bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the Nucleoid‐Associated Protein HU in E. coli Transcription
Authors: Bugliosi Sara , Jessen Erik , Landick Robert
Published: November 2023

Transcription by RNA polymerase (RNAP) is the most basic level by which the cell can regulate gene expression, allowing for dynamic responses to environmental changes. Interactions between RNAP and chromatin‐organizing proteins is one method organisms have evolved to regulate transcription, and is best understood in the context of eukaryotic RNAPs and nucleosomes. In prokaryotes, a set of functionally analogous nucleoid‐associated proteins (NAPs) form interactions with the bacterial chromosome. We are studying how HU, the most abundant NAP in exponentially growing E. coli cells1, coexists on DNA with elongating RNAP. Genome‐scale ChIP data revealed that RNAP and HU are spatially co‐localized along a subset of transcription units (TUs). To investigate why HU was bound only to this subset, additional ChIP experiments against R‐loops were performed. HU has been shown to have high affinity for single‐stranded DNA2. R‐loops are formed upstream of elongation complexes when the nascent RNA transcript invades upstream DNA, displacing a single strand of DNA and making it available for binding by HU. These data showed that whereas R‐loops were exclusively found at TUs bound by HU, deletion of HU had no influence on R‐loop distribution. Analysis of R‐loop‐enriched TU sequences revealed higher G content on the antisense DNA strand and an increased frequency of G‐quartets (GGGG), motifs that have been shown to promote R‐loop formation3. To investigate the physiological significance of these results we tested the viability of an HU deletion strain with an oxygen‐dependent growth defect. We showed that the HU deletion strain is hypersensitive to mutagenic reactive oxygen species (ROSs). Taken together, these results lead us to propose a model in which HU binds to R‐loops upstream of elongating RNAP to protect the exposed non‐template DNA strand from mutagenesis by ROSs.
Characterization and Expression Analysis of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle
Authors: Orsech Hannah , Waddell David
Published: November 2023

Skeletal muscle atrophy is a serious medical condition resulting from an array of physiological conditions such as aging, corticosteroid use, cancer and loss of innervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel genes showing differential expression in response to neurogenic atrophy. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, several fragments of the proximal regulatory region were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with a myogenin expression plasmid, resulting in myogenin dependent induction of the Zfp593 reporter genes. Bioinformatic analysis of the promoter region of Zfp593 revealed two conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Myogenin is a member of the myogenic regulatory factor family of transcription factors, which are important in muscle cell differentiation and are also significantly induced in response to neurogenic atrophy. Interestingly, Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Therefore, in order to determine if Zfp593 is localized to the nucleus of muscle cells, the Zfp593 cDNA was amplified from C2C12 cells and sub‐cloned into an expression plasmid that produces a GFP‐Zfp593 fusion protein. The GFP‐Zfp593 expression plasmid was then transfected into proliferating C2C12 cells followed by visualization using confocal microscopy. Cells transfected with the GFP‐Zfp593 expression plasmid showed fluorescence that is exclusively localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.

Support or Funding Information

University of North Florida Transformational Learning Opportunity Grant
The RexA Protein of λ Phage: a New Member of a Classic Genetic Switch
Authors: Schiltz Carl , Thomason Lynn , Hosford Chris , Johnson Virginia , Pan Hao , Court Don , Chappie Josh
Published: November 2023

The rexA and rexB genes of lambda phage act as a putative phage exclusion system to prevent superinfection of lambda lysogens by other lytic phages. The Rex exclusion system was thought to act as an internal suicide mechanism by which soluble RexA would bind RexB localized on the inner membrane, causing RexB to open as an ion channel, depolarizing the membrane and leading to cell death. Genetic data suggests that RexA can function independently of RexB to induce the lytic promoters of the lambda lysogen. Additionally, RexA must exist at a higher concentration than RexB in vivo to produce the lytic response. Through genetic data and in vitro binding assays, we have also shown that RexA interacts with cI repressor protein, the factor needed to repress lytic promoters in the lambda lysogen. Intriguingly, RexA is capable of stably binding lambda operator DNA in vitro. We have also solved the structure of RexA to 2.05Å resolution. The structure identifies RexA as existing as a constitutive dimer, each monomer consisting of a split dimerization and globular domain. An electrostatic potential map of the structure shows a region of high positive charge at the interface of the two globular domains, indicating a potential region for DNA binding; however, there is not sufficient space between the dimers to facilitate binding in this crystal form. In addition, RexA shares some structural similarity with bacterial recombination‐associated RdgC protein. Structural superposition with RdgC and normal mode analysis in addition to the electron density map hints at a possible conformational change in RexA upon cI and/or DNA binding. RexA thus appears to function as a master regulator of the classic lambda phage genetic switch. With this in mind, we are now attempting to parse out the specific interactions between RexA and cI and/or DNA that allow for induction of the lytic promoters.
Most stable G‐quadruplex structures are selectively avoided at the codon level
Authors: Basu Soumitra , Arachchilage Gayan Mirihana , Piontkivska Helen
Published: November 2023

