bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic Characterization of Novel Genes in the Novel Mycobacterium Phage Butters
Authors: Juarez Michelle C. , Grajales Melanie , Mageeney Catherine M. , Kenna Margaret A. , Ware Vassie C. 
Published: November 2023

The SEA‐PHAGES program at Lehigh University is a collaborative undergraduate research enterprise focused on isolating and characterizing phages that infect Mycobacterium smegmatis to gain a better understanding of phage genome structure, gene function, and phage biology in general. One specific long term objective of the research program is to characterize phage gene products that function in lytic infection and to evaluate their potential as biocontrol agents to target pathogenic bacteria. Among the phages isolated at Lehigh is Mycobacterium phage Butters – a temperate N cluster phage with one of the smallest known mycobacteriophage genomes (41,491bp) among the >6000 mycobacteriophages isolated and characterized thus far (www.phagesdb.org). Among 66 annotated and primarily uncharacterized genes in Butters, two (gp30, gp31) are “orphams” with no known mycobacteriophage family members and one is N‐specific (gp57). To test the hypothesis that specific genes are essential for phage infection, gene deletions were made using a Bacterial Recombineering Electroporated DNA (BRED) technique to determine if mutant phage could be recovered after gene targeting. Here we focus on gp30 and gp57. Initial identification of mutant plaques was based on notable changes in plaque size phenotype and the presence of deletion‐sized PCR products. Mutant plaques retained the characteristic turbidity of wildtype plaques, indicating little, if any, perturbation of the lysogenic cycle for this temperate phage and a likely requirement for gp30 and gp57 in lytic growth. In most instances, mutant phage plaques (recovered after mixed BRED infection with wildtype phage DNA) could not be individually propagated after multiple plating rounds, suggesting that an essential component (required for bacterial infection) is missing in the population of mutant phages. We report progress in characterizing mutant phages by: 1) analysis of lysogens recovered from mutant plaques that harbor lytic‐incompetent phages, 2) cloning and sequencing of deletion‐sized PCR products recovered from mutant prophage genomes, and 3) plating mixed phage populations on gene complementation strains to recover mutant phages.Support or Funding InformationLehigh University, Department of Biological Sciences; Howard Hughes Medical Institute Science Education Alliance
Transcriptomic analysis of host response to the Karp strain of Orientia tsutsugamushi in inbred mice blood
Authors: Miller Stacy‐Ann , Chao Chien‐Chung , Yang Ruoting , Chan Teik‐Chye , Zhang Zhiwen , Chen Hua‐Wei , Ching Wei‐Mei , Hammamieh Rasha , Jett‐Tilton Marti 
Published: November 2023

Scrub typhus is caused by the obligate intracellular Gram negative bacterium, Orientia tsutsugamushi (OT). Previous studies have shown mRNA expression profile was altered in PBMC upon in vitro infection by OT. A combined expression profile based on a few selected mRNAs appeared unique to OT infection. To better understand the host response upon infection, we infected C3H mice and monitored gene expression profile in blood at different time post infection.C3H mice were infected with sham (i.e. PBS only) or Karp strain of OT (250 mLD50) by an artificial route. Mice were sacrificed at different time post infection. RNA was isolated from blood using the automated PAXgene blood miRNA kit on the Qiacube. Agilent SurePrint G3 Mouse GE 8×60K Microarray slides were used. The experimental and universal reference RNA were labeled with CY5 and CY3, respectively using Agilent Low Input Quick Amp Labeling Kit and Two‐Color Spike‐Mix, following by fragmentation and hybridization using the Agilent Gene Expression Hybridization Kit. Arrays were scanned using the Agilent Microarray G4900DA scanner system. Microarray data was extracted using Feature Extraction v 11.0.1.1. Further analysis was performed using functional analysis clustering toolbox (FACT) and customized R code.Using FACT, we were able to dissect the most important gene expression patterns. Lots of changes (1667 probes) occurred only at 6 h, 12 h (796 probes), then 2 days (828 probes) post infection. Multiple pathways with highly up‐regulated expression occurred at 8 – 12 h. Pathway‐specific qPCR is being performed to confirm these altered mRNAs.Using a mouse model with an artificial route of infection, we are able to show various genes involved in pathways responded to OT infection. While further validation of these altered genes is needed, the results demonstrated a massive change of gene expression early during the infection progression.Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the author(s) and do not constitute endorsement by the U.S. Army.
The molecular model of PKC life cycle explained through Systems Biology Approach
Authors: Aslam Naveed , Alvi Farah 
Published: November 2023

