bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Structure of EmrE and Its Role in Antibiotic Resistance
Authors: Kenana Jaida , Langat Brenda , Kalicki Colin , Inthavong Emily , Kanna Arrihant 
Published: November 2023

Antibiotic resistance occurs when bacteria evolve different methods of surviving in the presence of antibiotics such as quaternary cationic compounds (QCC). One mechanism that bacteria utilize to attenuate the impact of antibiotics is the use of efflux systems. These systems allow bacteria to expel toxins and other compounds. The bacteria Escherichia coli (E. coli) uses Ethidium multidrug resistance protein E (EmrE) as an efflux system. EmrE is a 110 amino acid protein classified as a small multidrug resistance transporter (SMR). It can be encoded on a chromosome, plasmid, or integron. EmrE transports substrates into the periplasm. In order to do that, EmrE exchanges protons to channel substrates. Those substrates can then be taken up by other efflux systems such as resistance‐nodulation‐division (RND) transporters to be exported to the outer membrane. EmrE is a dual‐topology membrane protein with the functional structure of a homodimer. However, the most functional orientation of the monomers, having either a parallel or antiparallel structure, within the dimer is under debate. Recent studies have concluded that the antiparallel structure is more stable. This structure consists of four transmembrane helical bundles. Helices 1, 2, and 3 reside at the core, where there is a single binding pocket in which all toxic molecules are transported. Some amino acids within the core are highly conserved, indicating that amino acids such as glutamate 14 and asparagine 104 of EmrE are present through several different bacteria. Glutamate 14 is essential to the function of EmrE because mutations at this position can result in differing rates of efflux. When glutamate 14 is mutated into cysteine, EmrE activity is abolished. Also, when glutamate 14 is mutated to asparagine, EmrE does not confer drug resistance. Instead, it slows the rate of EmrE and transports compounds to liposomes. Helix 4 of EmrE is lipid facing and protrudes from the core. Lipid facing amino acids are less conserved compared to those in helices 1, 2, and 3. Often times mutations to the lipid facing amino acids, such as tyrosine 40 being mutated to glycine, occur. This can result in EmrE having modified specificity to different substrates. Though the specific structure of EmrE has been controversial, its antiparallel structure and functionality has been confirmed to be vital in antibiotic resistance as an efflux system.Support or Funding InformationThis is a SMART Team project supported through the contributions of Dr. Joanna Slusky of the University of Kansas, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.
Functional Analysis of Toxoplasma gondii cGMP‐Dependent Protein Kinase Isoforms Using an Auxin‐Inducible Degron System
Authors: Brown Kevin M. , Long Shaojun , Sibley L. David 
Published: November 2023

Toxoplasma gondii is an important obligate intracellular pathogen and model apicomplexan parasite. To cause infection, T. gondii utilizes an active form of invasion that is regulated by cGMP‐dependent protein kinase (PKG). In T. gondii, two isoforms of PKG are produced from the PKG transcript due to alternative translation initiation methionines (M1, M103) (Donald and Liberator 2002). The full length 994 aa type I isoform (PKG I) associates with the plasma membrane via an N‐terminal dual acylation motif. The 892 aa type II isoform (PKG II) lacks the first 102 amino acids and is cytosolic. Whether these isoforms are both required and if they are redundant or functionally distinct has not been determined. To date, functional analyses of T. gondii PKG have relied on small molecule inhibitors of its activity. However, PKG inhibitors are not isoform‐specific and may have off‐target effects in both the parasite and host (Donald et al 2006). To study PKG and other essential genes, we have adapted the auxin‐inducible degron (AID) system (Nishimura et al 2009) for use in T. gondii. Using this system, we are able to target AID‐tagged proteins for proteasomal degradation by addition of auxin (indole‐3‐acetic acid) in strains that heterologously express the plant auxin receptor TIR‐1. Using CRISPR/CAS9 genome editing, we introduced a miniature‐AID (mAID) tag to the C‐terminus of PKG, allowing us to rapidly deplete PKG‐mAID isoforms simultaneously with auxin, resulting in parasite death. By complementing the PKG‐mAID strain with PKG isoform‐specific plasmid constructs, we were able to define unique roles for PKG I and II. Importantly, PKG I is necessary and sufficient for parasite fitness while PKG II is only partially able to support growth and is fully dispensable in the presence of PKG I. Interestingly, these roles could be reversed based the presence or absence of the 15 amino acid N‐terminal dual acylation motif of PKG I. Taken together, our results provide strong evidence that PKG I activity at the plasma membrane is required for T. gondii infectivity. Furthermore, the auxin/AID system offers a new and robust avenue for exploring essential gene functions in T. gondii.Support or Funding InformationPostdoctoral Fellowship (KMB), American Heart Association, (15POST22130001)
