mismatches that arise by deamination of 5‐methylcytosine (mC). TDG also has a critical role in active DNA demethylation; it excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), epigenetic bases that are generated via oxidation of 5mC by Tet (ten eleven translocation) enzymes. TDG also excises other lesions, including uracil (U) and 5‐hydroxymethyluracil (hmU). Like most DNA glycosylases, TDG exhibits very tight binding and slow dissociation from its reaction product, abasic DNA. In contrast, TDG has negligible affinity for, and is not inhibited by, the nucleobases that it excises from DNA. Intriguingly, it was reported that TDG retains the hmU base that it excises from a G:hmU substrate in a ternary product complex (TDG‐DNA‐base). While a ternary product complex was observed in crystal structures of some glycosylases (UNG, TAG), a binary (Enzyme‐DNA) complex was observed for MUG, the most‐closely related to glycosylase to TDG. We solved high‐resolution crystal structures (up to 1.6 Å) of TDG product complexes generated from several different substrates (G:hmU, G:U, G:T, G:fC, G:caC) and find no evidence that the excised base is retained in the active site. Moreover, NMR chemical shift perturbation (15N‐HSQC) experiments collected for multiple TDG product complexes give the same result. Taken together, our findings indicate that TDG has little affinity for the bases that it removes from DNA. Implications for catalysis by TDG and other glycosylases will be discussed.
α2A‐Adrenergic Receptor: Expression in Insect Cells and Detection of Phosphorylation
Authors: Varney Matt , Hebert Aaron , Vijay Jesus , Kaszubowski Joe , Beer Jacob , Donahue Ryan , Sterne‐Marr Rachel
Published: June 2021

The α2A‐adrenergic receptor (α2AAR) is a member of the G‐protein coupled receptor (GPCR) superfamily that responds to catecholamines such as epinephrine. In contrast to β‐adrenergic receptors, α2‐adrenergic receptors act to decrease blood pressure and control pain perception during the “flight or fight” response. GPCR kinases (GRKs) are responsible for deactivating these receptors by phosphorylating their third intracellular loop or carboxyl terminal tail peptide. The phosphate group attracts proteins known as arrestins that terminate the signal. GRK2 has been shown to have a critical role in desensitization of the β2‐adrenergic receptor and other receptors but its role in regulating the α2AAR is less understood. Phosphorylation occurs on four consecutive serines (296‐299) in the third intracellular loop of the wild type (WT) α2AAR. Previously in the lab, we generated a phosphopeptide antibody and therefore our goal was to determine the specificity of its recognition. First we showed that we could express WT α2AAR using a baculovirus infection system. We then prepared a mutant baculovirus expressing α2AAR with serines 296‐299 converted to alanine. The phosphorylation site antibody recognized the WT but not the mutant receptor. Furthermore, phosphatase treatment of phosphorylated WT α2AAR removed the signal. This signal was maintained when the same experiment was carried out in the presence of phosphatase inhibitors thus demonstrating the specificity of the antibody for phosphorylated receptor. With a bona fide α2AAR phosphorylation site antibody, GRK2 and its interaction with and phosphorylation of the α2AAR can now be investigated.
New Insights into DNA Binding and Dimerization Dynamics of E. Coli Histone Like Protein HU.
Authors: Arora Kanika , Guptasarma Purnananda
Published: June 2021

Histone like protein HU is the major nuceoid‐associated protein that plays important role in the architecture and dynamic compaction of bacterial chromosome. In E. coli, HU exists as 2 isoforms, HUα and HUβ. It forms homo‐dimers (HUα2, HUβ2) and hetero‐dimer (HUαβ) during different phases of growth. Mechanism and regulation of chain switching between HUα2and HUβ2 forming HUαβ is not known. Absence of Trp and Tyr residues in HU, makes it difficult to study both dimerization properties and DNA‐protein interactions using highly sensitive fluorescence spectroscopy methods. For in vitro experiments, a purification method was developed to purify HU without genomic DNA contamination. Three single Trp mutants were created by replacing Phe residues at positions 47, 51 and 79. Of three mutants, two showed similar structural (secondary structure and stability) and functional properties (DNA binding) like wild type HU. Difference in fluorescence properties of the mutants gives insights into local environment of the Trp residue in these mutants. On DNA binding these mutants showed significant changes in the Trp fluorescence and anisotropy. To look at the kinetics of hetero‐dimerization, Fluorescence Resonance Energy Transfer studies between the two subunits were carried out, showing that hetero‐dimerization is a spontaneous process. Dimerization dynamics was also studied by looking at the differences between thermodynamic properties of homo‐dimers and the hetero‐dimer indicating that hetero‐dimer is more stable than homo‐dimers. Our studies indicate that hetero‐dimerization in E. coli HU is a thermodynamically and kinetically favorable process giving some insights into chain switching mechanism.