The coding region (CDS) of mRNA is known to be less structured compared to the untranslated regions (UTRs) primarily to reduce any structural interference with the translation process. Translational rate and open reading frames have been shown to be affected by the secondary structures present within the CDS. However a systematic and comprehensive analysis of secondary structure avoidance is difficult as some of the common secondary structures such as hairpins can be formed by a wide range of sequence combinations. Using a G‐quadruplex (GQ) as the secondary structure we studied the evolutionary selectivity against the GQ structures within the CDS. G‐quadruplexes are well‐known secondary structures adopted by RNA and have been shown to control translation. Since the GQs have defined sequence requirements, they provide an excellent platform for investigation of the selectivity of such structures at the codon level. Using a bioinformatics approach we studied the frequencies of GQs within the CDS of a variety of species. We discovered that the most stable GQs are significantly avoided within the CDS by evolutionary pressure. Further we showed that the avoidance of GQs at the codon level correlates to the stability of such structures. Finally we established that the translation is affected by the stable GQs that are present within the CDS. This work not only provides a comprehensive analysis on how the stable secondary structures are avoided within the CDS throughout the evolution but also broadens the current understanding of codon usage as they relate to structure‐function relationship of RNA.
Thermodynamic Examination of the U6 RNA and DNA Constructs
Authors: Hanson Jared Anker
Published: November 2023

The spliceosome is a large ribonucleoprotein complex that assembles on pre‐messenger RNAs (pre‐mRNA) to catalyze the excision of introns and ligation of exons to produce a mature mRNA. Once assembled, the spliceosome undergoes a large conformational transition to become catalytically active. The intramolecular stem‐loop (ISL) located in the U6 subunit unfolds and refolds resulting in the activation of the spliceosome. The U6 ISL contains a highly conserved 1X2 asymmetrical bulge that coordinates Mg2+ that is implicated in catalysis. This bulge contains an A79 C67 non‐canonical base pair and an unpaired U80 residue. In pH below 6.5, an A+ C base pair forms, resulting in the ISL forming a helical structure. This rearrangement causes displacement of the U80 residue and prevents the binding of Mg2+, thus, inhibiting catalysis. This study examines the role of sequence, ions and pH effects on interactions in the ISL. Various RNA and DNA constructs were designed and thermally denatured in in 1M KCl and 10mM Mg2+ at pH 5.5 and 7.0. RNA wild type construct showed an increase of 1.0 kcal/mol of stability at a pH of 5.5, indicating the formation of the A+ C base pair. Constructs in buffer containing Mg2+ also demonstrated increased stability. DNA wild type construct's stabilities did not change upon altering pH or ionic conditions, indicating that the A+ C base pair is not formed in DNA. DNA constructs were also less stable than equivalent RNA constructs. Detailed thermodynamic analysis of 1×2 bulge region of U6 ISL will be presented.
GSK3β Modulates Excess Nutrient‐Induced Autophagy Impairment in Human Aortic Endothelial Cells via FOXO1
Authors: Weikel Karen , Ruderman Neil , Ido Yasuo
Published: November 2023

Dysregulated autophagy, the process by which damaged proteins and organelles are turned over, and decreased AMP‐activated protein kinase (AMPK) activity are each associated with type 2 diabetes and atherogenesis. In a human aortic endothelial cell (HAEC) model of poorly‐controlled type 2 diabetes, we observed that the same concentrations of excess nutrients that increased inflammation and apoptosis also decreased AMPK activity and autophagy. Our current objective was to determine whether glycogen synthase kinase 3β (GSK3β), an enzyme whose hyperactivity has been associated with diabetes, atherogenesis and inhibition of AMPK activity, attenuates autophagy and AMPK activity in nutrient‐laden HAECs.

AMPK activity was measured by protein levels of p‐AMPKThr172 and the SAMS peptide assay. GSK3β activity was measured by protein levels of p‐GSK3βSer9. GSK3β activity was increased by treating cells with Akt inhibitor VIII (Akti) and GSK3β activity was suppressed by infecting HAECs with lentivirus knocking down GSK3β (shGSK3β) or expressing the kinase‐dead (K85A) mutant. Changes in Lysotracker staining reflected changes in lysosome acidity. Autophagosome formation was evaluated by measuring bafilomycin‐induced changes in protein levels of LC3‐II, by both Western blot and immunofluorescence. P‐FOXO1Thr24 and mRNA levels of PI3KC3 and SOD2, FOXO1 target genes, were used to evaluate FOXO1 signaling.

A combination of 25 mM glucose and 0.4 mM palmitate (excess nutrient conditions) inhibited basal autophagy and AMPK activity and also increased GSK3β activity. Treatment of HAECs with Akti decreased Lysotracker staining under both control and excess nutrient conditions. Suppression of GSK3β activity attenuated excess nutrient‐induced losses in Lysotracker staining. Under both control and excess nutrient conditions, suppression of GSK3β activity increased LC3‐II protein levels and knockdown of GSK3β increased FOXO1 signaling.

These data suggest that increases in GSK3β activity may impair autophagy in our HAEC model of poorly‐controlled type 2 diabetes by decreasing lysosome acidity and autophagosome formation. The mechanism by which GSK3β affects each of these aspects of autophagy remains to be clarified, but may involve modulation of FOXO1 signaling.