Genesis of Antibiotic Resistance XXIII: The River Ganga (RG)‐Segment IVB, secede Hardy‐Weinberg‐Castle Equilibrium (HWCE), and concoct as cataclysmic epicenter of Antibiotic Resistance Pandemic (ARP)
Authors: Martinez Francisco , Fuentes Esai , Montoya Hector , Flores Jorge , Carbajal Carlos , Wickham Cheyenne , Lopez Christian , Johnson Dennie , Kannan Subburaj 
Published: November 2023

An effort has been made to compare the water quality of the City of Eagle Pass Water Works and RG to determine the mechanism of induction of antibiotic resistance (AR) in bacteria (e.g. Fecal coliform bacteria (FC)) known to cause AR infectious diseases. For this purpose, a correlation – regression analysis was carried out to determine the dependency among the following variables: i. Nephelometric Turbidity Units (NTU), ii. Saprobic index (SI) Pantle and Buck 1955) and iii. Incidence of Fecal Coliform (MPN/100ml), F.C 160,000 and iv. Morbidity and Mortality rate due to AR pathogen induced infectious diseases. According to water quality data of RG Segment‐IVB[1], a positive correlation coefficient was observed between increases in NTU (~95.53NTU) paralleled with higher FC count (F.C. (MPN/100ml): ~ higher than 3000 – 6.5×104). Regression coefficient analysis (where y= a + bx) showed a slope=1. This phenomenon is attributed to the above normal Biological Oxygen Demand (BOD) ~ 95.04TPD due to the influx of an untreated sewage 1780 million liter/ day (MLD) (54 sewage drains) along with 22 Grossly Polluting Industries (GPI) discharging an average of 87 MLD(15.6 BOD), pose unknown quantities of mutagenic agents. SI, S=5.44[2](strongly polluted water) showed a positive correlation with FC count and NTU. Data on mortality and morbidity rate (M&M) due to incidence of AR pathogen induced infectious diseases in RG‐IVb by both Government of West Bengal and Government of India is incoherent. As a result, no correlation was found. Data analysis from this study shows that an alarming trend of ever increasing pollution loads with an unknown mutagenic potential. Taken together, in comparison to CEPWW water quality data, it is concluded that the aforesaid factors in RG IVb mount a cause for a serious concern as an a major contributing factor prompting an “Infinite allelic” model in AR gene(s), rustling a “Founder Effect” in FC with an allelic fix accentuating an ARP. Data analysis to be presented in EB 2017.Support or Funding InformationProfessional development funds to Subburaj Kannan
Genesis of Antibiotic Resistance XXV: Mitigation of antibiotics resistance pandemic (ARP) by immediate moratorium on effluent discharge practices from antibiotics manufacturing pharmaceutical industry
Authors: Carbajal Carlos , Fuentes Esai , Wickham Cheyenne , Lopez Christian , Carrizales Diana , Carrizales Tania , Jauregui Suzzette , Thomas Ryan , Lechler Raul , Beautnagel Joanathan , Johnson Dennie , Kannan Subburaj 
Published: November 2023

To date, no substantive coefficient and regression analysis has been performed to explore and determine correlative data that is concerned with the cause and effect relationship of the incidence of antibiotic resistance in bacteria and the concentration of antibiotics in the effluents from antibiotic manufacturing processes in the pharmaceutical industry. In order to actualize correlative data, four variables were identified and compared to ascertain the cause and effect relationship as may contribute to ARP. The following variables were prosecuted: j Concentration of the Active Pharmaceutical Ingredients (API), break down of intermediate / metabolite(s), decomposition isomer(s) in the effluents discharged by the antibiotics manufacturing industries (Global Scale: India, China, United States, Turkey, France, Nigeria, and Brazil); k Evolution of AR resistance pathogen as determined by Culture‐Sensitivity(C‐S lab) (phenotypic/genotypic); l Epidemiological data on morbidity‐mortality rate due to the incidence of AR pathogen(s) induced infectious diseases (county‐village/city/health