Protein kinase C (PKC) is a family of kinases which is poised to transduce signals emanating from the cell surface. Membranes of most cells provide the regulatory platform for supporting the life cycle and function of PKC enzymes. Maturation, activation and termination are three key regulatory steps of PKC life cycle. These regulatory steps precisely control the level of signaling competent PKC enzyme in the cells and any dysfunction in this regulation results in pathophysiological disease state such as cancer, where PKC levels are observed to be altered. Here, we describe a molecular model of PKC life cycle based on computational approach. Our model explains the fate of PKC during denovo synthesis and lipid mediated activation cycle. Through our systems biology approach we show that life cycle of PKC is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types 1: Maturation processing of newly synthesized enzyme; 2. Activation of enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and is stored in cytosol as a signaling competent inactive molecule. First step in this maturation sequence of PKC is PDK1 mediated phosphorylation at activation loop (Thr500 in PKCβII). Once phosphorylated at activation loop PKC molecule binds with mTORC2 complex and undergoes constitutive auto‐phosphorylations at turn and hydrophobic motifs (Thr641 and Ser660 in PKCβII). Upon stimulation by extra‐cellular stimuli the generation of diacylglycerol (DAG) and Ca+2 at membrane engages the PKC molecule through binding at C2 and C1 domains respectively. This activates the enzyme. Our model explains all these steps in PKC lifecycle through a quantitative approach. Our model also shows that once active PKC molecule is prone to de‐phosphorylation (PHLPP‐mediated) and degradation. This model also explains the stabilizing role of HSP‐60 in rescuing the de‐phosphorylated molecule and assisting its re‐phosphorylation, thus re‐building the pool of signaling competent enzyme after activation cycle. This model shows that for small to moderate activation stimuli the pool of PKC molecule is effectively unchanged however, larger stimuli can down‐regulate the enzyme through degradation pathways. Our results also show that blocking the PHLPP de‐phosphorylation pathway could recover the PKC pool in a dose dependent manner even after a large amplitude stimulus has been applied. Our results also points toward a possible role of HSP‐60 overexpression in recovering the PKC signaling competent pool after large amplitude stimuli has been applied. The results presented in this communication unravel some interesting aspects of PKC life cycle through a computational approach. Enhancing the understanding of PKC life cycle could be instrumental in translating new drugs for this important transducing molecule for diseases like breast cancer.Support or Funding InformationBioSystOmics
Quantifying HbF Using a Modified Kleihauer‐Betke Assay
Authors: Singh Rhena , Randolph Tim R 
Published: November 2023