Research support: CPSDE‐MHRD, IISER Mohali.
Investigation of FSH Responsiveness in a Granulosa Cell Line
Authors: Zeltsman Sara , Cohen Brian
Published: June 2021

The follicle stimulating hormone receptor (FSHR) is a g protein‐coupled receptor found in granulosa cells and Sertoli cells in females and males, respectively. FSH binds to its receptor on the surface of granulosa cells leading to proliferation and maturation of follicles towards ovulation. Understanding this pathway is significant because it can lead to developments in regulating fertility. Previously, our lab studied FSHR using an HEK293 cell line stably transfected with FSHR cDNA. More recently we have investigated an immortalized human granulosa cell line (hGrC1) to study FSHR signaling in a more physiologically relevant context. Due to the novelty of the hGrC1 cell line, not much is known about the cells, specifically what stage of follicular development they are most closely related to. Based on previous datafrom our lab regarding FSHR responsiveness, the question arose of whether hGrC1 cells undergo developmental changes in culture. It was hypothesized that this developmental change is analogous to follicular maturation in vivo. The change is significant because FSHR is not present in granulosa cells until later stages of follicular development. hGrC1 cells were grown in culture for a variable number of days before treatment with FSH, after which the presence of phosphorylated CREB and phosphorylated p44 were determined by western blot analysis as measures of FSH dependent signaling. An FSH dependent increase in signaling from both pathways was seen after cells were treated 4 days post‐confluency suggesting that there is a developmental shift leading to more hFSHR protein the longer the cells were grown in culture. This is significant because it suggests that hGrC1 cells mimic granulosa cell differentiation in vitro and may be a viable model for testing both FSHR function as well as understanding the factors driving folliculogenesis.
Multi‐Color, Live Cell CRISPR‐Based 3‐D Mapping of the Human Nuclear Genome
Authors: Ma Hanhui , Neseri Ardalan , Reyes‐Gutierrez Pablo , Zhang Shoajie , Wolfe Scot , Pederson Thoru
Published: June 2021

Understanding the locations and dynamics of interphase chromosomes is a major goal in the field of nuclear organization and function. Fluorescence in situ hybridization (FISH) has been a major tool and, as important as it is for a static picture, there is a parallel need to determine the locations and dynamics of interphase chromosomes in live cells. Here we report a multi‐color CRISPR‐based method for labeling individual chromosomes in human cells. It is based on computational mining for chromosome‐specific, tandemly repetitive sequences combined with optimized single guide RNAs for three different dCas9 orthologs. Using this method we have labeled the complete human karyotype in live cells. In confirmation of FISH results, we found that the gene‐rich chromosome 19 occupies a central position whereas the gene‐poor chromosome 18 is located at the nuclear periphery. We also were able to trace the dynamics of rDNA repeats (Nucleolus Organizer Regions) on the short arms of acrocentric chromosomes during the cell cycle. Our recent refinements of these methods have led to a barcoding approach to determine the 3‐D contours of each chromosome in the interphase nucleus, as a live cell technology complementing FISH, with applications underway for tumor cell diagnosis and for probing the pathogenesis of neurodegenerative diseases arising from trinucleotide expansions, for which we will present results pointing to selective CRISPR targeting of the mutant allele.
Salt‐induced Changes to the Stability of Poly(dA:dT) Tract DNA and Implications for Nucleosome Formation
Authors: Jensen Jaimi , Grilley Dan
Published: June 2021