Support or Funding Information

This research was supported by NIH T32 Multidisciplinary Training in Cardiovascular Research HL007224.
A Novel Thermotolerant β‐glucosidase from Aspergillus nidulans Has Activity Across a Broad pH Profile and a Likely Bacterial Origin
Authors: Auta Richard , Radecka Iza , Hooley Paul
Published: November 2023

This study aims at cloning, expression and biochemical characterization of recombinant β‐glucosidases from Aspergillus nidulans AN1804 gene for biofuel production. Purification was conducted using ammonium sulphate precipitation and anion exchange chromatography on a DEAE‐Sephadex A‐50 column. The enzyme was purified 2.25 fold from the crude extract with a 33.33% recovery yield. The protein migrated homogeneously as a single band on SDS‐PAGE stained with Sterling rapid silver stain and the molecular weight was determined to be approximately 100 kDa. It was optimally active at 50 °C and pH 5.5, though it had a broad pH range of pH 3.0 – 10.0. The presence of CoCl2, CaCl2, FeCl3, FeCl2 and ZnCl2 slightly enhanced the activity of β‐glucosidase but was slightly decreased in the presence of HgCl2. β‐glucosidase showed very high affinity to para‐Nitrophenyl β‐D‐glucopyranoside (pNPG). Km and Vmax for the hydrolysis of pNPG by β‐glucosidase was calculated as 0.59 mM and 2.04 μmole/ml/min respectively. An initial bioinformatics studies shows a rare occurrence of introns in AN1804, providing additional support to the argument of horizontal transfer from a bacterial source. The spectacular thermo‐tolerance and very broad pH profile of AN1804 β‐glucosidase could be the reason why the fungus has kept the bacterial gene.

Support or Funding Information

Personal funding
Hepatitis B immunoglobulin produced from long‐term expansion of B lymphocytes from donors with high titer HBsAb
Authors: Yang Yonglin , Jie Cai , Xu Jiling , Cai Xubing
Published: November 2023

Background

In China, the demanding of hepatitis B immunoglobulin (HBIg) is increasing rapidly in recent years due to the passive immune and the prevention of reinfection after liver transplantation. The HBIg supply is severely limited because HBIg is produced only from volunteers who vaccinated hepatitis B virus vaccine.

Objective

To test an effective method to produce HBIg in vitro by activating and maintaining the expansion of B lymphocytes from donors with high titer hepatitis B surface antibody (HBsAb).

Method

Peripheral blood mononuclear cells were collected from donors with high titer HBsAb, among which B lymphocytes were purified by magnet beads coated with anti‐CD19 antibody. The rest cells were irradiated by g‐ray and then co‐cultivated with purified B lymphocytes in the medium containing the cytokines including recombined human CD40L, IL‐4, IL‐10 and IL‐2 for 30 days. The levels of total immunoglobulin and HBsAb were measured at different time points by enzyme‐linked immunosorbnent assay.

Result

B lymphocytes were expanded more than 2000 times at 30 day. The mount of total immunoglobulin was increasing stably in the whole culture period and reached the peak level (167±51 μg/ml) at 21 day. At 30 day, the mount of total immunoglobulin was 22.6±6.8 mg obtained from 1×105 B cells. Meanwhile, the HBIg was 0.47±0.29 mIU and the HBsAb titer was not changed obviously.

Conclusion

With autologous cells and cytokines, purified B lymphocytes which contain hepatitis B surface antigen‐specific lymphocytes can be effectively activated and expanded to produce general and specific immunoglobulin in vitro, indicating it may be a potential method to produce a large mount of HBIg to meet clinic demanding.

Support or Funding Information

Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (No. ZKX12045; QRX11240).
Investigating The DNA Binding Constraints Of The I‐Sob2593 Meganuclease
Authors: Ruff Ray , Kaiser Brett
Published: November 2023

Meganucleases are DNA cleaving enzymes present in microbial genomes that are considered “selfish” elements because they facilitate their spread to other genomes. They are prime candidates as genome editing reagents because they recognize unusually long DNA sequences (18–22 bp). However, redirecting their specificity towards novel targets remains a challenge since the same surface of the protein mediates DNA and binding and cleavage. A recent study of unicellular green algae identified the widespread insertion of meganuclease homologs at conserved sites within mitochondrial and chloroplast genomes. Analysis of a subset of these homologs demonstrated that the enzymes are capable of cleaving an identical (or nearly identical) DNA target sequence despite having limited sequence identity (34–60%). These results suggest that the enzymes have evolved unique solutions to recognizing a common DNA target. We have focused on characterizing the meganuclease I‐Sob2593. We have solved the x‐ray structure of I‐SobI2593 bound to DNA, allowing molecular level comparisons of binding mechanics to a previously solved meganuclease, I‐CreI. We will also determine I‐Sob2593's tolerance to polymorphism along its target sequence using in vitro cleavage assays. In future experiments we will expand this analysis to other homologs of I‐Sob2593. Completion of these experiments will give insight into the rules that govern DNA recognition by these enzymes and will serve as a starting point to redirect their specificity towards desired DNA targets.