district/state/country/continental); and m Detection of API, decomposition isomers (active vs. inactive@ based on Fenton Reaction) in the water resources [e.g.: ground water table, surface, stagnant, storm water runoff, bioremediation by chlorination/membrane filtration/ Ozonization / Flocculation / advanced oxidation / portable water – purified / nonpurified]. Based on the correlation coefficient analysis, a linear – positive correlation was observed between variable j and k. Though the data for variable l was limited, no correlation was observed between variable j and l (where r = 0). A similar trend was observed between the variables j and m (where r = 0). The regression coefficient among the variables was not symmetric as bxy≠byx. Data sources were Appl Envir Micro 64(9)3499–3502(1998); 76, 3444–3451(2010); Water Res. 42(1–2) 307–317(2008); Env. Toxi.Chem. 28(12)2522–2527(2009). Until the “Best Available Techniques” for detoxification and reduction in the discharge of API from Ab manufacturing industries are implemented on a global scale, current practices pose plausible cause for concern for API. Data analysis to be presented in EB2017.Support or Funding InformationProfessional development funds to Subburaj Kannan
O‐GlcNAc is Critical for the Regulation of Energy Metabolism
Authors: Tan Ee Phie , McGreal Steven , Graw Stefan , Tessman Robert , Koppel Scott , Dhakal Pramod , Zhang Zhen , Machacek Miranda , Soares Michael J , Zachara Natasha , Koestler Devin , Peterson Kenneth R , Thyfault John P , Swerdlow Russell H , Kasturi Partha , Apte Udayan , Slawson Chad 
Published: November 2023

O‐linked N‐acetylglucosamine (O‐GlcNAc) is the attachment of a single β‐N‐acetylglucosamine to serine/threonine amino acid residues of nuclear, cytoplasmic, and mitochondrial proteins. The modification is sensitive to changes in the cellular environment and is dynamically regulated by the opposing functions of two specific enzymes; O‐GlcNAc transferase (OGT) adds the modification, whereas, O‐GlcNAcase (OGA) removes it. Disruptions in O‐GlcNAc cycling contribute to diseases such as neurodegeneration. Dysfunctional mitochondria lead to neurodegenerative diseases, and importantly, O‐GlcNAcylation regulates mitochondrial function. Previously, we found in a proteomic‐based study that OGT or OGA overexpression disrupted mitochondrial protein expression, including electron transport chain (ETC) and TCA cycle proteins. Here, we hypothesized that prolonged elevations in cellular O‐GlcNAc levels would decrease mitochondrial function. We elevated cellular O‐GlcNAc levels by either treating SH‐SY5Y cells with low levels of Glucosamine (GlcN), the metabolic substrate of OGT, or OGA inhibitor Thiamet‐G (TMG). We found that mitochondrial respiration and ATP production rates are lower in these cells. Additionally, these cells produce significantly less reactive oxygen species (ROS). We found that mitochondria were longer and mitochondrial fusion/fission protein expressions were decreased. Next, we performed RNA‐Seq analysis and found that 21 mitochondrial‐encoded genes are affected by the treatment. We found the nuclear factor erythroid 2 (NRF2) pathway that controls cellular ROS response was down regulated in these O‐GlcNAc elevated cells. Next, we found that C57B/6J mice dosed with TMG display a similar phenotype as SY5Y cells. Mitochondria isolated from mice brain had lower respiration and ROS levels, and decreased NRF2 response compared to control. Altogether, our results point towards prolonged elevations of O‐GlcNAc modulating mitochondrial function by influencing mitochondrial transcriptional programs, and that O‐GlcNAc affects ROS production likely via adjusting ETC activity and skewing its substrate (NAD+/NADH) availability. Furthermore, prolonged elevations of O‐GlcNAc impacts mitochondrial ROS production influencing the NRF2 antioxidant response. Importantly, these findings demonstrate the critical role for O‐GlcNAc cycling in the regulation of metabolism and will provide new insights into metabolic diseases such as Alzheimer's.Support or Funding InformationBiomedical Research Training Program Fellowship, Mabel A. Woodyard Fellowship, National Institute of Diabetes and Digestive and Kidney Diseases R01DK100595 to C. Slawson
Inflammatory stress induces a biphasic Nrf2 activation in neuronal cells
Authors: Krepel Stacey A , O'Hara Lars , Mangum Sally , Hogan Mary B , Kuhn Thomas B 
Published: November 2023

