bluuebunny/crossref_metadata_2025_split huggingface dataset:
Manipulating the Bone Marrow Microenvironment to Prevent Survival of AML Cells
Authors: Sterner Rosalie M. , Kremer Kimberly N. , Dudakovic Amel , Westendorf Jennifer J. , van Wijnen Andre J. , Hedin Karen E. 
Published: November 2023

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication.Support or Funding InformationThis study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.
ATG5 knockout leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2
Authors: Lee Michael , Hwang Sung‐Hee 
Published: November 2023

We previously demonstrated that modulators of autophagy might be used beneficially as adjunctive therapeutic agents during the treatment of cancers with Src tyrosine kinase specific inhibitor PP2. Here, we investigated the role of autophagy in cell growth by using established ATG5 knockout (KO) cell line with CRISPR/Cas9 system. The conversion of non‐autophagic LC3‐I to autophagic LC3‐II and RT‐PCR confirmed the functional gene knockout. Interestingly, the growth of ATG5 KO cells was significantly enhanced compared to wild type (WT) cells. In particular, autophagy‐deficiency in NIH 3T3 cells enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and soft agar colony formation assay. Furthermore, ATG5 KO cells were more resistant to PP2 treatment than were WT cells. Additionally, the proportion of apoptotic cells significantly decreased when treated with PP2 in ATG5 KO cells compared with WT cells. Interestingly, we found that expression levels of p53 were much stronger in ATG5 KO cells, which showed lower apoptosis rates than did WT cells. ATG5 knockout is thought to be lead to a compensatory increase in p53 proteins due to a decreased apoptosis rate. We also found the marked up‐regulation of p‐ERK and p‐MEK in ATG5 KO cells, suggesting that the promotion of PP2 resistance by ATG5 knockout was associated with elevated levels of p‐MEK and p‐ERK. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to anti‐cancer therapy.Support or Funding InformationGrant Funding Source: Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016R1D1A1B03930193)
(A3)Beating Cancer: Impact of APOBEC3B on 5‐Fluorouracil Treatment
Authors: Fine Sydney R 
Published: November 2023

The APOBEC3 family of cytosine deaminases consists of seven members that function as part of the innate immunity. One family member, APOBEC3B (A3B), has been implicated as an endogenous source of mutation in cancer cells, specifically in head/neck, lung, cervical and breast cancers. A3B increases the rate of mutation in DNA through its ability to deaminate cytosine into uracil. The aim of this study was to determine if A3B over‐expression synergizes with 5‐Fluorouracil (5‐FU), an FDA‐approved chemotherapeutic agent, to promote cell death. 5‐FU promotes cell death through two mechanisms. First, 5‐FU inhibits thymidylate synthase, an enzyme that converts deoxyuridylate to deoxythymidylate. Second, 5‐FU becomes incorporated into DNA, which causes significant DNA damage. To investigate the hypothesis, A3B was over‐expressed in a mammary epithelial cell line, MCF10A. Then, the cells were treated with various concentrations of 5‐FU. Cell viability in response to 5‐FU treatment was assessed by clonogenic survival assays, which measure cells' ability to proliferate. It was determined that up‐regulation of A3B had little to no effect on the efficacy of 5‐FU. This research has implications in regards to personalized treatment in cancer patients with A3B‐high tumors because treating these tumors with 5‐FU may widen the therapeutic window and allow for more effective disease treatment.Support or Funding InformationThe Harris lab is supported by grants from the NIH. Dr. Harris is an Investigator of the Howard Hughes Medical Institute. The NIH provides funding for the Cancer Research Education & Training Experience.
Evaluation of the immune response induced in mice by a recombinant form of SPO‐1, a Schistosoma mansoni modulatory protein
Authors: de Oliveira Santos Bernardes Wilma Patricia , Alves Clarice Carvalho , Pereira Rosiane Aparecida , Fonseca Cristina Toscano 
Published: November 2023