Poly(dA:dT) tracts are major determinants of nucleosome positioning in eukaryotic cells. These DNA segments, which contain all dA nucleotides on one strand and all dT nucleotides on the other strand, are depleted of nucleosomes in a manner that depends on the length and purity of the A‐tract. These A‐tracts differ from generic sequence DNA in that their equilibrium structure contains a narrowed minor groove. This narrowed minor groove allows a network of ordered water molecules to mediate hydrogen bonds across base steps. The narrowed minor groove also shortens the inter‐strand distance between the negatively charged phosphates. We hypothesized that ions that match the geometric constraints of the narrowed minor groove would stabilize the unique A‐tract structure. To test this hypothesis we have used circular dichroism to monitor the effect of a series of monovalent cations on the global DNA structure. Using this measurement we have identified specific cations that appear to specifically promote A‐tract structure. Using UV‐monitored temperature melts, we have found that these same ions have preferentially stabilized A‐tract structure relative to generic sequence DNA. When DNA is wrapped around nucleosomes it is deformed away from its equilibrium configuration. Our results imply that nucleosome formation will be dependent on the species of cations that are present in solution. To further illustrate this, we have used competitive nucleosome reconstitution assays that will test how cation identity differentially affects the ability of A‐tracts to form nucleosomes relative to generic sequence DNA. Our work demonstrates that A‐tracts respond differently to specific cations, and that relative nucleosome stability depends on the identity of the cations in solution.
The Developmentally Important RNA‐binding Protein, Zygote arrest (Zar), Regulates mRNA Translation
Authors: Cook Jonathan , Charlesworth Amanda
Published: June 2021

Zygote arrest (Zar) proteins, Zar1 and Zar2, are required for successful fertilization and embryogenesis. Zar proteins bind mRNA sequence specifically, via the Translation Control Sequence (TCS), a cis‐element in the 3′UTR of mRNAs that regulates translation during early development. The TCS represses translation in the immature egg and activates translation and cytoplasmic polyadenylation in the maturing egg.
Our objective is to show that Zar proteins are bona fide translation factors and are candidates for mediating translational regulation by the TCS. A dual‐luciferase reporter tethered assay was used to measure the effect on translation by Zar proteins. RNA ligation‐coupled RT‐PCR was used to evaluate Zar‐mediated cytoplasmic polyadenylation. GST affinity purification and mass spectrometry were used to identify proteins that interact with Zar and interactions were verified by co‐immunoprecipitation (Co‐IP).
We show that when tethered to mRNA reporters, both Zar proteins repressed translation up to 50% and this repression was relieved during egg maturation, although no translational activation and no polyadenylation of the mRNA reporters were observed. Zar1 required a polyA tail to repress translation, whereas Zar2 did not. Zar1 and Zar2 interacted with overlapping but distinct sets of proteins and some interactions changed during egg maturation. Proteins recovered from co‐IPs included many known translation factors, such as: CPEB, Rap55, DDX6, and embryonic polyA‐binding protein.
Together, these data suggest Zar proteins have a role as translation regulators and they may mediate repression by the TCS, but are not likely to mediate translation activation and cytoplasmic polyadenylation. They also suggest mechanistic differences between Zar1 and Zar2.
The role of miR‐450a in oral squamous cell carcinoma carcinogenesis
Authors: Hsing En‐Wei , Shiah Shine‐Gwo , Hsiao Jenn‐Ren , Lyu Ping‐Chiang , Chang Jang‐Yang
Published: June 2021

microRNAs in tumor progression are crucial in head & neck cancer. Our research group found that miR‐450a were significantly higher in 46 clinical OSCC specimens than those in corresponding adjacent non‐cancerous specimens (p<0.001). However, its role in oral carcinoma has not been elucidated yet. miR‐450a is highly conserved among mammals. In homo sapiens, duplicated miR‐450a were located at adjacent loci in X chromosome. Gene expression microarray was performed to identify potential miR‐450a mediating genes. Differential gene expressions in pre‐miR‐450a‐transfected DOK and SCC15 were analyzed. Using Gene Set Enrichment Analysis, 16878 & 17000 down‐regulated genes were clarified in DOK & SCC15, respectively. To narrow down these miR‐450a targeting genes, two gene lists were independently overlapped with target predicted program microRNA.org. These two gene lists were further reduced to 419 genes in DOK & 465 genes in SCC15, and 256 genes were common in both two gene sets. We used RT‐PCR to validate a suspected membrane protein, TMEM182. The expression of TMEM182 was down‐regulated in RNA and protein levels. We verified that this TMEM182 down‐regulation was under a post‐translational level mediated by miR‐450a. Up‐regulation of miR‐450a in OSCC would enhance cell detachment ability. But, the cell proliferation was higher in miR‐450a overexpressed cells. We suggested that miR‐450a regulates cell adhesion through down‐regulation of TMEM182, and further influences cell migration in oral cancer.
Characterization of a Set of Caulobacter Crescentus Transcription Factors Deletion Strains and Their Role in the Oxidative Stress Response
Authors: Peterson Celeste , Perchuk Barrett , N'Gadjaga Djamila , Gora Kasia , Laub Michael
Published: June 2021