Persistent inflammatory and oxidative stress (IOS) is largely responsible for the progressive loss of neuronal integrity and connectivity underlying the continuous decline of cognitive function associated with most chronic CNS disorders as well as normal aging. The transcription factor Nrf2 plays a pivotal role in the antioxidant defense of non‐neuronal and neuronal cells. Nrf2 remains sequestered in the cytosol of cells under basal conditions tightly bound to its repressor Keap1. Upon oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus to stimulate transcription of antioxidant defense mechanisms. It is not yet clear how duration and extent of IOS impact Nrf2 activation and nuclear translocation in neuronal cells. Exposure of SH‐SY5Y human neuroblastoma to 200 ng/ml TNFa caused a time dependent Nrf2 translocation (2 fold increase) into the nucleus peaking 4 h after addition. However, nuclear Nrf2 decreased and remained at 50% of peak maximum for a prolonged time period. A presence of 50 ng/ml TNFa resulted in the highest accumulation of Nrf2 in the nucleus—giving a 53% increase of translocation relative to the control—compared to higher concentrations (100 to 400 ng/ml) even over prolonged periods of time. Immunocytochemistry against Nrf2 corroborated these findings. Additionally, prolonged exposure of SH‐SY5Y cells to TNFa (24 h) at concentrations 200 ng/mL or higher resulted in significant cell death due to oxidative stress. At high concentrations of 200 ng/mL and 400 ng/mL, even short treatment times of 1 hour resulted in the respective 52% and 112% reduction in Nrf2 translocation relative to the peak translocation achieved at 50 ng/mL. Together these findings suggest that Nrf2‐mediated antioxidant defenses in response to high concentrations of either chronic or acute TNFa exposure were insufficient and even depressed. One of the many health benefits of nutrition rests on the capacity of distinct botanicals, in particular polyphenols, to directly act on biochemical mechanisms rather than through passive antioxidant capacities. We demonstrated that supplementation of SH‐SY5Y neuroblastoma cells with extracts obtained from Alaska wild bog blueberries (5 mg/ml) increased neuronal viability in the prolonged presence of 200 ng/mL TNFa. Surprisingly, blueberry extracts prevent Nrf2 nuclear accumulation upon exposure to TNFa. Understanding the regulation of Nrf2 through botanicals could provide vital insight for developing neuroprotective strategies applicable to an array of neurodegenerative diseases.Support or Funding InformationSupported in part by NIH grants RL56M118990, 1UL16M18991, TL4GM118992, and generous donations from the Alzheimer's Resources of Alaska. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health
Lipid Stress Alters Cell Distribution, Traffic, and Desensitization Properties of Melanocortin‐4 Receptor, a GPCR Involved in Appetite Control
Authors: Cooney Kimberly , Molden Brent , Russell Susan , Baldini Giulia 
Published: November 2023

Melanocortin‐4 Receptor (MC4R) is a G‐protein coupled receptor expressed in the brain hypothalamus where it regulates food intake and energy expenditure. MC4R signals in response to its natural agonist, α‐MSH, by increasing intracellular cAMP. Obesity is often associated with dyslipidemia, a condition where the concentration of circulating Non‐Esterified Fatty Acids (NEFAs), such as palmitic acid, is increased. It is not clear how a high‐fat diet effects the function of MC4R. We have modeled effects of lipid stress on MC4R function by exposing neuronal Neuro2A cells and immortalized hypothalamic mHypoE‐42 neurons to elevated concentrations of palmitate within the physiological range. Using confocal microscopy we found that lipid stress changes the cell shape of Neuro2A cells and inhibits internalization of transferrin receptor both in these cells and in the mHypoE‐42 neurons. Super‐resolution fluorescence microscopy indicates that exposure to lipid stress inhibits transit of tagged HA‐MC4R‐GFP to early endosomes by halting exit of the receptor from clathrin‐coated pits. In addition, lipid stress blunts desensitization of MC4R in response to the synthetic agonist Melanotan II (MTII). A single intraperitoneal injection of MTII decreases weight in obese male C57BL/6J mice exposed to high fat diet, but not in lean mice. These studies provide evidence that lipid stress may unexpectedly promote an increased response to anti‐obesity treatments targeted to MC4R by changing traffic of the receptor.Support or Funding InformationThis work was funded by NIH Grants R01‐DK102206 (to G.B.) and by Intramural Funding Support from the UAMS Research Council
′Ome is where the Wound is: Biomarkers of Healing in Chronic Diabetic Foot Ulcers
Authors: Glucksman Marc , Philibert Keith , Shao Xiaomeng , Yang Charlie , Ortiz Jacque , Wu Stephanie 
Published: November 2023