Support or Funding Information

Murdock Trust
DIGITOXIN TREATMENT REDUCES NASAL EPITHELIAL CYTOKINES IN CF SUBJECTS
Authors: Lee Seakwoo , Walker Douglas , Zeitlin Pamela L.
Published: November 2023

Objectives

Cystic fibrosis (CF) is characterized by progressive lung inflammation. Proinflammatory proteins such as IL‐8 are highly expressed in CF airways secretions, and cardiac glycosides, such as digitoxin, can suppress hypersecretion of IL‐8 from cultured CF lung epithelial cells. The specific mechanism of digitoxin action in vitro is to inhibit the TNF‐α/NF‐κb signaling pathway. We tested the hypothesis that digitoxin could suppress this pathway in vivo, using CF nasal epithelial cells obtained during a Phase II, randomized, double‐blind, placebo‐controlled trial of two dose levels of digitoxin and placebo in CF.

Methods

Twenty four CF subjects were randomized to take digitoxin 50 μg (n=8), 100 μg (n=8) daily or placebo (n=8) 100 μg for 28 days. Nasal epithelial cells were collected from patients pre‐ and post‐treatment and placed in Trizol for RNA isolation. Quality control (QC) was performed before microarray analysis. Expression levels were analyzed by using Affymetrix 1.0 Human Exon ST arrays at the Johns Hopkins Microarray Core Laboratory. Statistical analysis was performed with the Partek Genomic Suite and visualization of the data was performed in Spotfire.

Results

Three dimensional principal component analysis (PCA) plots revealed that the patient groups receiving placebo and 50 μg/day digitoxin fall into similar patterns which are distinct from the patient group taking 100 μg/day digitoxin. We identified 43,613 genes and 173 genes are significantly up‐ or down‐regulated in the patient group taking 100 μg digitoxin/daily. Interestingly, mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells, including CCL2, CXCL1, CXCL2, CXCL3, IL‐1R, MHC class II, integrin α4, CD48, and CD300a in the patient group taking 100 μg digitoxin/daily.

Conclusions

Digitoxin treatment in cystic fibrosis patients was associated with reductions in nasal epithelial cytokines and cell surface molecules found on immune cells and higher dose cohort (100 μg/daily) shows more significant changes in gene expression. Longer term exposures and microarray studies of circulating immune cells as compared with airways epithelial cells would be useful.

Support or Funding Information

R01 FD003456
A Robust Computational Pipeline to Identify Phenotype‐Controlling Candidate Genes Within a Quantitative Trait Loci
Authors: Winnett Alexander , Gulick Trudi , Morey Stuart , Kesseli Rick
Published: November 2023

Current methods of determining which genes affect an organism's phenotype through Quantitative Trait Loci (QTL) mapping require extensive and lengthy physical experimentation. We develop an efficient, computational pipeline for the prediction of these phenotype‐controlling genes within a QTL by exploiting the functional impact of non‐synonymous single nucleotide polymorphisms (SNPs) within different ecotypes of a species of choice. After a QTL region has been identified, the analysis begins by constructing in silico protein coding sequences for ecotypes by exchanging reference sequence nucleotides for SNPs. These sequences are then translated and homologs for each sequence are identified within an all‐plant database. The hits are then aligned and phylogeny is constructed, which with theoretical physiochemical properties of the amino acid sequences, predict the functional impact of non‐synonymous mutations for each position in the protein. Ecotype sequences are then parsed for the presence of high‐impact non‐synonymous SNPs to identify the most likely phenotype‐controlling genes within the QTL.

Support or Funding Information

We would like to acknowledge the collaborators in the Composite Genome Project (http://compgenomics.ucdavis.edu/), the faculty of the UMass Biology Department for assistance and direction of this project, and the CSM Student Success Center.

This research was possible through financial support provided by the Beacon Student Success Fellowship, the Oracle Computational Research Fellowship, the Initiative for Maximizing Student Development (NIH Grant R25GM076321), and the NSF Plant Genome (DBI 0820451) program.
Sicpin, a multifunctional immunomodulatory salivary protein from black flies significantly reduced T and B cell proliferation and directly binds to soluble CD4 receptor
Authors: Calvo Eric , Chagas Andrezza Campos , Ribeiro Jose
Published: November 2023

Sicpin, the first immunomodulatory salivary protein described in black flies significantly reduced T and B cell proliferation and directly binds to soluble CD4 receptor Hematophagy is key to blood feeding arthropods reproductive success and an important link in pathogen transmission cycles. Salivary gland homogenates from blackflies have been shown to contain immunomodulatory activity on murine splenocytes. However, the molecule(s) responsible for this salivary activity remains elusive thus far. Here, we report the first immunosuppressive protein from blackfly salivary glands. Sicpin (Simulium cell proliferation inhibitor) was produced in E. coli and purified using size exclusion and ion exchange chromatography. Purified Sicpin was LPS‐decontaminated and its sequence verified by N‐terminal sequencing and LC‐MS/MS analysis. Sicpin inhibited cell proliferation in a dose‐response manner independently of the mitogen utilized (ConA, LPS, CD3/CD28 and Pokeweed). LPS or ConA stimulated cells had a significant lower proliferation rates (P<0.001) in the presence of Sicpin (IC50=0.5μM) with 10uM completely abrogating cell proliferation. Flow cytometry analysis showed that Sicpin inhibited proliferation of CD19+ B‐cells and CD4+/CD8+ T‐cells. Sicpin did not induce apoptosis or necrosis in mitogen‐induced proliferative responses by murine splenocytes as determined by flow cytometry. Sicpin also inhibits antigen‐specific cell proliferation without inducing apoptosis in resting or mitogen‐induced splenocytes. We demonstrate that the production IFN‐α, IL4, IL5, IL6 and IL10 by splenocytes stimulated by ConA or LPS was dose‐dependently reduced by Sicpin. Reduction of cytokines in presence of Sicpin could lead to a retardation of B and T cell activity. Carrageenan‐induced paw‐edema model showed that the intensity of edema significantly decreases when Sicpin was administered at 5 and 15 μg/animal. The molecular mechanism of Sicpin on cell proliferation inhibition is currently under investigation; however, initial binding experiments using SPR analysis showed a direct binding to soluble CD4 receptor with a calculated KD of 17.77 nM. Direct binding of Sicpin to CD4 could inhibit the subsequent TCR ligation‐induced T cell signaling at the earliest steps including tyrosine phosphorylation of the receptors, downstream effector proteins, and lipid raft reorganization. The immune suppressive and anti‐inflammatory properties of Sicpin should be explored as a strategy to modulate immune responses in infection and tumor proliferation as well as its involvement in parasite transmission