Transcriptional factors control gene expression cascades, often in response to environmental conditions. Here we have investigated the role of these cascades in orchestrating the oxidative stress response in the model system C. crescentus. Expanding the suit of high‐through put tools available in the bacterium, we made a collection of deletions of all the known and predicted transcription factors in the organism and screened these mutants for phenotypes related to general growth defects and the oxidative stress response. We characterized a gene, CC3697 (an OxyR homolog) which regulates the transcriptional response to oxidative stress. Using microarrays, we identified three classes of genes that it regulates as both positive and a negative regulator and used gel shift assays to confirm that some of this transcriptional control was direct. Further characterization of the cascade revealed CosR, a small RNA which facilitates survival during oxidative stress. Thus our collection results shed light on the mechanisms involved in the ability of C.crescentus to adapt to oxidative stress.
Thermodynamic Examination of the U6 ISL RNA Bulge
Authors: Lewis Daniel , Grover Neena , O'Connell Allison
Published: June 2021

Spliceosome is large complex made up of five snRNA and nearly 70 proteins that assembles on pre‐mRNA to excise the introns. The U6 subunit of the spliceosome is required for spliceosome assembly and activation. Spliceosome undergoes number of structural rearrangements that disrupt U4/U6 complex and form U2/U6 complex to become activated. The association of U2/U6 complex results in the formation of a highly conserved internal stem loop (ISL) in U6 RNA. The formation of a 1x2 nucleotide bulge that binds catalytically active Mg2+ at U80 position is essential in the RNA cleavage. The 1x2 bulge contains an A79•C67 non‐canonical base pair, which forms a stronger A+•C base pair in lower pH conditions. Upon the A+•C base pair formation, the ISL adopts a conformation closer to an A‐form helix and the unbound U80 in the bulge is forced out into the major groove. This conformation is mutually antagonistic to the binding of the catalytic Mg2+ ion, and therefore prevents splicing activity. Thermal denaturation experiments were performed on RNA constructs derived to mimic 1x2 bulge of the U6 RNA. We tested constructs containing the conserved bulged nucleotides as well as those with modifications that disrupt spliceosomal function. All constructs were examined in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. The thermodynamic contributions of the bulged RNA were compared to those of the helical RNA and DNA constructs. Thermodynamics of RNA with modification in and around the 1x2 bulge loop will be presented and influence of pH and ionic conditions will be discussed.
Rnp1p Plays a Role In P‐body Assembly in S. cerevisiae
Authors: Mackay Katherine , Segal Scott
Published: June 2021

Control of translation is an important response to physiological stress. Upon physiological stress, mRNA becomes unbound by ribosomes and shifts to cytoplasmic structures such as P‐bodies or stress granules (SG). P‐bodies and SG differ in that P‐bodies are thought to be sites of mRNA degradation, whereas SG are sites where mRNA are stored. In yeast, Pat1p, Dhh1p, and Sbp1p, nucleolin‐like factor are implicated in translational repression. Sbp1p also localizes to both P‐bodies and SG.