Sickle cell anemia is one of the most commonly inherited diseases, annually affecting approximately 275,000 births worldwide. The disease is most prevalent in African countries and countries in which a large proportion of the population is of African descent, such as the Caribbean nation of Haiti. Hydroxyurea is the only FDA approved drug that is used to mitigate sickle cell symptoms by reducing the percentage of HbS and increasing HbF. Since hydroxyurea is also mildly carcinogenic, HbF levels must be monitored during treatment to determine the minimum effective dosage or to stop the drug in non‐responders. The objective of this study is to develop and validate an inexpensive and simple method of measuring HbF levels in sickle cell patients undergoing Hydroyurea treatment. This method uses a modified Kleihauer‐Betke (KB) principle to elute HbA and HbS in a weak citric acid solution while retaining HbF. Normal blood samples (source of HbA) were collected from the research team, de‐identified cord blood samples (source of HbF) were provided by the St. Louis Cord Blood Bank at Cardinal Glennon's Children's Hospital and de‐identified sickle cell blood samples (source of HbS) were provided by Cardinal Glennon Children's Hospital. The procedure involves the mixing of 20 uL of type specific cord blood/normal blood, or cord blood/sickle blood mixtures that were standardized to 35% hematocrit to 4 mL of citrate solution (0.07 M sodium citrate in a 0.06 M sodium phosphate buffer solution). Samples were incubated at 370C for 6 minutes, centrifuged for 30 seconds and the supernatant read spectrophotometrically at 395 nm. As HbA or HbS levels increase (decreasing levels of HbF), the absorbance also increases showing a strong positive linear correlation (r2 = 0.99, N=5). This simple and cost‐effective method shows promise to be used in underdeveloped countries for HbF quantification.Support or Funding InformationRS is supported by the DeNardo Education and Research Foundation.
Phenotypic studies of PHO13Δ in Saccharomyces cerevisiae
Authors: Kellogg Courtney , Mills Kimbria J , O'Handley Suzanne , Gehret Austin U. 
Published: November 2023

PHO13 in Saccharomyces cerevisiae (bakers yeast) is a para‐nitrophenylphosphatase (pNPPase) family of the haloacid dehalogenase (HAD) superfamily. The natural substrate for PHO13 was previously unknown and a knockout phenotype has only been observed in strains engineered to grow on xylose; however, the purified enzyme has been identified in our lab to specifically hydrolyze 2‐phosphoglycolate, classifying it as a phosphoglycolate phosphatase (PGPase). In photosynthetic organisms, 2‐phosphoglycolate is generated by the oxygenation reaction of RuBisCO in the Calvin Cycle. To be recycled back into general metabolism, photosynthetic PGPase converts 2‐phosphoglycolate (2‐PG) to glycolate. In non‐photosynthetic yeast, 2‐PG must be generated by some other mechanism, possibly by oxidative damage to DNA. Regardless of its origin, 2‐PG must be catabolized due to its ability to inhibit triose phosphate isomerase (TPI). Yeast cells lacking functional Pho13p (pho13Δ) are being investigated for growth a phenotype(s) that supports TPI inhibition including the inability to grow in the absence of exogenous inositol. To increase intracellular levels of 2‐PG, cells are being treated with hydrogen peroxide. While many conditions are currently being explored, no phenotype has been apparent under normal growth conditions or in the presence of hydrogen peroxide in complete medium.Support or Funding InformationFunded by NIH AREA Grant GM066715‐01
Entamoeba spp. as Models for the Effects of Environmental Stress in Protists
Authors: Hackey Meagan , Leitao Joshua , Espinosa Avelina 
Published: November 2023

The Entamoeba lineage includes protists residing in the gut of organisms including komodo dragons, turtles, snakes, and humans. Some Entamoeba varieties exhibit pathogenicity. In vitro assays were performed to test environmental conditions such as temperature and pH for impact in growth and survival of Entamoeba. Trophozoites incubated at high temperatures and extreme pH showed significantly reduced cell surface area, indicating stress. Slightly increased temperature and decreased pH values resulted in little change or slightly improved cell health, based on cell shape, confluency, cell density, aggregation, and accumulation of waste. We suggest that one consequence of climate change could be the increase of pathogenic protists such as E. histolytica, E. invadens VK‐1: NZ, and E. invadens IP‐1, which pose a significant threat to human and animal health worldwide. The secretion of small molecular weight proteins in cultures incubated above 37°C was detected. Protein gel electrophoresis and mass spectrometry analysis identified heat shock (HSP70) and chaperonin proteins similar to those identified in aggregation studies. These proteins have been reported in E. invadens, E. histolytica, Giardia lamblia and Trichomonas vaginalis as down regulated under stressful conditions. We are currently exploring how Entamoeba varieties respond to changes in salt concentration. The identification of cell‐signaling proteins that decrease Entamoeba aggregation in response to extracellular conditions could aid in finding alternatives to managing pathogenic Entamoeba spp. in the changing environment.Support or Funding InformationSupported by the Rhode Island National Science Foundation EPSCoR Summer Undergraduate Research Fellowship.
Glycome Sequencing; Are We There Yet?
Authors: REINHOLD VERNON NYE 
Published: November 2023

