bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationPersonal 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

BackgroundIn 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.ObjectiveTo 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).MethodPeripheral 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.ResultB 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.ConclusionWith 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 InformationKey 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 InformationMurdock Trust
DIGITOXIN TREATMENT REDUCES NASAL EPITHELIAL CYTOKINES IN CF SUBJECTS
Authors: Lee Seakwoo , Walker Douglas , Zeitlin Pamela L. 
Published: November 2023

ObjectivesCystic 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.MethodsTwenty 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.ResultsThree 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.ConclusionsDigitoxin 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 InformationR01 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 InformationWe 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 transmissionSupport or Funding InformationThis 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 Glycine Rich Proteins in Amblyomma americanum
Authors: Bullard Rebekah , Karim Shahid 
Published: November 2023

BackgroundThe 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).MethodsRNA‐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.ResultsNine 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.ConclusionThis 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 InformationThis 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 InformationThis 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 InformationUAMS 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 InformationThis work was supported by the National Basic Research Program of China (2012CB517606, 2013CB530805, 2014CBA02005 and 2013FY114100).
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.