Interestingly, SBP1 has a paralog, RNP1, whose function is not fully known. Sbp1p and Rnp1p both have two RRMs, but Rnp1 lacks the RRG box found in Sbp1p. Given the similarities between the two proteins, we determined whether Rnp1p plays a role in translational repression or P‐body/SG assembly. To determine Rnp1p role in these processes, rnp1Δ cells were stressed at high OD and P‐body assembly was ascertained. High OD exposure resulted in markedly reduced P‐body assembly as compared to control. Overexpression of Rnp1p in unstressed cells increased P‐body assembly. These results implicate Rnp1p in driving P‐body assembly or translational repression. To discern between these possibilities, further work will be done to localize Rnp1p, directly look at the translation of Rnp1p, and furthermore look at SG assembly.
The tRNA Methylome of Trypanosoma brucei, the Causative Agent of African Sleeping Sickness
Authors: Lazatin Justine , Chen Leanne , Ackerman Sarah , Wang Jie , Valentine Erika , Mandarano Alexandra , Militello Kevin
Published: June 2021

Trypanosoma brucei is the causative agent of African Sleeping Sickness. The regulation of gene expression in T. brucei primarily occurs at the posttranscriptional level, indicating that RNA processing and modification events are extremely important for the parasite's life cycle. Yet, there is little information regarding the collection of covalent RNA modifications that comprise the epitranscriptome in this organism or any protozoan parasite. We focused on cytosine base modifications of tRNAs, as tRNAs are well known to contain a range of modified RNA bases, including 5‐methylcytosine. Small RNAs were isolated from insect‐stage T. brucei parasites and unmodified cytosines were converted to uracils using sodium bisulfite treatment. Sodium bisulfite‐treated small RNAs were analyzed by RNAseq. To confirm the RNAseq data, sodium bisulfite‐treated RNA samples were amplified by RT‐PCR from either total RNA or small RNA fractions, and analyzed by digestion with HpaII or via Sanger sequencing. Overall, our data indicate that tRNA molecules contain between zero to four 5‐methylcytosine residues. The most common location of 5‐methylcytosine in T. brucei tRNA is at the junction between the variable region and TψC arm at positions C48, C49, and C50, although different tRNAs have different numbers of 5‐methylcytosines in this region. There was little evidence for methylation of other sites in the tRNA, including C38 methylation in the anticodon loop. Overall, our data indicated that T. brucei tRNAs contain 5‐methylcytosine at some, but not all standard eukaryotic positions, and the levels of 5‐methylcytosine vary in different tRNA molecules.
Probing Disordered Region Influence on Pdx1 Binding to Natural Promoters and Near‐Consensus Sites
Authors: Bastidas Monique , Showalter Scott
Published: June 2021

Pdx1 is a well‐known protein in the set of β‐cell‐specific transcription factors (TFs), involved in the production of insulin and islet amyloid polypeptide (IAPP) hormones. Like many TFs, Pdx1 contains regions of disorder whose function in transcription has not been adequately studied. It is known that the Pdx1 homeodomain (HD) binds to a core DNA recognition sequence containing the tetranucleotide TAAT, for which its consensus binding site is reported as 5´‐CTCTAAT(T/G)AG‐3´. Naturally, insulin is expressed in much higher levels than IAPP, perhaps owing to the affinity of Pdx1 to its target sites. Alignment of human insulin and iapp promoter elements highlights the single nucleobase substitution from 5´‐CTAAT‐3´ to 5´‐TTAAT‐3´, respectively. We hypothesize that the interactions mediated by the disordered tails provide additional target specificity by interacting with DNA sequences upstream of the TAAT motif. Here we report a combination of ITC, NMR spectroscopy, and multi‐microsecond MD calculations of Pdx1 variants that define its interactions with a panel of natural promoter and consensus‐derived sequences. Our results point to a small preference of the Pdx1 HD for 5´‐CTAAT‐3´. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. More significantly, our computational studies suggest that interactions between the disordered region of Pdx1 and the major groove of the DNA are responsible for sequence identification upstream of the core motif. Taken together, our results suggest a molecular mechanism where the disordered regions of Pdx1 are responsible for establishing differential affinity to elements from the insulin and iapp promoter sequences.
Binding of F‐actin to the Cardiac Myosin Binding Protein C (MYBPC3) M Domain
Authors: Patton Connor , Van Heidi , Hoffman Matthew , Dhe‐Paganon Sirano , Gilbert Richard
Published: June 2021