The S. mansoni tegument (Smteg) represents an important source of antigens to vaccine formulations. However the tegument is also composed of molecules that are involved in the modulation of the host immune system to favour parasite survival. Using immune proteomic tools we have identified proteins from the tegument that were recognized by sera from Smteg‐immunized mice and thus might be involved in the induction of the protective immunity observed in these animals. One of these proteins is SPO‐1, also known as Sm16. This protein has been described as a potent anti‐inflammatory response inducer in the host. The aim of this study was to characterize SPO‐1 expression in the parasite and the immune response trigger by the recombinant form of SPO‐1(rSPO‐1) in mice. To obtain the rSPO‐1, the gene sequence coding for amino acid 23‐90 were sub cloned into the expression vector Pet21a. Escherichia coli BL21 strain were transfected with this construction and the expression was induced using 1mM of Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). A recombinant protein of approximately 12 kDa fused with a histidine tag was obtained and purified using a niquel column. Using an approved protocol from Brazilian Ethical Committee (LW25/15), Balb‐c mice were immunized with rSPO‐1 and their sera were obtained to be used in Western Blot assay to determine SPO‐1 expression levels through parasite life stage. Protein extracts of the different parasite life stage were also obtained. The results showed that SPO‐1 is mostly expressed in cercariae, 3‐hour schistosomulum and 7‐day schistosomulum. To evaluate the immune response induced in mice by rSPO‐1, 7 mice were intraperitoneally inoculated with 25 μg of rSPO‐1 and one week later their spleen were collected to determine lymphocyte activation status and subtypes. A lower frequency of IL‐10‐producer B cells (p<0.02) and activated CD8+T cells (p<0.02) in SPO‐1‐inoculated animals was observed in comparison to saline‐inoculated mice. Moreover, there was no difference in the frequency of regulatory T cells (CD4+CD25+Foxp3+) or activated CD4+ cells (CD4+CD25+) between groups. An increased percentage of CD4+ cells producing interferon gamma and IL‐4 was observed after in vitro SPO‐1 stimulation in both groups. However, rSPO‐1 was unable to trigger IL‐10 production by CD4 T cells. In conclusion, despite being described in the literature as a regulatory protein, Spo‐1 was unable to induce this profile in the adaptive immunity. Thus, it is important to characterize the effect of SPO‐1 inoculation in myeloid lineage cells.Support or Funding InformationFapemig, CNPq, Capes, CPqRR, Proep/CPqRR
Polar Lipid in Human Blood Regulates Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum
Authors: Gerdt Joseph P. , Brancucci Nicolas M.B. , Wang Charley , Adapa Swamy R. , Zhang Min , Adams John H. , Jiang Rays H.Y. , Marti Matthias , Clardy Jon 
Published: November 2023

For Plasmodium falciparum to infect hundreds of millions of people annually, it must first be transmitted by a mosquito vector. Only the parasite cells in human blood that have matured into sexual precursor cells (gametocytes) are capable of transferring to mosquitos. Therefore, understanding the regulation of gametocyte development in humans will provide avenues to halt the spread of malaria. Toward this end, we developed a new in vitro gametocytogenesis assay and used it to test the hypothesis that environmental factors exist in human blood that regulate P. falciparum sexual commitment. Our hypothesis was confirmed when we identified a polar lipid extracted from human blood that is sufficient to block gametocytogenesis at physiological concentrations. Fluorescence microscopy and metabolomics analysis revealed that the parasites readily uptake and metabolize this lipid. We furthermore found that parasites that are starved of this lipid upregulate alternative metabolic pathways to restore it. Therefore, we conclude that asexual blood stage P. falciparum senses its environment in the human host; under lipid starvation, the parasite converts to the transmissible gametocyte stage. This finding can be leveraged to identify molecular targets against the spread of malaria.Support or Funding InformationJ.P.G. was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award F32 GM116205 from NIGMS. This research was also supported by NIGMS award R01 GM086258 to J.C.
Anti‐fluoroquinolone resistance activity of E558: A natural product
Authors: Iyanda‐Joel Wisdom Oluwayemi , Omonigbehin Emmanuel Adedayo , Iweala Emeka Eze J. , Chinedu Shalom Nwodo 
Published: November 2023

Fluoroquinolone resistance (FQR) has paralleled the abuse of this class of antibiotics in Nigeria ever since the patent rights expired in 2003. However, nature has a potent way of uncoupling unfavourable synergies targeted against the serenity of the biosphere. In this preliminary study, we examined the antibacterial activity of E558, a medicinal plant product against multidrug‐resistant gram‐negative isolates from immunocompromised patients with acute respiratory infections. The agar‐well diffusion method was employed to screen for antibacterial activity against strains of Klebsiella pneuminiae, Escherichia coli and Pseudomonas aeruginosa. The organisms were initially subcultured on McConkey agar followed by streaking standard innoculum of the bacterial strains on Mueller‐Hinton agar plates. A known concentration of crude extract was loaded into the well bored on the plates and then incubated at 37 °C for 24 hours. Antibiotic susceptibility testing (AST) was carried out to monitor resistance of isolates. Protein profiling with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as well as restriction endonuclease mapping with agarose gel electrophoresis was employed to study alterations in the genomic DNA of selected organisms. A reverse mutation was observed in the morphology of organisms exposed to E558 compared to the organism without exposure. AST result further revealed that strains in the reverted zone, which were originally resistant to fluoroquinolones and other antibiotics had become susceptible with very wide zone of inhibition (≥ 40 mm; CLSI‐sensitive). Restriction mapping and SDS‐PAGE respectively revealed there are major differences in the genomic composition and protein profile of the reverted strains when compared to the original organisms. It can therefore be deduced that E558 possibly contains bioactive compound(s) with potent antitumour and anti‐fluoroquinolone resistance activity.Support or Funding InformationOrganization for Women in Science for the Developing World (OWSD), Trieste, Italy.Covenant University, Ota, Nigeria
Genesis of Antibiotic Resistance (AR) XXX‐Mitigation of AR Pandemic (ARP) through Rational Measures.
Authors: Blackmon Sarah , Francisco Martinez , Montoya Hector , Flores Jorge , Anaya Edson , Martinez Juan , Calzoncit Melissa , Picazo Antonio , Jauregui Suzzette , Lechler Raul , Beautnagel Joanathan , Gutierrez Sonia , Johnson Dennie , Kannan Subburaj 
Published: November 2023