Of the approximate 30 million Americans that suffer from Type 2 diabetes, 25% will develop a foot ulcer during their lifetime that often exhibit impaired healing with a cost of diagnosis and treatment exceeding $250 billion per year. These chronic wounds require continuous care and treatment to promote healing and prevent infection. To provide insight into the biochemical and molecular mechanisms underlying diabetic foot ulceration that are currently unclear, we sought to define biomarkers as an objective biologic measurement and evaluation of the normal versus pathological biological processes leading to wound healing. The objective is to discover and validate proteins that may predict what patient will heal or not. These biomarkers can thus be used for diagnosis, prognosis and for targeted therapies. Our approach was to obtain patient plasma samples from current diabetic and non‐diabetic patients with foot ulcers and utilize both genomic and proteomic approaches to discover lead molecules.Debridement of the wound yielded the sample, blood collected (aliquot of whole blood or serum was for RNA analysis), centrifugation and protease inhibitors added, aliquoted and frozen. One aliquot was used for Luminex 18 flow cytometry‐based cytokine analyses. Another aliquot was immunodepleted of the 12 most abundant proteins comprising 95% of total plasma protein content and separated and identified by electrospray ionization mass spectrometry. Bioinformatics was then used for protein identification and quantification of samples. Patients returned bimonthly for medical assessment: wound size, cardiovascular, diabetes, and physical data with follow‐up samples obtained and processed accordingly.The proteomics and genomics discovery phases yielded an increase in RANTES (CCL5), CXCL2 (GRO2), and Leucine Rich Alpha‐2‐ glycoprotein in non‐healing patients whereas there was an increase in interleukin IL‐8, granulocyte colony stimulating factor (G‐CSF), colony stimulating factor‐2, matrix metalloprotease 7, Alpha‐2‐HS‐glycoprotein, and Kininogen 1 in healing patients. We are currently validating these results in archived patient samples and assaying more patients to yield a further understanding of the factors for prediction of wound healing in diabetic foot ulcers. Identification of biomarkers of wound healing in DFU will allow for better clinical diagnosis, treatments and new targets, to prevent complications and non‐traumatic amputations to improve patient quality of life and reduce financial burdens.Support or Funding InformationSupported by a Pilot Grant from RFUMS/DePaul University and NIH OD010662 (MJG).
Regulation of Hepatic Inflammation by Folic Acid in Non‐alcoholic Fatty Liver Disease (NAFLD)
Authors: Sid Victoria , Shang Yvette , Siow Chris , Karmin O 
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a multifaceted liver disorder that often exists with the comorbidities of obesity and type 2 diabetes. The histopathology of NAFLD is characterized by steatosis, inflammation, fibrosis, and liver injury. Chronic hepatic inflammation is an important pathogenic mediator of NAFLD that contributes to disease severity. The progression of NAFLD may be associated with development of cardiovascular disease which is the primary cause of death in patients. Dietary supplementation of vitamins is important for health maintenance, and has been suggested to be a beneficial strategy for NAFLD management. Folic acid is a water soluble B vitamin that has been demonstrated to have lipid‐lowering and anti‐oxidant effects. The objective of our study was to investigate the effects of folic acid supplementation on hepatic inflammation and to identify the underlying mechanisms. Male C57Bl/6J mice were fed a control diet (10% kcal fat), a high‐fat diet (60% kcal fat), or a high‐fat diet supplemented with folic acid (26mg/kg diet) for a 5 week period. High‐fat diet feeding induced a significant body weight gain and increased aggregation of inflammatory foci in the liver. Folic acid supplementation did not alter the body weight of mice fed a high‐fat diet. However, folic acid supplementation reduced the number of inflammatory foci as well as lipid vacuoles in the liver of high‐fat diet fed mice. This correlated with reduced expression of pro‐inflammatory cytokines mediated through attenuation of NF‐κB transcription activity, an inflammatory transcriptional regulator. Our results suggest that folic acid supplementation can alleviate the hepatic inflammatory response induced by chronic consumption of high‐fat diets, which may contribute to the hepatoprotective effect by folic acid.Support or Funding InformationThis study was supported, in part, by NSERC and CIHR.