A common feature in most forms of heart failure is an abnormality of the intricate machinery of sarcomeric protein interactions. Impaired expression or dysfunction of one such protein (MYBPC3) has been associated with hypertrophic cardiomyopathy and an altered regenerative response to injury. Compelling evidence exists that MYBPC3, acting principally through the phosphorylation of its M domain and associated effects on myofilament binding, plays an important role in the regulation of cardiac mechanics. MYBPC3 interacts with titin and light meromyosin (LMM) via its C‐terminus, whereas interactions of the N‐terminus with actin and myosin S2 sub‐fragments are controversial. Given the proximity of the thin filament to the MYBPC3 N‐terminus, it has been hypothesized that actin binding to the N‐terminus domain has fundamental importance in the regulation of sarcomere mechanics. N‐terminal fragments of MYBPC3, containing the M domain, were cloned with histidine tags and expressed and purified in E. coli. Actin was purified from rabbit skeletal muscle acetone powder to form F‐actin and G‐actin monomers, and cross‐linked G‐actin dimers. Employing a Ni‐NTA pull‐down protocol, we demonstrated that the N‐terminal domain fragment of MYBPC3 lacking the C0 subunit binds F‐actin, but not monomeric G‐actin. We conclude that dynamic and selective binding relationships involving the N‐terminus of MYBPC3 and various conformations of actin are fundamental to the control of sarcomeric interactions and may play a role in the pathogenesis of the failing heart. The work represented was supported by NIH RO1 DC 011528.
Wide‐angle X‐ray Solution Scattering Studies of Slow Correlated Motions in Proteins
Authors: Makowski Lee , Zhou Hao
Published: June 2021

Slow correlated motions in the submicrosecond to second range are particularly important for protein function but remain challenging to study. Wide‐angle x‐ray solution scattering (WAXS) generates information about the length scale of intramolecular motions difficult to obtain by other methods; is highly sensitive to these motions, being able to detect changes due to single amino acid replacements, ligand binding or alterations in solution conditions; and is sufficiently fast as to make practical the screening of hundreds of conditions/ligands/mutations to identify those that impact protein motions.

Direct comparison of the results of WAXS and molecular dynamics (MD) is accomplished through introduction of the sigma‐r plot, a plot of the standard deviation of interatomic vector lengths as a function of that length ‐ derivable from WAXS data and MD trajectories. MD simulations generate extraordinary detail about protein trajectories; WAXS provides experimental measures of length scale of protein motion under a large number of conditions. Direct comparison of WAXS with MD will enable detailed molecular interpretations of WAXS data using the power of MD; while making possible the extrapolation of MD results to understand molecular behavior of a large number of mutants in multiple ligation states and solution conditions. Application of this approach to HIV protease provides new insights into the role of protein dynamics in its enzymatic function.
Naturally Occurring Single Nucleotide Mutations Alter the Function of a Highly Conserved Hsp70 Gene in Humans
Authors: Hess Kyle , Siracusa Matthew , Nikolaidis Nikolas
Published: June 2021

Molecular chaperones and in particular the 70‐kD heat shock proteins (Hsp70s) are key regulators of the cellular stress response and alterations in their function have direct physiological consequences. To determine how natural mutations alter the function of Hsp70s we used single nucleotide polymorphisms (SNPs) found on HSPA1A the major stress inducible Hsp70 gene in humans. Specifically, the wild‐type (WT) HSPA1A sequence was subcloned into both bacterial and mammalian expression vectors and the mutated gene variants were generated using site directed mutagenesis. Recombinant proteins corresponding to the WT and mutated variants were then generated and tested for their ability to hydrolyze ATP. These experiments revealed that these mutations significantly change the rate by which the protein hydrolyzes ATP. The mutant proteins' ability to properly localize intracellularly within mammalian cells was also tested using immunofluorescence and confocal microscopy. These experiments demonstrated that the mutants and the WT protein had similar subcellular localization. However, live‐dead and cell proliferation assays revealed that some of these mutations significantly change cellular survival after heat stress. Given that these natural variants are either population‐specific or clinical we believe that the observed functional differences alter the ability of cells and the individuals carrying them to cope with stress or disease.
Comparative chaperone activities of trigger factors from mesophilic and psychrophilic bacteria
Authors: Baranova Ancha , Melkina Olga , Goryanin Ignatiy , Zavilgelskiy Genadii , Wall J.
Published: June 2021