Comprehensive and bolstered analysis of data available in domestic and global quantitative data bases provides the following reasonable and implementable measures that have the potential to mitigate Antibiotic Resistance Pandemic (ARP). The establishment of a centralized national and global data base and infrastructure that will: i. Monitor the amount of antibiotics manufactured per year, ii. Require periodic reporting (Quarterly/semi‐annual/annual) of the volume of antibiotics utilized on a regional, national and global scale, iii. Evaluate the volume of prescription antibiotics recovered by the “National Drug Take Back Program” sponsored cooperatively by the United States Drug Enforcement Agency, Department of Justice, and the Food and Drug Administration (DEA, DOJ, and FDA), iv. Survey and audit the process of disposal of unused prescription antibiotics by the DEA, DOJ, and FDA to ensure that employed destructive practices are conventional and appropriate. Particular oversight and monitoring must be given to incineration and solid waste facilities, v. Detection and evaluative analysis of metabolic breakdown, intermediate, and mother formulation of antibiotics in ground water, portable water systems, raw sewage and waste water, water captured from bio‐remediation processes, rivers, standing storage water reservoirs, storm runoff, and bio‐solids, vi. Collect and maintain epidemiological data on morbidity and mortality rates attributed to AR pathogenic induced infectious diseases on regional, national, and global scales, vii. Collection of data concerned with the effectiveness of antibiotics timeout (ATO) from private practice physicians, institutional reports from mental health facilities and hospitals, both private and public, nursing homes, hospice care, and infant and adult day care facilities, viii. Arrange and contribute to periodic updates concerned with ARP threats discovered or supposed by the United States Department of Health and Human Services Centers for Disease Control and Prevention (CDCP), ix. Research, collection, and reporting of data attentive to the hoarding of antibiotics and partitioning data to regional, national, and global levels, x. Data surveillance of the efficacy of antibiotics, and xi. Elimination of ineffective antibiotics(antibiotics with an increased morbidity and mortality rate) from the pharmacopeia and clinical practice. Taken together, effective universal adoption and implementation of aforesaid measures in health care facilities, and regulatory agencies across the globe, would potentially mitigate the ARP.Support or Funding InformationProfessional development funds to Subburaj Kannan
Gyrase Inhibition by Toxin‐Antitoxin Modules
Authors: Bourne Christina R , White John C , Dabadi Shivani , Muthuramalingam Meenakumari 
Published: November 2023

Toxin‐Antitoxin (TA) modules are interacting cytosolic protein pairs found in most prokaryotes that can mediate metabolic adaptation by interacting with essential cellular machinery. TA modules are organized into families with highly similar structures yet poorly conserved sequences. Among these, the RelE/ParE superfamily has conserved protein folds but disparate cellular functions. Interactions of RelE‐type toxins with the ribosome, resulting in translational inhibition, have been well characterized. In contrast, the interactions of ParE toxins with their target, DNA gyrase, have remained elusive.The objective of this study was to determine the potency of individual ParE toxin proteins with respect to gyrase inhibition. This was determined by changes in cell morphology when the toxins were over‐expressed, and directly using in vitro inhibition assays. Through these studies we have identified a severely truncated ParE toxin in Agrobacterium tumefaciens that maintains an ability to inhibit DNA gyrase. We have also discovered a sub‐family member within this organism that closely matches the E. coli YoeB translation‐inhibiting toxin, however, it effectively inhibits DNA gyrase. Continuing studies are evaluating the propensity of RelE/ParE superfamily members to interact with DNA gyrase. Among over‐expressed ParE toxins, characteristic patterns of filamentatous cell morphology are consistent with phylogenetic sequence analyses. In conclusion, these results unexpectedly revealed that selected members of this superfamily can carry out overlapping functions. Ongoing studies are pursuing shared sequence features dictating these resulting phenotypes, as well as the structural interactions between the toxins and their cellular target.Support or Funding InformationThis research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103640.
Streptococcus mitis and Streptococcus oralis Mutate an ‘Essential’” Gene Upon Exposure to Daptomycin
Authors: Adams Hannah , Joyce Luke , Guan Ziqiang , Akins Ronda , Palmer Kelli 
Published: November 2023