Support or Funding Information

This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Characterization of a New Ferritin Protein from the Polychaete Chaetopterus Sp.
Authors: Hamlish Noah , De Meulenaere Evelien , Deheyn Dimitri
Published: November 2023

The marine polychaete worm Chaetopterus sp. secretes a sticky mucus that exhibits a soft blue long‐lasting bioluminescence. Iron (both ferrous and ferric) and riboflavin have been found abundant in the mucus and identified as potential cofactors involved in the control of the light production. The Deheyn lab has recently identified a novel ferritin protein (ChF) from fractions of the worm mucus still able to produce bioluminescence after purification by chromatography. Ferritin proteins are ubiquitous across the animal kingdom and exhibit ferroxidase activity, converting ferrous iron into a ferric form that is stably stored and soluble in the ferritin. Here, ferritin may serve as a source of biological iron for the worm through a process of iron acquisition, storage, and release during the light production process. This study addresses these options by assessing foundational data that characterize the ferroxidase activity of recombinant ChF with respect to human heavy‐chain ferritin (HuHF). ChF exhibits faster initial rates of iron oxidation than HuHF, but reaches an equilibrium state with detectable levels of ferrous iron still in solution; in contrast this was was not observed for HuHF that oxidizes all available iron in solution. This may support the hypothesis that ChF has a reducing activity. This could involve the release of ferric iron, which may be reduced by flavin molecules found in the mucus; the resulting ferrous iron could then subsequently undergo a Fenton reaction, acting as a source of electrons for long‐lasting mucus bioluminescence.
Characterization of Glycine Rich Proteins in Amblyomma americanum
Authors: Bullard Rebekah , Karim Shahid
Published: November 2023

Background

The feeding habits of ticks require extended attachment of the tick on the host. This extended interaction with the host's immune system requires the tick to shield itself by modulating the host immune response with the use of multiple families of pharmacologically active compounds. Glycine‐rich proteins are secreted from the salivary glands into the host where they may have multiple functions including: attachment (proteinaceous cement matrix surrounding the mouthparts), anti‐hemostasis (block blood clotting factors), anti‐inflammatory (prevent pro‐inflammatory signals or induce anti‐inflammatory signals), or anti‐microbial (prevent the growth or establishment of microbial organisms).

Methods

RNA‐seq transcriptome was used to select GRPs of interest. The gene expression for each GRP was measured throughout the bloodmeal. RNA interference of a GRP gene was used to determine gene function and to measure compensatory mechanisms. In vitro feeding apparatus was developed and comparative structural information of the tick cement cones was collected using SEM and FT‐IR.

Results

Nine GRPs selected from the transcriptome showed varying levels of expression throughout the full bloodmeal. Knockdown of a single GRP resulted in no phenotypic change due to suggested compensatory mechanisms. In vitro feeding apparatus supports tick feeding through female engorgement. Comparison of cement cones reveals differences in structure but not composition.

Conclusion

This study shows the redundant nature of GRPs found in the tick sialome. Although no changes are noted in the knockdown ticks, a network of GRP compensation is revealed. Further analysis of the tick cement cone gives new insight into the secondary structure of the cement proteins. Allowing for a better understanding of their composition.

Support or Funding Information

This work was funded by NSF MS‐EPSCoR Seed Grant EPS 0903787 and EPS 1006883.
Refinement of the ProMOL Algorithm and Determination of Function Based on Homology
Authors: Madha Shariq , Lambrecht Mitchell , Mills Jeffrey L. , Bernstein Herbert J. , Craig Paul A.
Published: November 2023

ProMOL is a plugin for the molecular visualization program PyMOL and is designed to align proposed catalytic residues from proteins of unknown function to those of known and catalogued functions. Roughly 4% of the structures in the Protein Data Bank (PDB) are proteins with unknown functions. The focus of this study is a test of the reliability of function prediction by ProMOL, based on analysis of several thousand structures of known function. Initial structural alignments using standard settings within ProMOL found that approximately 44% of these 10,000 structures of known function were properly identified using 296 motif templates in the ProMOL library if homologs are selected solely by EC class. However, we found that if we define homologs as those structures that share both EC class and Pfam family, the identification rate improved significantly from 44% to 66%. We are further refining the algorithm in ProMOL to include sequence alignment to further improve the true positive rate.