The refolding level of thermally inactivated bacterial luciferases by trigger factor (TF) of the mesophilic bacterium Escherichia coli (TFEc) and the psychrophilic bacterium Psychrobacter frigidicola (TFPf) was measured. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luuciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. Refolding of heat inactivated luciferases reached a maximum of 25‐30% in E. coli ΔdnaKdnaJ cells containing plasmids with a tig gene (encoding TF) and luxAB genes (encoding heterodimeric (αβ) luciferase from). However, while the activity of TFEc was characterized by a significant reduction in refolding with an increase in TF concentration, the chaperone activity of the TFPf remained at a plateau at higher concentrations. TFEc and TFPf effectively assisted in refolding dimeric forms of luciferase but were unable to refold an enzyme variant in monomeric form. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). It was shown that TFPf but not TFEc is a target for the action of Lon protease.
Nucleotide Dependency of Endogenous Hsp90 Chaperone Protein Machinery and Its Association with Baculovirally Expressed Glucocorticoid Receptor
Authors: Molenda Deanna , Kaemingk Gabe , Murphy Patrick
Published: June 2021

Hsp90 and hsp70 are essential eukaryotic chaperone proteins required for facilitating functional activity of mammalian transcription factors, including the glucocorticoid receptor (GR). Both chaperones exist in ATP‐ and ADP‐bound states, and understanding the relationship between nucleotide‐bound forms of hsp90 and hsp70, their affiliated cochaperones, and GR activation has signiﬁcant pharmacological implications. The goal of this study was to investigate the extent to which hsp90 and hsp70 nucleotide interactions affect production of a GR‐hsp90 heterocomplex capable of steroid binding activity. Reticulocyte lysate (RL) containing high endogenous concentrations of hsp90 and hsp70 was incubated in the presence or absence of an ATP regenerating system and fractionated by size exclusion chromatography (SEC). ATP incubation did not appear to alter total global protein elution, as detected by A280 spectrophotometry. However, SDS‐PAGE and western blot analysis identified nucleotide‐associated alterations in hsp90 SEC elution profiles. In order to further compare nucleotide dependency and chaperone effects, full‐length GR, produced in baculovirally infected insect cells, was immunoadsorbed, stripped of endogenous chaperones, and reassembled into a heterocomplex with hsp90 ± ATP. SDS‐PAGE and western blotting showed hsp90 derived from RL−ATP samples were tightly bound to the GR, while hsp90 from RL+ATP failed to stay bound to the GR. The difference in SEC elution profiles and co‐immunoadsorption imply a notable role of nucleotide alterations in the hsp90 chaperone machinery and functional interactions with the GR.
Combined Inhibition of Hsp90 and Heme Oxygenase‐1 induces apoptosis and endoplasmic reticulum stress in Melanoma
Authors: Barbagallo Ignazio , Parenti Rosalba , Vanella Luca , Zappalà Agata , Castruccio Castracani Carlo , Li Volti Giovanni
Published: June 2021

Heat shockproteins are ubiquitous molecular chaperones involved in post‐translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment because it is a key regulator of various oncogene products and cell‐signalling molecules. Heme oxygenase‐1 (HO‐1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO‐1 activation may play a role in tumour development and progression. In the present study we investigated the antitumor effects of combining Hsp90 inhibitor (NMS973) and HO‐1 inhibitor (SnMP) on A375 melanoma cells. Hsp90 inhibitor treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1a, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS973 treatment resulted in a significant HO‐1 overexpression. Interestingly, the combination of NMS973 and SnMP produced an increase of ROS and reduced cell viability respect NMS973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by BFAR mRNA expression and lower phosphorylation of Akt when compared to NMS973 alone. In conclusion, these data suggest that combined inhibition of Hsp90 and HO‐1 is a promising strategy for melanoma treatment.
A Highly Charged Region in the Middle Domain of Plant ER‐Localized HSP90 is Required for ER Stress Resistance
Authors: Zhao Rongmin , Chong Lisa , Wang Yao , Gad Nanette , Anderson Nathaniel , Shah Bhavank
Published: June 2021