Viridans Group Streptococci (VGS) are Gram‐positive opportunistic pathogens which are part of the normal gastrointestinal and oropharyngeal flora. Clinically, VGS are causative agents of infective endocarditis and bacteremia in immuno‐suppressed and neutropenic patients. Multidrug resistance (MDR) is on the rise within the VGS, as many isolates are now resistant to ampicillin, fluoroquinolones and macrolides. As with most pathogens, novel treatment methods are needed to combat VGS infections. Daptomycin (DAP) is a lipopeptide antibiotic with bactericidal activity against Gram‐positive bacteria. DAP has been shown to be effective against MDR infections, including methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant enterococci (VRE). Previously, Akins et al obtained DAP‐resistant VGS strains after overnight exposure to DAP in a simulated endocardial vegetation model. Using two Streptococcus oralis strains (1647 and 1648) and one Streptococcus mitis strain (1643), we demonstrated that DAP resistance up to 256 μg/mL could be obtained overnight in standard laboratory conditions with a single 4 μg/mL DAP exposure. Using whole genome sequencing, we identified loss‐of‐function mutations in the gene cdsA for all three VGS strains. These mutations reverted when the resistant strains were serially passaged until susceptibility was observed. cdsA is essential in better studied streptococci such as S. pneumoniae and S. pyogenes. CdsA is responsible for catalyzing the conversion of phosphatidic acid (PA) into cytidine diphosphate‐diacylglycerol (CDP‐DAG), which is a key intermediate in the biosynthesis of major phospholipids found in bacterial membranes, including phosphatidylglycerol (PG) and cardiolipin (CL). Using liquid chromatography/mass spectrometry (LC/MS), we confirmed CdsA loss‐of‐function mutations in each of the DAP‐resistant strains, as evidenced by the absence of PG and CL, and the accumulation of PA in these strains. In addition, we have identified a new glycolipid in our S. mitis strain. These data show that DAP resistance proceeds through a novel adaptive pathway in S. mitis and S. oralis as compared to MRSA and VRE; that cdsA is non‐essential in these VGS and that adaptation to DAP results in altered membrane lipid composition in these organisms.Support or Funding InformationStart‐up funds from UT‐Dallas to KP and GM069338 and EY023666 from NIH to ZG.
Biochemical characterization of Type II Toxin‐Antitoxin module from Pseudomonas aeruginosa
Authors: Muthuramalingam Meenakumari , White John C , Bourne Christina R 
Published: November 2023

To date numerous putative Toxin‐Antitoxin (TA) loci have been reported, and these consist of a dicistronic operon encoding two genes; one for antibacterial toxic protein and another for antitoxin which neutralizes its specific toxin (Leplae et al., 2011). TA systems have been reported to play numerous physiological roles, including formation of persister cells (Maisonneuve et al., 2011 & Li et al., 2016), stress resistance (Christensen et al., 2001), protection from bacteriophages (Koga et al., 2011) and regulation of biofilm formation (Wang et al., 2011). Further, overexpression of a Hig‐type toxin affects the virulence factor production that is crucial for modulating Pseudomonas infection (Wood TL, Wood TK. 2016).The goal of this study is to characterize a highly conserved member of the ParE/RelE superfamily from Pseudomonas aeruginosa. Toxin and antitoxin genes were cloned into the pHERD shuttle expression vector for propagation in both E. coli and P. aeruginosa. Heterologous over‐expression of the toxin in E.coli promoted a filamentous phenotype and decreased viability based on enumerating the colony forming unit, while overexpression of the antitoxin did not affect the cell count or the phenotype. We developed purification strategies to purify RelE toxin in the absence of any binding partners, as well as the RelBE protein complex. Gel filtration purification revealed mixtures of dimers of both proteins in complex (RelB2E2) and also fractions with molecular weight matching the free RelE monomer mass. Upon examining the sequence of this RelE toxin, the longer helices associated with a ParE gyrase inhibiting toxin were evident. Experimental testing has clearly demonstrated the ability of this toxin to inhibit DNA gyrase, and studies are underway to evaluate potential ribonuclease function as expected from its annotation. However, based on the longer helices, the region important for specificity for cellular targets, we expect that this RelE toxin is instead a ParE toxin. Additionally we have been successful in obtaining crystals of RelE and evaluated their diffraction properties at the OU Macromolecular crystallography facility, which yielded weak reflections to 4.5 Å. Initial crystallization condition for the RelBE complex have been identified and current efforts are focused on optimizing these for analysis by crystallography. The determination of the structure of this TA module will be the first from P. aeruginosa. Our overall goal of combining structural features and functionality of this TA module will facilitate understanding its role in bacterial physiology, a promising approach for combating this multidrug resistant pathogen.Support or Funding InformationThis work was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health.