Support or Funding Information

This work was supported in part by NIH GM078077.
Proteomic Profiles of Vulvar Squamous Cell Carcinoma Variants
Authors: Holthoff Emily R , Byrum Stephanie , Kelly Thomas , Tackett Alan , Quick Charles M , Post Steven R
Published: November 2023

Vulvar squamous cell carcinoma (vSCC) is a gynecologic malignancy diagnosed in nearly 4500 women in the US each year. High risk of recurrence and nodal metastasis lead to increased morbidity and mortality in these patients. In previous studies, we identified 2 major variants of vSCC. Some tumors exhibit a pushing invasive pattern and lymphoplasmacytic (LPC) stromal response; others display an infiltrative invasive pattern and a fibromyxoid (FMX) stromal response. We also showed that infiltrative tumors with FMX stroma are more likely to recur and metastasize to the lymph nodes, contain perineural invasion, and undergo an epithelial mesenchymal transition (EMT). Molecular tumor features that contribute to these outcomes have not been well defined in vSCC and may be due to differential expression of key proteins involved in biological processes that aid in tumor growth and invasion.

In this study, label‐free high resolution LC‐MS/MS was used to determine the proteomic profiles of FFPE tissue sections from 10 infiltrative vSCC with FMX stroma and 10 pushing vSCC with LPC stroma. Of these 20 tumors, 11 were associated with recurrence and 8 contained lymph node metastases. Proteomic profiles were associated with morphologic tumor features, tumor recurrence, and nodal metastases to identify proteins linked to increased risk of adverse outcomes.

When comparing infiltrative and pushing tumors, 205 proteins exhibited significant expression changes between the two groups. Importantly, 42 proteins were uniquely identified in infiltrative tumors. Included in these was thrombospondin‐4, a glycoprotein that mediates cell‐matrix interactions and may play a role in macrophage recruitment. In addition, 96 proteins were identified only in the pushing tumors, including STAT1, which may play a significant role in mediating effects of the inflammatory stromal response through IFNγ signaling. Expression of 105 proteins was either significantly different or uniquely identified in recurrent tumors, and the expression of 115 proteins was either uniquely identified or significantly varied in tumors with nodal metastases. Included in these 115 significant proteins was SRPX2, an important regulator of FAK signaling and cell motility, which could contribute to increased metastatic potential in these tumors.

Overall, our results identify a proteomic profile in vSCC that implicates a role for specific pathways in differentiating aggressive vulvar squamous cell carcinoma as defined by morphologic tumor features, recurrence, and nodal metastases.

Support or Funding Information

UAMS TRI grants UL1TR000039 and KL2TR000063; SPaT institutional training grant T32GM106999
Host urinary proteins changed significantly in mice infected with malaria
Authors: Li Xundou , Deng Weiwei , Wang Heng , Gao Youhe
Published: November 2023

Malaria is one of the most prevalent infectious diseases in tropical and subtropical regions. Effective biomarkers are urgently needed. Biomarker is measurable changes associated with physiological and/or pathophysiological conditions. Urine can be better source for biomarker discovery because it accumulates the changes of body while body removes them from blood by homeostatic mechanisms. In this study, we attempted to hunt for urinary biomarkers for malaria using mice infected with Plasmodium yoelii. The urine of mice was collected before infection and 3, 5 days after infection, separated by SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), digested in‐gel based on molecular weight and identified using nanoLC‐MS/MS (liquid chromatography‐mass spectrometry). More than 70% of red blood cells were damaged by plasmodium 5 days after infection so it is expected that there might be big changes in urine. Unexpectedly, no reliable parasite proteins were identified, indicating that extrinsic protein may be mostly degraded in blood rather than draining into the urine. 427 urinary proteins of mice were identified and 22 proteins were significantly changed (n=3; p<0.05; and fold change>2) and 19 of those proteins were increased and 3 were decreased after infection. Some of these proteins participate in the defense against parasites and the regulation of tissue inflammation. CHIA and CLCA1 were further validated by western blot in another eight mice. Urinary peptidome might be worth more attention because in which fragments of parasite proteins may be found there if they exist. As a result, although there are no parasite proteins identified in the urine, the host (mice) urine proteins may be fairly good biomarker candidates for malaria.

Support or Funding Information

This work was supported by the National Basic Research Program of China (2012CB517606, 2013CB530805, 2014CBA02005 and 2013FY114100).
Proteomic Analysis of Ewing's Sarcoma Cell Lines
Authors: Thompson Rhese , Kruchten Anne
Published: November 2023