Heat shock protein HSP90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven HSP90 isoforms (HSP90.1 to HSP90.7) that are localized in the cytoplasm/nucleus, mitochondrion, chloroplast and endoplasmic reticulum (ER) where protein folding actively takes place. In this study, we analyzed the sequence of ER‐localized Arabidopsis HSP90.7 and the other ER GRP94 proteins from plants and animals, and identified a short, charged region that is specifically present in the middle domain of plant‐derived GRP94 proteins. To understand the role of this charged region, we analyzed transgenic plants that express a mutant protein HSP90.7Δ22, which has this charged region deleted. We showed that seedlings expressing HSP90.7Δ22 have significantly enhanced sensitivity to ER stress induced by tunicamycin or high concentration of calcium, although its general chaperone activity in preventing model protein from heat‐induced aggregation was not significantly affected. We also analyzed the ATP‐binding and hydrolysis activity of both wild type and mutant HSP90.7 proteins, and found that they have slightly different ATP‐binding affinities. Finally, using yeast two‐hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions. This work was supported by an NSERC Discovery Grant (371789‐2009) awarded to RZ.
Characterisation Of Whole‐Cell Properties In Chondrocytes From An In Vitro Model Of Arthritis
Authors: Lewis Rebecca , Jones William , Wegg Michaela , Barrett‐Jolley Richard
Published: June 2021

Chondrocytes express a rich complement of ion channels with a range of functions including regulation of membrane potential (Vm) and cell volume. Transcript studies show changes in ion channel expression with osteoarthritis (OA) and in vitro studies show that pro‐inflammatory cytokines can induce cell apoptosis. Using an in vitro model of OA (DMEM + 10ng/ml TNFα and Il1β) we examined changes in cellular properties with patch‐clamp and intracellular Ca2+ measurements. Healthy chondrocytes have depolarised Vm (~‐10mV) so, using a whole cell tail current voltage protocol, we investigated if Vm was affected in our model of OA. Cells received a 1s hyperpolarising step with 10mV increments for 10 cycles. Cells from the model had a whole cell reversal potential (Vrev) 16±2mV more positive than healthy cells (n=5, p<0.05). Healthy cells had a greater whole‐cell conductance than cytokine treated cells (1.8±0.6nS vs 0.9±0.1nS; n=4 and 5, respectively; p<0.05). Given the positive shift in Vrev, we investigated Ca2+ handling of these cells using hypotonic challenge to increase intracellular Ca2+ entry. Healthy cells responded to the challenge with a peak Ca2+ increase of 115±15nM (n=16). When applied to cytokine treated cells the same challenge caused a greater Ca2+ increase (328±45nM; n=11; p蠄0.01). This Ca2+ increase was inhibited by a TRPC channel antagonist, PYR3 (n=69). Intracellular Ca2+ increases greater than 300nM have been shown to lead to cell apoptosis; the changes we observe here could implicate important pathological changes in cytokine‐stimulated chondrocytes. Further work is necessary to identify the mechanism by which this Ca2+ increase occurs. Funded by DBOARD (EU FP7/2007‐2013).
Dimer Intermediate in the Assembly of Influenza A M2 Transmembrane Domain in Lipid Membranes
Authors: Georgieva Elka , Borbat Peter , Norman Haley , Freed Jack
Published: June 2021

Influenza A M2 is a single‐pass transmembrane protein, that assembles into tetrameric proton channels. This protein is a pharmacological target and knowing its structural and functional properties is of high importance. We studied the assembly of M2 transmembrane domain of (M2TMD) in DOPC/POPS membranes. To this end, we employed one of the modalities of Double Electron Electron Resonance technique (DEER), specifically spin counting based on DEER modulation depth. DEER signals were collected for a series of samples of a spin‐labeled M2TMD peptide and a progressive increase of peptide‐to‐lipid molar ratio (P/L) in the range from 1:18,800 to 1:160 and pH's 8 and 5.5. We found that DEER modulation depth strongly depends on P/L. A thorough analysis revealed a two‐stage equilibrium of M2TMD self‐association that includes initial formation of dimers at P/L smaller than 1:2,300, which then associate to form a dimer‐of‐dimers or M2 tetramers at higher P/L's. Strikingly, at P/L close to native for influenza membrane, i. e. below 1:2,500, M2TMD exists predominantly as a very stable dimer. This points to the essential role of the M2 dimer in the active channel assembly. The data obtained in detergent‐reconstituted M2 were similar to those in lipid; however oligomers were formed only at much higher peptide‐to‐detergent molar ratios. Hence, bilayer properties play a significant role in M2 self‐association and the stabilization of its functionally relevant forms. This cascade mechanism of M2 self‐oligomerization as revealed in this study could be relevant to other single‐spanning membrane proteins.