Sequence challenges provided by the uncoded glycomes of proteins and lipids are, in principle, comparable to the glycosaminoglycans and proteoglycans. But, in neither case, are there protocols or technologies comparable to those of genes and proteins.But the demands of personalized medicine and the early successes of carbohydrate antibodies and vaccines strongly suggest this is a research limitation that must be addressed soon. Described in this brief presentation is an overview of a “bottoms‐up” sequencing approach that extends from the glycans of proteins and lipids to the glycosaminoglycans. After sample preparation all sequencing is dependent on ion fragmentation and spatial resolution in the ion trap (MSn). Here two fundamental fragment ions are basic, the common B‐ion and the more elusive A‐ion. This latter ion is time dependent and is generated by combinations of electron rearrangement and group elimination which importantly varies with linkage point. Such detailed accounting provides a comprehensive spatially acquired sequence.Support or Funding InformationPEG; Common Fund
Location and Partners of Polysialic Acid in the Dorsal Horn and Trigeminal Nucleus of Adult Rat
Authors: Shahbazian Shila , Lee Albert , Everest‐Dass Arun , Bokiniec Phill , Packer Nicolle , Goodchild Ann 
Published: November 2023

Polysialic acid (PSA) is a large cell‐surface glycan predominantly attached to the neural cell adhesion molecule (NCAM) in the mammalian brain. PSA has discrete expression patterns to specific areas of the CNS, postulated to have high levels of synaptic plasticity. The regulation, expression and interacting partners of PSA is complex and remains poorly understood. In this study, we aimed to examine the cellular distribution of PSA by immunohistochemistry in the dorsal horn and trigeminal nucleus of adult Sprague Dawley rats. Both regions receive significant sensory input thus are likely to have high levels of plasticity. We further investigated interacting partners of PSA in these regions using co‐immunoprecipitation (IP) followed by label‐free liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis.We found that PSA was highly expressed in the superficial laminae of the dorsal horn, and within the trigeminal complex distribution was restricted to only the superficial layers of nucleus caudalis (TNc). Using LC‐MS/MS analysis, 14 proteins were identified in the dorsal horn as potential binding partners of PSA‐carrying NCAM. Three candidates (Receptor expression‐enhancing protein 5 (REEP5), Guanine nucleotide‐binding protein G(o) subunit alpha (GNOA1), and Clathrin heavy chain 1 (CLTC) were validated by IP and western blot analyses. Moreover, co‐IP of PSA from TNc preparations followed by western blot analyses of the three candidates confirmed their PSA status within TNc. This is the first study to demonstrate the detailed cellular distribution of PSA in the rat trigeminal nucleus and the identities of three potential interacting partners. These findings will improve our understanding of the mechanisms by which PSA affects synaptic plasticity and may reveal novel PSA functions.Support or Funding InformationAnti‐polysialic acid antibody (mAb735) was kindly provided by prof. Rita Gerardy‐Schahn.
Axin Is Localized within Mitochondria and Regulates Energy Metabolism through Alterations of Oxidative Phosphorylation and Glycolysis
Authors: Shin Jee‐Hye , Kee Sun‐Ho 
Published: November 2023