Ewing's Sarcoma is a pediatric bone cancer that affects approximately 3 in 1,000,000 children each year. The prognosis for a Ewing's patient can be very poor due to the extremely aggressive and metastatic nature of Ewing's Sarcoma cells. Treatment for patients with this cancer typically includes surgery, radiation, and/or chemotherapy. EWS‐Fli1, the chromosomal translocation product responsible for the development of Ewing's Sarcoma, has been shown to upregulate the transcription of the Aurora kinases, a group of serine kinases. The actin‐binding protein cortactin is an actin‐binding protein that has been shown to be involved in cellular migration and metastases in cancer, and its phosphorylation is expected to be of importance in Ewing's Sarcoma metastases. Here we report on the proteomic analysis of Ewing's Sarcoma cell lines RD‐ES‐1 and SK‐ES‐1. We have used 2‐D gel electrophoresis to analyze the phosphorylation states of cortactin in these cells, as well as the overall proteomic patterns of Ewing's Sarcoma cells in response to various treatments. These results provide an increased understanding of protein modification states in metastatic cells, and future experiments will focus on identifying the protein modifications using mass spectrophotometry.
Integrin β1 and ICAM‐1; enrich markers for isolating rat mesenchymal stem cells from bone marrow
Authors: Suto Eriko Grace , Mabuchi Yo , Suzuki Nobuharu , Ogata Yusuke , Akazawa Chihiro
Published: November 2023

The therapeutic applications of MSCs are under intensive exploration because of its multi‐potency. MSC‐purifying markers are defined in mice and humans, but not other species. Some groups reported rat MSC (rMSC) markers, though these markers were identified after culturing whole bone marrow (BM) cells for about one month. MSCs gradually undergo differentiation, accompanying with morphological and functional change from the parent stem cells, under long‐term culture. rMSCs are widely used for regeneration experiments, such as regenerating bone and cartilage tissues, but these rMSCs are not purified. In order to analyze intravital function of MSCs, it is required for identifying cell surface markers of rMSCs.

In this study, we improved the rMSC isolating procedure and identified its purification markers. Many groups have tried to isolate rMSCs from BM with the flush out method, but the yield of rMSCs is very low. We used collagenase to dissociate BM cells effectively, and commercially available 9 antibodies, whose antigens are candidates as rMSC markers. First, fibroblast colony‐forming units were performed for identifying candidate markers. We found that integrin β1 (CD29)+/ICAM‐1 (CD54)+ cells had high colony forming and proliferation ability, and differentiated into adipocytes, osteocytes and chondrocytes. Further, we carried out a transplantation experiment using these isolated cells. Cells from each subset were differentiated into cartilage‐like pellets, and then were transplanted into the rat subcutaneous tissue. Two weeks after from the transplantation, a pellet from CD29+/CD54+ cells was engrafted in transplanted area and formed hyaline cartilage, whereas pellets from whole BM, CD29−/CD54+ and CD29+/CD54− cells were infiltrated with recipient cells and had poor chondrogenesis.

Our finding will facilitate better understanding of rMSC properties and developing of the effective translation of MSC for clinical therapeutics.
Protein‐protein interactions reduce Ca2+‐requirements for Transglutaminase 2 mediated post‐translational modifications of α‐synuclein
Authors: Zeczycki Tonya Nicole , Viscomi Jessica S
Published: November 2023

The combined projected increase in the incidence of Alzheimer's and Parkinson's disease is nearly 40% over the next ten years as the “baby‐boomer” population advances in age. In the majority of late onset neurodegenerative diseases, the underlying cause of neuron damage is the accumulation of toxic protein aggregates. The Ca2+‐dependent, post‐translational modification of tau, α‐synuclein and β‐amyloid by transglutaminase 2 (TG2) contributes to the development of protein aggregates in the diseased brain. Vulnerable to the toxic effects of aggregated proteins, damaged neurons exhibit increased Ca2+‐influx, further activating TG2. Degeneration leads to neuronal death, which gives rise to cognitive decline and dementia. The pathological TG2 protein cross‐linking activity in neuronal cells is actually beneficial to normal cellular apoptosis when regulated properly. The advancement of TG2 as a therapeutic target is hampered by the lack of fundamental understanding of how TG2 cross‐links pathogenic protein substrates. In contrast to previous in vitro mechanistic studies, which rely heavily on the use of artificial substrate analogs (i.e. Cbz‐Gln‐Gly), we present here the kinetic characterization of the TG2‐mediated, Ca2+ deamidation and polyamidation of recombinant α‐synuclein. The apparent Km for α‐synuclein is nearly 10‐orders of magnitude lower than that for Cbz‐Gln Gly and Ca2+ requirements for activation (KA ~0.34 μM) are significantly reduced. In addition, we explore the effects the protein‐protein interaction between TG2 and CRT, a low affinity, high capacity Ca2+ binding protein, have on Ca2+‐activation and protein cross‐linking activity.

Support or Funding Information

Office of Research and Graduate Studies, Brody School of Medicine
Avoiding the Carbapenem Trap: KPC‐2 β‐lactamase Sequence Requirements for Carbapenem Hydrolysis
Authors: Mehta Shrenik C , Samanta Moumita , Chow Dar‐Chone , Palzkill Timothy
Published: November 2023