Axin is a scaffolding protein that antagonizes Wnt signaling and controls various cellular events, involving cell proliferation and death. Tankyrase inhibitors, such as XAV939 stabilizes the axin protein, provide a therapeutic strategy for cancers.To study effects of axin expression on mitochondria, we established two axin‐expressing HeLa cells models: doxycycline‐inducible axin expression and enhanced axin expression by treatment with XAV939. Axin‐expressing cells showed localization of axin in mitochondria, especially the oxidative phosphorylation (OXPHOS) complex IV. Axin reduced complex IV subunits expression and altered supercomplexes formation. Moreover, axin led to functional alteration containing the reduction of cell proliferation, ATP level, complex IV activity, and basal respiration. Despite the respiratory effects, the glycolysis was increased and mitochondrial membrane potential was maintained, which even preserved in the presence of mitochondria‐affecting stimuli in axin‐expressing cells.In conclusion, our results suggest that axin was expressed in complex IV of mitochondria, which modulated mitochondrial function and glycolysis.
MboII Restriction Fragment Length Analysis Of Estrogen Receptor 1 Gene
Authors: Saibu Morounke G , Ayorinde James , Adu Oluwatosin , Faduyile Francis , Wusu Dorcas , Iyapo Oluwadamilare , Kazeem Mutiu , Soyemi Sunday , Izuagie Sekinat 
Published: November 2023

IntroductionCervical cancer is the commonest genital tract malignant neoplasm that affect women worldwide. It is most notable in the lower‐resource countries of sub‐Saharan Africa. Understanding the restriction digestion pattern of Human estrogen receptor‐α gene (ESR‐1) would provide information on the possible mutations on the gene that can serve as potential diagnostic and therapeutic markers.The present work is focused on determining the MboII restriction fragment length on esr1 gene of cervical cancer cells. The elucidation of this is vital in the diagnosis of the cervical cancer diseaseMaterials and MethodPCR technique was used to amplify the promoter region of exon1 of the esr1 gene for ten samples. MboII restriction enzyme specific for the promoter site was used to carry out restriction fragment analysis.Result and DiscussionThe result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme. It is important to note however, that there is need for similar work using large sample size in order to conclusively ascertain the presence of polymorphism in the ESR 1 promoter region and the restriction fragment length of the ESR1 gene. Seven out of the ten samples analyzed for restriction fragment length exhibited the presence of restriction site specific to the MboII restriction endonuclease. These restriction sites are vital for diagnostic purposes.ConclusionThe result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme.Support or Funding InformationNo conflict of Interest Disclosure
The Bioactive Properties of Acetone Extract of Cola Acuminata in Breast Cancer Cells
Authors: McGovern Janachia , Gray Wesley 
Published: November 2023

Breast cancer (BC) is the leading cause of death in women in the United States. Currently there are many treatment options, however the side effects associated with these treatments usually result in a decrease in the standard of life. Previously we reported the identification of a bioactive fraction from the natural product Cola Acuminata (Biz‐3) which is capable of inhibiting cancer cells. In this study we tested the hypothesis that bioactive substances in the Cola Acuminata (Biz‐3) extract contains compounds that may provide a natural treatment option for BC. Thus, our objective was to isolate the bioactive compound from the Biz‐3 and reveal which ones are responsible for inhibiting the MCF 7 breast cancer cell growth. The Biz‐3 extract was isolated using SPE reverse phase chromatography and the resulting analytes of interest were characterized using a combination of thin layer chromatography and UV‐Vis chromatography and a MTT cell inhibitory assay. Elution of the SPE column with solvents of increasing polarity resulted in three distinct ethanol peaks with the majority of the analytes appearing in the 70% ethanol fraction. UV‐Vis spectrum reveals that Biz‐3 contains at least five different compounds absorbing between 210 and 265nn. To determine in which of the SPE peaks the cell inhibitory activity co‐eluted, MCF‐7 cells were induces with different concentrations of the SPE peaks for 1, 3 and 5 days. Our preliminary data suggests that the growth inhibitory activity co‐elutes with the 30% SPE ethanol peak. However, we observed a stimulation of the MCF‐7 cell growth in the presence of the 70% and 100% SPE ethanol peaks. Our overall results demonstrate that the bioactivity present in Biz‐3 can be isolated using SPE and it's likely that two different bioactive compounds are present in Cola Acuminate.Support or Funding InformationNIH grant, Grant No. NIH R15AT006821