β‐lactamases are bacterial enzymes that mediate antibiotic resistance through hydrolysis and inactivation of β‐lactam antibiotics. The hydrolysis reaction is a two‐step process involving cleavage of the β‐lactam amide bond to form a covalent acyl enzyme intermediate followed by deacylation through an activated water molecule. Traditionally, the carbapenem class of β‐lactam antibiotics formed the last line of therapy for treating bacterial infections. β‐lactamase enzymes, such as TEM‐1, that are responsible for resistance to most β‐lactam antibiotics are inefficient at providing resistance to carbapenem antibiotics because they become trapped in a deacylation deficient conformer. The emergence of β‐lactamases capable of hydrolyzing carbapenems, such as KPC‐2, in the bacterial population has resulted in increased mortality rates and healthcare costs. While TEM‐1 and KPC‐2 share a high degree of structural similarity (RMSD = 0.9 Å), KPC‐2 can evade the inhibitory trap and hydrolyze carbapenem antibiotics. In the absence of a KPC‐2/carbapenem co‐crystal structure, the molecular basis of carbapenem hydrolysis is not well understood. In this study, steady‐state and pre‐steady state kinetic analysis on several KPC‐2 active‐site mutants was used to decipher the mechanism of carbapenem hydrolysis. The results indicate that the sequence constraints for hydrolysis of imipenem and meropenem (carbapenem substrates) are more stringent as compared the requirements for hydrolysis of ampicillin (penicillin substrate) and cephalothin (cephalosporin substrate). Active site residues T235 and T237 were found to be critical for both imipenem and meropenem hydrolysis and mutating them to alanine resulted in inefficient or reduced carbapenem turnover. Residue R220 was found to be critical for meropenem hydrolysis but not imipenem hydrolysis. Surprisingly, mutating positions R220 and T237 to alanine resulted in a catalytic efficiency for cephalothin hydrolysis similar to the wild‐type enzyme suggesting that these positions are not important for cephalosporin hydrolysis. However, the T235A mutant was found to be critical for cephalothin hydrolysis. In addition, for ampicillin hydrolysis, each mutant displayed an increase in catalytic efficiency as compared to wild‐type enzyme suggesting the wild type residues at these positions are detrimental for penicillin hydrolysis. Thus, the KPC‐2 β‐lactamase active site has evolved to hydrolyze carbapenem antibiotics at the cost of its penicillinase activity.

Support or Funding Information

Work supported by NIH RO1 A132956 to T.P.
The Role of Lys42 on the Catalytic Efficiency of the ADP‐glucose Pyrophosphorylase from Escherichia coli
Authors: Mahaffey Angela L , Aiyash Saleh , Ballicora Miguel A
Published: November 2023

The synthesis of α‐1,4 polysaccharides (glycogen and starch) via NDP‐glucose pyrophosphorylases (NDP‐Glc PPases) is pertinent for energy storage in prokaryotic (bacteria), eukaryotic (plants) and mammalian organisms. Of this NDP‐Glc PPase superfamily, the ADP‐glucose Pyrophosphorylase (ADP‐Glc PPase) enzyme functions in the “first committed step” of glycogen synthesis for Escherichia coli, in the presence of a divalent magnesium ion (cofactor), ATP (substrate) and Glc‐1P (substrate), by production of the glucosyl donor (ADP‐glucose). The 200 kDa homotetrameric (α4) enzyme differs from the heterotetrameric (α2β2; S‐subunits and L‐subunits; 50–52kDa; 60kDa) plant counterpart as it has a conserved Lysine residue (Lys42 in E. coli ADP‐Glc PPase), present in the bacterial ADP‐Glc PPases and bacterial NDP‐Glc PPases, but not the Large subunit (non‐catalytic) of the eukaryotic plant ADP‐Glc PPase. This prompted our investigation into any catalytic properties of the Lys42 residue in E. coli ADP‐Glc PPase. By site‐directed mutagenesis, enzymes characterization and homology modelling, we found (1) Lys42 plays a critical role in the Mg2+ (cofactor) binding network (Lys42/Asp142/Asp276) – observed by both modelling and kinetics ‐a decreased cooperativity of the Mg2+ binding curves of the mutants (2) Modelling indicates the Lys42 amine side chain interacts with the oxygen between the α and β‐phosphate groups of the ATP. Possibly via a “general acid‐catalysis” mechanism the PPi product is stabilized becoming a better “leaving group” and (3) Mutagenesis of this site lowers the catalytic efficiency (kcat/S0.5) for the ADP‐Glc PPase enzyme substrates by 3–4 orders of magnitude. Conclusively, we postulate Lys42 is critical for the catalysis of ADP‐Glc PPase in E. coli.

kcat and kcat/S0.5 for ATP and Glc‐1P (substrates) of WT and Lys42 mutant E.coli ADP‐Glc PPase enzymes, in the synthesis direction.

Enzymea
kcatb
ATP
Glc 1P

‐fold decreasec
kcat/S0.5
kcat/S0.5

s−1

s−1mM−1
s−1mM−1

WT
103 ± 11
1
515
3814

K42Q
0.140 ± 0.006
736
0.7
2

K42A
0.098 ± 0.002
1051
0.2
2

K42R
0.053 ± 0.001
1932
0.2
0.9

K42L
0.041 ± 0.002
2512
0.5
0.6

K42C
0.040 ± 0.002
2575
0.3
0.6

K42E
0.032 ± 0.002
3312
0.3
0.7

K42T
0.026 ± 0.002
3962
0.3
0.2

Enzymes were purified to homogeneity and assays were performed at 37 °C as described under “Experimental Procedures.”

kcat values were calculated from curves via assays performed in the presence of saturated substrates (ATP and Glc‐1P) and activator (FBP). Assays were averaged from three independent experiments.

Fold decrease was calculated using wildtype enzyme activity as a reference.